CA2457287C - Production and use of human cd124-and cd116-positive tumor cell lines in the production of allogenic or semi-allogenic immunotherapy agents - Google Patents

Abstract

Disclosed is a method for the production and use of CD124+ and CD116+ cell lines in the production of effective dendritic cells (DC) with the aid of stimulatory molecules.

Description

Production and use of human CD124- and CD116-positive tumor cell lines in the production of allogenic or semi-allogenic immunotherapy agents Description The invention describes the production and use of CD124+
and CD116+ cell lines for the production of effective den-dritic cells (DC) using stimulatory molecules, their use in the production of allogenic or semi-allogenic immuno-therapeutic agents and the use thereof in the treatment or prophylaxis of immune diseases. Furthermore, the invention describes the use of CD124+ and CD116+ tumor cell lines, preferably also being CD34+, as model and test systems for testing the DC biology and for testing substances having an impact on the immune system and on the conditioning thereof.
Dendritic cells (DC) play an important role as antigen-presenting cells (APC). They transmit co-stimulatory sig-nals required for T cell activation and induce primary im-mune responses by presenting antigens to CD4 and CD8+
T cells (Banchereau et al. 1998, Nature 392 (6673), 245-252). DCs develop from hematopoietic precursor cells in the bone marrow, going through sequentially different stages of differentiation (intermediary precursor cells in blood and .
immature DCs in peripheral tissues and organs) (Banchereau et al. 2000, Ann. Rev. Immunol. 18, 767-811). Once having reached the tissue, immature DCs (iDC) assume an important sensor function which is characterized by a high active up-take of antigens from the surrounding medium. Following stimulation by external signals ("danger signals") such as bacterial or viral infections or inflammatory processes, the DCs migrate into the peripheral lymphatic organs, there

- 2 -undergoing differentiation into mature DCs, and activating T cells by presenting antigens.
According to previous methods for the in vitro production of DCs, two major populations of DC precursor cells are ob-tained: CD1a+/CD14- cells developing into Langerhans cells (LC), and CD1a.1CD14+ cells differentiating into intersti-tial DCs. Following culturing with GM-CSF and IL-4, mono-cytes can develop a phenotype which is similar to that of immature DCs (iDC). Further differentiation and maturing is achieved by various stimuli such as bacterial lipopolysac-charides (LPS), TNFalpha, PGE2, CD40 ligand or polyIC.
Well-defined culturing systems available so far have been used to investigate the DC biology. However, their use in large-scale experiments is limited, depending on the avail-ability of donor material and the variability thereof. In the murine system, cytokine (GM-CSF)-dependent dendritic cell lines were found to be highly valuable when studying the DC differentiation and development in in vitro and in vivo disease models. Such cell lines were obtained by im-mortalization of murine lymphatic or cutaneous tissues.
They represent an immature DC phenotype, which is invari-able, and therefore do not allow investigations concerning various factors involved in DC differentiation. Further-more, due to the heterogeneity of DCs in the murine and hu-man systems, statements as to the DC biology in humans, if at all, are only possible to a very limited extent.
It has been observed that tumors of lymphoid or myeloid origin have features in common with APC in the ontogenesis.
Studies on PBMC from patients suffering from chronic mye-loid leukemia (CML) and acute myeloid leukemia (ANL) have shown that cytokines in subpopulations of CML and AML
blast cells achieve a somewhat DC-similar differentiation exhibiting enhanced APC function in part. Consequently, at-tempts have been made to use established leukemic cell

- 3 -lines as in vitro model systems in investigations regarding the DC biology. However, no success has been achieved in this respect because all of the investigated cell lines were only capable of reaching specific stages of DC devel-opment beyond which further differentiation thereof was not possible, thus failing to reflect the DC biology as de-sired. This is due to the fact that the capability of such malignant cells of responding to cytokine stimuli depends on the expression of specific and functional receptors.
However, many leukemia cell lines do not respond to cyto-kine treatment. Other leukemia cell lines examined so far only respond to treatment with particular cytokines and cannot be developed into effective DCs by sequential DC
differentiation. While pharmacological agents mobilizing intracellular calcium and thus avoiding corrupted receptor signal pathways can be used to induce a DC-like phenotype in myeloid cells - the activation of protein kinase C by PMA induces a DC phenotype in the human myeloblast cell line KG-1 - manipulation of intracellular signal pathways by means of such agents results in APCs failing to cover the full DC function. Thus, in the case of cytokine-stimulated KG-1, no differentiation without immediate ma-turing has been observed, for example.
As a consequence, all of the cell lines investigated up to now have only limited suitability for use in investigations directed to the DC biology. They are not suitable for immu-notherapeutic uses and in test systems for testing sub-stances having an impact on the immune system. The state of the art therefore implies that leukemic cell lines or other tumor cell lines would not be capable of differentiating into immature DCs by corresponding stimulation, said DCs -depending on the stimulation - being similar either to in-terstitial DCs or Langerhans DCs, and subsequently into po-tent mature DCs, specifically either DC type 1 or DC type 2.

- 4 -At present, DCs are being used in various procedures and approaches to treat various diseases, including e.g. tumor diseases, infectious diseases, and autoimmune diseases. The results indicate success and are promising. In all such treatments, however, DCs recovered from primary cells have to be used at present because, despite great efforts, no success has been achieved in generating and identifying cell lines that would allow production of DCs stimulating an effective immune response. For example, the disadvan-tages of DCs from primarily cells are the following: DCs or their precursor cells can be obtained from patients or do-nors only in very low quantities, thus severely limiting the use of these cells; their recovery requires a high in-put of time and work; the amount of recovered DCs is ex-ceedingly small, so that nowhere near proportional amounts could be employed in humans that achieve greatest success of treatment in murine models. The DCs have to be obtained from precursor cells, such as CD34-positive stem cells or monocytes, maturing in vitro by suitable stimulation with stimulatory molecules to form DCs, said precursor cells be-ing extremely rare both in blood and tissue. Their contri-bution to the PBMC is estimated to be about 1%. Further-more, culturing thereof is difficult, being severely re-stricted by the amount of monocytes recovered from the PBMC
and frequently impaired by progenitor cell impurities. The resulting large variance in the efficiency of purification, stimulation and effectiveness of autologous DC precursor cells massively impedes the standardization of methods for use in immunotherapeutic treatment. In addition to the variance within a patient, there is a variance from one particular individual to another.
To develop immunotherapeutic agents based on effective DCs, it is advantageous to generate precisely characterized cell lines which either represent effective DCs or can be trans-formed in vitro into such by means of appropriate stimula-

- 5 -tion using suitable signal molecules, which DCs can then be used alone or in combination with other substances to provide effective immunotherapeutic agents.
The object of the invention is therefore to provide a method of producing cell lines or cells allowing generation of effective dendritic cells (DC) therefrom which can be used particularly as immunotherapeutic agents or as part of immunotherapeutic agents in the treatment of immune diseases.
The invention solves this technical problem by providing a method of producing effective dendritic cells or cell lines, wherein cells from CD124- and CD116-positive cell lines are contacted simultaneously or in a sequentially deferred fashion with at least one stimulatory molecule, thus obtaining the effective dendritic cells or cell lines.
In one aspect, the present invention relates to a method for the production of effective dendritic cells or cell lines, the method comprising:
(a) providing cells from the CD124- and CD116-positive cell line MUTZ-3; and (b) contacting the cells simultaneously or in a sequentially deferred fashion with at least one stimulatory molecule which is: GM-CSF, TNFa, LPS, PGE2, CD40 ligand, polyinosinic-polycytidylic acid (polyIC), calcium, PMA, TGF01, IL-7, IL-13 or IL-4;
thereby producing the effective dendritic cells or cell lines.
In another aspect, the present invention relates to an effective dendritic human MUTZ-3 cell or MUTZ-3 cell line, produced by the above mentioned method.

- 5a -In another aspect, the present invention relates to a pharmaceutical composition for immunotherapy, comprising the effective dendritic human MUTZ-3 cells or the MUTZ-3 cell line mentioned above, and a pharmaceutically acceptable carrier.
In another aspect, the present invention relates to the use of the MUTZ-3 cell or cell line mentioned above for immunotherapy or for the manufacture of a medicament for immunotherapy.
In another aspect, the present invention relates to the use of the MUTZ-3 cell or cell line mentioned above for the prophylaxis or treatment of infectious, tumor and/or autoimmune diseases, or for the manufacture of a medicament for accomplishing same.
In another aspect, the present invention relates to the use of the MUTZ-3 cell or cell line mentioned above for the for use in the processing and/or presentation of antigens.
In another aspect, the present invention relates to the use of the MUTZ-3 cell or cell line mentioned above the processing and/or the presentation of antigens.
In another aspect, the present invention relates to a test system comprising the MUTZ-3 cell or cell line mentioned above.
In another aspect, the present invention relates to the use of the test system mentioned above for testing immunoactivity-inhibiting and/or -modulating substances.
In another aspect, the present invention relates to the use of the test system mentioned above for testing tumor vaccines or for testing the influence of substances, pharmacological agents, cosmetics or foodstuffs on the immune system.

, - 5b -In another aspect, the present invention relates to a method for producing immature dendritic cells or cell lines, the method comprising culturing a MUTZ-3 cell line with the stimulatory molecules: (a)GM-CSF, TNFa and IL-4; (b) GM-CSF, TNFa and IL-13; or (c) GM-CSF, TNFa, and TGF[31; until the immature dendritic cells or cell lines are obtained.
In another aspect, the present invention relates to a method for producing mature dendritic cells or cell lines from immature dendritic cells or cell lines generated from a MUTZ-3 cell line, the method comprising culturing the immature dendritic cells or cell lines with the stimulatory molecules:
IFNy, dexamethasone, TNFa, LPS, CD40 ligand, polyinsosinic-polycytidylic acid (polyIC), or any combination thereof, until the mature dendritic cells or cell lines are obtained.
In another aspect, the present invention relates to a dendritic cell or cell line produced by any one of the above mentioned methods.
In another aspect, the present invention relates to a pharmaceutical composition for immunotherapy comprising the above mentioned dendritic cells or cell line, and a pharmaceutically acceptable carrier.
In another aspect, the present invention relates to the use of the above mentioned dendritic cell or cell line for immunotherapy or for the manufacture of a medicament for immunotherapy.
In another aspect, the present invention relates to the use of the above mentioned dendritic cell or cell line for the prophylaxis or treatment of infectious, tumor and/or . =
, - 5c -autoimmune diseases, or for the manufacture of a medicament for same.
In another aspect, the present invention relates to the use of the above mentioned dendritic cell or cell line for the processing and/or the presentation of antigens.
In another aspect, the present invention relates to a test system comprising the above mentioned dendritic cell or cell line.
In another aspect, the present invention relates to the use of the above mentioned test system for testing immunoactivity-inhibiting and/or -modulating substances.
In another aspect, the present invention relates to the use of the above mentioned test system for testing tumor vaccines or for testing the influence of substances, pharmacological agents, cosmetics or foodstuffs on the immune system.
In another aspect, the present invention relates to a MUTZ-3 cell line for the manufacture of a pharmaceutical composition for immunotherapy.
In another aspect, the present invention relates to a MUTZ-3 cell line for the manufacture of a pharmaceutical composition for immunotherapy.
In another aspect, the present invention relates to a MUTZ-3 cell line for use in producing dendritic cells or cell lines.
In another aspect, the present invention relates to the use of a MUTZ-3 cell line for producing dendritic cells or cell lines.

- 5d -In another aspect, the present invention relates to an immature dendritic cell or cell line generated from a MUTZ-3 cell line.
In another aspect, the present invention relates to a mature dendritic cell or cell line generated from a MUTZ-3 cell line.
In connection with this invention the following terms will be used as follows:
Cell lines from which effective dendritic cells (effective DC) are obtained according to the invention include all tumor cell lines, preferably leukemia cell lines, such as myeloid, lymphoid and plasmacytoid lines, as well as cell lines of non-leukemic origin, but bearing CD124 and CD116, and preferably CD34 as well, including such cell lines which are not tumor cell lines in the strict sense. Also possible are cell lines lacking CD124 and/or CD116, but expressing functional recombinant CD124 and CD116 as a result of incorporating genes, thus enabling production of effective DCs. Preferably, the cell lines from which effective DCs are obtained are also CD34-positive, and the CD34 can also be incorporated by means of genes. Such cell lines, from which effective DCs are produced, can be obtained from tumor cells or primary cells. This is effected by means of per se conventional methods such as transformation, immor-

- 6 -talization, cell fusion with tumor cells and/or culturing in vitro or in vivo with or without cloning of cells of preferably homogeneous cell lines. Those methods are pre-ferred wherein CD124- and CD116-positive cells are accumu-lated and cloned by means of magnetic ball techniques or cell sorting in an FACS according to per se known proce-dures. Patients suffering from chronic myeloid leukemia or acute myeloid leukemia are preferred as donors of tumor cells which, according to the invention, are transferred into cell lines by stimulatory molecules, from which effec-tive DCs are obtained; however, the invention is not re-stricted thereto. Primary cells from which suitable cell lines are obtained are preferably of myeloid, lymphoid, plasmacytic or monocytic origin. To obtain effective DCs from cell lines and/or increase the effectiveness of the DCs obtained, one or more genes can be incorporated in the cell lines, tumor cells or primary cells according to per se known methods, which genes encode and/or express e.g.
receptors for or inhibitors of stimulatory molecules. It is also possible to introduce one or more immunotherapeutic agents in the form of genes. Introduction of the immuno-therapeutic agent genes at this stage of the cell line is advantageous in that the genes can be characterized as a cell line and do not have to be introduced subsequent to maturing into dendritic cells for further use as immuno-therapeutic agents. Another way of introducing genes is fu-sion of the cell lines with other cells or cell lines ac-cording to per se known methods.
According to the invention, effective DCs are understood to be such cells or cell lines which, as a result of stimula-tion of cell lines with stimulatory molecules, differenti-ate into cells acting like dendritic cells, activating, in-hibiting or modulating humoral and/or cellular portions of the immune system. Such effective DCs are used as immuno-therapeutic agents. To this end, the effective DCs, the

- 7 -precursor cells thereof at a suitable stage of differentia-tion, or the cells of the cell lines are loaded with at least one antigen. Such loading is effected according to per se known methods, e.g. by loading with tumor antigens or infection antigens, synthetic or purified or partially purified from biological material, with cell lysates of tu-mor cells, tumor cell lines, infected cells or cell lines, by fusion with other cells or cell lines, by introducing at least immunotherapeutic gene, by infection with infectious particles or portions thereof. Optionally, the loaded cells or cell lines are subjected to further differentiation by stimulatory molecules. In general, the effective DCs will process the antigens, presenting them to the corresponding immune cells of the immune system via particular molecules, e.g. via MHCI or MHCII molecules, thereby correspondingly activating the humoral and/or cellular immune response which combats the disease or builds up an immunological memory preventing diseases in a prophylactic fashion. For this purpose, the effective DCs are used at at least one suitable activity and/or effector stage in the patient as immunotherapeutic agent.
According to the invention, stimulatory molecules are un-derstood to be those chemical and biological molecules which influence the differentiation of cells, such as cyto-kines (IL-4, TNFalpha), growth factors (e.g. GM-CSF), sur-rogate molecules for cytokines or growth factors inducing a biological effect comparable to that of the stimulatory molecules themselves, e.g. antibodies, other biological molecules (e.g. LPS, polyIC), and chemical agents. The molecules can be employed together at the same time or in a sequentially deferred fashion so as to achieve the corre-sponding desired differentiation stage of the cells and thus different activity and effector stages, e.g. DC type 1 or DC type 2 phenotype cells which can be employed for each of the various uses, depending on the suitability thereof.

- 8 -Using different stimulatory molecules, for example, it is possible to produce DCs of varying effectiveness from the same initial tumor cell line, which DCs e.g. have an in-hibitory or stimulatory effect on different components of the immune system and are thus used e.g. in the immunother-apy of infectious diseases, tumor diseases or autoimmune diseases. According to the invention, stimulatory molecules are also understood to include all danger signals, even those which are not molecules in a strict sense, such as mechanical stress, for example.
According to the invention, immunotherapeutic agents are understood to be those therapeutic agents which can be used against diseases in a prophylactic or curative fashion where the use of effective dendritic cells for treatment is possible, and suitable effective dendritic cells can in-volve varying stages of development and activation. The success of treatment can be complete or partial, and the agents can also be vaccines, for example.
According to the invention, semi-allogenic DCs are those effective DCs matching in one or more of the HLA molecules with the recipient of the immunotherapeutic agents, with the cells not being derived from the same person. Thus, this also includes those DCs which exhibit complete match-ing in the HLA molecules and are not derived from the same person.
According to the invention, allogenic DCs are those effec-tive DCs matching in none of the HLA molecules with the re-cipient of the immunotherapeutic agents.
According to the invention, CD124-positive cell lines or cells are understood to be those cells which are sensitive to treatment with IL-4.

- 9 -According to the invention, CD116-positive cell lines or cells are understood to be those cells which are sensitive to treatment with GM-CSF.
According to the invention, immune diseases are understood to include all those diseases allowing the use of dendritic cells for treatment, for instance:
- infectious diseases, - tumor diseases, - autoimmune diseases.
According to the invention, introduction of genes is under-stood to be transfection or viral infection or transforma-tion of cells or cell lines, thereby introducing genetic material into the cell or cell lines according to per se known methods. The genetic material can be DNA or RNA. The genetic material codes for the expression of at least one protein or peptide, or/and the RNA itself can have an in-hibitory or stimulatory effect, e.g. as an antisense RNA.
The proteins being expressed can be further processed and modified, e.g. by glycosylation. Genes can also be intro-duced by fusing cells or cell lines with other cells or cell lines.
According to the invention, immunotherapeutic agent genes are genes encoding proteins and/or peptides which play a role in the use of the effective dendritic cells as immuno-therapeutic agents, e.g. tumor antigens, viral antigens or antigens from parasites, bacteria or other microorganisms.
Cells or cell lines having immunotherapeutic agent genes incorporated therein will express the proteins or peptides of these genes, and these are presented to the immune sys-tem by the dendritic cells, so that the effective dendritic cells activate, inhibit or modulate corresponding immune responses, depending on the activity and effector stages of the effective dendritic cells. For presentation of the gene

- 10 -products, the expressed proteins or peptides are processed or directly used; furthermore, the expressed proteins or peptides can be modified, e.g. by glycosylation.
According to the invention, surrogate molecules are those molecules which are capable of replacing the stimulatory molecules as to the effect thereof; instead of cytokines, for example, it is possible to use antibodies or mimicry peptides which influence the cells in the same way as stimulatory molecules.
Cell apoptosis or necrosis to be caused according to the invention involves various methods as required, e.g. irra-diation, thermal shock, mechanical stress, oxidative stress, ultrasound, induction of suicide genes, induction by chemical and biological molecules, glycerol, zinc, buti-linic acid, sodium butyrate, leptomycin B with STI571 and/or Fas ligand. The cells may also form mixed popula-tions, part of which undergoing apoptosis or necrosis. This method can be used to make sure that effective dendritic cells are not viable in the organism.
According to the invention, tumor antigens are peptides, proteins, lipids, lipopeptides, lipoproteins, carbohy-drates, glycolipids, glycopeptides, glycoproteins, phospho-rylated proteins, phosphorylated peptides, proteins or pep-tides otherwise modified following translation, which, com-pared to normal tissue, are overexpressed in the cells of the tumor, underexpressed, expressed de novo, mutated, dif-ferentially modified after translation, differentially processed, differentially situated, differentially folded, or otherwise modified.
According to the invention, infection antigens are pep-tides, proteins, lipids, lipopeptides, lipoproteins, carbo-hydrates, glycolipids, glycopeptides, glycoproteins, phos-

- 11 -phorylated proteins, phosphorylated peptides, proteins or peptides otherwise modified following translation, which are derived from an infectious particle.
According to the invention, infectious particles are infec-tious moieties causing diseases, or portions derived there-from, including e.g. viruses, bacteria, parasites, and pri-ons. The infectious particles which, according to the in-vention, serve in the production and use of effective den-dritic cells are not capable of propagating in vivo, i.e., in the patient.
Furthermore, the invention describes the production and use of CD124+ and CD116+ tumor cell lines, preferably also be-ing CD34+, as model and test systems for testing the DC bi-ology and for testing substances having an impact on the immune system and on the conditioning thereof.
According to the invention, model and test systems for testing the DC biology are understood to be test systems having as component a CD124+ and CD116+ tumor cell line, which is preferably also CD34+, and allowing the elucida-tion of processes during the differentiation of dendritic cells and of cells maturing into dendritic cells, and/or allowing the elucidation of processes influenced by the DCs or the precursor cells thereof during activation, inhibi-tion or modulation of the immune system and its immune re-sponse. The elucidation of these processes also includes the elucidation of other influences, such as the influence of stimulatory molecules and/or their effect in time, e.g.
during differentiation of the DCs and activity modulation of the immune system. Such model and test systems can be used in the form of kits and/or high-throughput systems, for example. The specific types of tests and the implemen-tation thereof are well-known to those skilled in the art.

- 12 -According to the invention, model and test systems for testing substances having an effect on the immune system are understood to be test systems having as component a CD124+ and CD116+ tumor cell line, which is preferably also CD34+, and allowing tests as to whether substances have an impact on the immune system and/or on the conditioning thereof. Inter alia, this also includes test systems serv-ing in the development of immunotherapeutic agents, e.g.
testing of suitable tumor vaccines and formulations thereof, as well as test systems allowing tests as to the influence of substances on the immune system, which are not immunotherapeutic agents, such as chemical substances, pharmacological agents, cosmetics or precursors thereof, or foodstuffs or components thereof. Consequently, such test systems can be used in the product development of e.g. im-munotherapeutic agents and other products which may have an influence on the immune system. For example, these model and test systems can be used in the form of kits and/or high-throughput systems. The specific types of tests and the implementation thereof are well-known to those skilled in the art.
Cells positive to CD124 bear the receptor for IL-4, the CD116+ bear the receptor for GM-CSF, and the CD34+ bear the marker for hematopoietic stem cells and progenitor cells.
Another preferred embodiment of the invention is a method for the identification of peptides presented by the effec-tive dendritic cells according to the invention, comprising the steps of (a) propagating the inventive dendritic cells according to per se known methods, said dendritic cells being imma-ture cells;
(b) adding antigens or immunogens or portions thereof or cell lysates, whereby the immature dendritic cells (iDC) develop into mature dendritic cells (mDC), proc-

- 13 -essing the antigens or immunogens or portions thereof or the cell lysates and presenting suitable peptides in the context of class I MHC or class II MHC molecules;
(c) recovering the presented peptides from the dendritic cells according to per se known methods; and (d) identifying/determining the removed peptides according to per se known methods.
The peptides obtained are preferably separated using per se known methods, e.g. by means of high-pressure liquid chro-matography (HPLC). In a particularly preferred fashion, the separated peptides are identified by mass spectrometry, and most preferably the peptides are subjected to sequencing.
The method according to the invention preferably allows for validation of the identified/determined peptides. More preferably, the identified/determined peptides obtained ac-cording to the method of the invention are produced by syn-thesis according to per se known methods. Most preferably, the peptides produced by synthesis are added to immature and/or mature dendritic cells according to the invention, the dendritic cells being loaded ("pulsed") according to per se known methods. In one variant, the following step is carried out instead of step (b): Loading of cells matured into mDCs with MHC I and/or MHC II peptides. This step can be preceded by a step of removing existing MHC I and/or MHC
II peptides according to per se known methods. In a pre-ferred fashion, libraries of MHC I and/or MHC II peptides are presented to the mDCs.
Surprisingly, leukemic cell lines having a specific prop-erty have been found with the aid of this invention, which function in all aspects like an immortalized equivalent of CD34+ DC precursor cells and are suitable for use in inves-tigations of the DC biology, testing of substances influ-encing the immune system, and in immunotherapeutic agents.

- 14 -The specific properties of the tumor cell lines involve positiveness to CD124 (IL-4R) and CD116 (GM-CSFRalpha) and preferably CD34. As an example, the myeloid cell line MUTZ-3 will be described in more detail, which recently has been reported to down-regulate the expression of CD14 upon stimulation with IL-4 and GM-CSF. The investigations of this invention demonstrate that, compared to other well-known and tested leukemic cell lines and other tumor cell lines, MUTZ-3 cells are unique in their capability of at-taining an immature DC state. Moreover, they express the maturing marker CD83 upon further stimulation, and func-tional assays prove their capability of antigen processing and presentation. Therefore, they are suitable for immuno-therapeutic purposes. MUTZ-3 is the first human leukemia cell line which can be stimulated so as to undergo differ-entiation and formation of an immature DC phenotype, and which is suitable as an in vitro model for use in investi-gations relating to the molecular and physiological path-ways leading to differentiation and maturing of DCs and in investigations on the DC biology and in testing of sub-stances influencing the immune system.
Within the context of this invention it will be demon-strated in a surprising fashion that effective DCs can be generated from human tumor cell lines which, in particular, can be used as immunotherapeutic agents or as a component of immunotherapeutic agents in the treatment of immune dis-eases. Key features of the cell lines are their positive-ness to CD124 and CD116 which can be obtained from leukemic cells, for example, and the sensitivity to stimulatory molecules such as cytokines, whereas other investigated leukemia cell lines lacking these properties fail to pro-vide effective DCs in the meaning of the invention. Pre-ferred is a cell line by means of which it is possible to obtain DCs of different activation and effector stages from said cell line by sequential stimulation with stimulatory

- 15 -molecules. The individual activation and effector stages can be used as effective DCs to activate various portions of the immune system, activating CD8+ T cells via MHCI
presentation, activating CD4+ T cells via MHCII presenta-tion, or activating NKT cells via CD1. Activated DCs are mainly employed in immunotherapeutic agents used in the treatment of infectious diseases and tumor diseases. Fur-thermore, suitable DC activation stages may give rise to induction of anergies and tolerances and are also suitable in the treatment of autoimmune diseases.
With reference to the example of the human myeloid cell line MUTZ-3, the invention will be described in more detail below.
The human acute myeloid leukemia cell line MUTZ-3 is sensi-tive to those cytokines which are responsible for the gen-eration of DCs from monocytes and CD34 positive stem cells in in vivo and in vitro models. In all their properties, MUTZ-3 cells behave as immortalized equivalents of CD34-positive DC precursor cells. When stimulated using the re-spective suitable specific cytokine cocktail, they develop into cells having phenotypes corresponding to the pheno-types of e.g. interstitial DCs or Langerhans cells. As a result of maturation these cells express CD83. MUTZ-3 have the complete spectrum of antigen processing and presenta-tion processes for MHC-dependent and CD1d-dependent presen-tation and activation. Under suitable conditions, e.g. ad-ministration of interferon-gamma or dexamethasone, they are capable of specifically adopting a DC1 phenotype or a DC2 phenotype, thereby allowing controlled immune response.
Thus, it is evident that MUTZ-3 cells represent an unlim-ited source of CD34-positive DC precursor cells (progeni-tors) which can be used efficiently in (directed) stimula-tion of various immune cells and thus as effective DCs in the treatment of immune diseases.

- 16 -The component of immunotherapeutic agents, which is impor-tant in the meaning of the invention, is the cell line rep-resenting effective DCs itself or forming effective DCs upon treatment with suitable stimulatory molecules. In the meaning of the invention, the effective DCs can be combined with other components to form allogenic or semi-allogenic immunotherapeutic agents, and, if required, further matur-ing of the cells is possible, optionally using suitable stimulatory molecules. In Example 1 this will be described for the case of MUTZ-3, with MHCI-, MHCII- and CD1-mediated activation each time. However, the invention is not re-stricted thereto, but also comprises all therapeutic or prophylactic fields of use where DCs can be employed.
This also includes tumor therapeutic agents, for example.
These agents can be produced in such a way that e.g. the allogenic or semi-allogenic effective DCs are pulsed e.g.
with tumor antigens according to per se known methods and administered to patients. Such tumor antigens can be one or more well-defined molecules such as peptides, glycopep-tides, proteins, glycoproteins, glycolipids which are syn-thesized, purified, or used in the form of cell lysates;
another example is transfection of effective DCs with RNA, DNA or viral vectors encoding tumor antigens or portions thereof; another example is antigen loading of effective DCs by incubation with apoptotic and/or necrotic tumor cells or with thermal shock-treated cells; a further exam-ple is fusion with tumor cells. Clinical use of such DCs produced within the scope of the invention is effected in the form of allogenic or semi-allogenic DCs, prophylactic or as a curative therapy, e.g. in tumor therapy or follow-ing removal of such tumors e.g. by surgery, as an adjuvant therapy for the treatment of minimal residual diseases, in-cluding combating metastases or preventing formation of me-tastases or micro-metastases.

- 17 -A number of immunization strategies are possible, such as intranodal, intratumoral, intradermal, intramuscular, sub-cutaneous, intraperitoneal, or mucosal application of DCs, in the presence or absence of additional immunostimulants such as cytokines, chemokines or other immunostimulatory or immunomodulatory substances. The DCs produced according to the invention may also be part of a more complex immuniza-tion regimen wherein e.g. further components are adminis-tered simultaneously or in a deferred fashion.
Although DCs derived from MUTZ-3 no longer undergo division following differentiation, it is not impossible that DCs produced from other leukemia cells or lines will divide further. One preferred variant is therefore irradiation of such antigen-loaded DCs, treatment thereof with mitomycin C, or other measures preventing cell division in vivo. For example, one alternative would be incorporation of a so-called suicide gene, such as HSV thymidine kinase (TK) gene, allowing selective destruction of the HSV TK-bearing cells by means of gancyclovir.
The invention also relates to the production of cell lines which can be matured into effective DCs. The method of the invention involves isolation of CD34+, CD124+ and CD116+
cells from human material, preferably from leukemia pa-tients, according to per se known methods. For example, the cells can be recovered sequentially from peripheral blood or bone marrow of leukemia patients by accumulation of cells, which are CD34+, CD124+ and CD116+, using magnetic beads bearing antibodies for CD34+, CD124+ and CD116+. Al-ternatively, CD34+, CD124+ and CD116+ cells can be obtained by cell sorting using flow cytometry and C1J34-, CD124- and CD116-specific antibodies.
Another embodiment of the invention is a method of produc-ing a drug, comprising the steps of the method according to

- 18 -the invention and further comprising the step of formulat-ing the drug in a pharmaceutically tolerable form, the drug optionally being combined with an additional adjuvant as an active substance enhancer.
According to the invention, the term "drug" defines sub-stances and formulations of substances intended to cure, alleviate or avoid diseases, illness, physical defects or pathological affection by application on or in the human body. During the production process of the invention, medi-cal and/or pharmaceutical-technical adjuvants can be added to the compounds identified by means of the method accord-ing to the invention. According to the invention, medical adjuvants are substances used (as active components) in the production of drugs in a process according to the inven-tion. Pharmaceutical-technical adjuvants merely serve to formulate the drug and, if required during the process only, can even be removed thereafter, or they can be part of the drug as pharmaceutically tolerable carriers. Exam-ples of pharmaceutically tolerable carriers are given be-low.
Drug formulation is optionally effected in combination with a pharmaceutically tolerable carrier and/or diluent.
Examples of suitable pharmaceutically tolerable carriers are well-known to those skilled in the art and include phosphate-buffered saline solutions, water, emulsions such as oil/water emulsions, various types of detergents, ster-ile solutions, etc..
Drugs comprising such carriers can be formulated by means of well-known conventional methods. Those routes of appli-cation are preferred where the inventive effective den-dritic cells in a pharmacological formulation are delivered to sites within the body where they assume their function

- 19 -in the best way possible. Such sites and routes of applica-tion are well-known to those skilled in the art, e.g. in-travenous, intraperitoneal, subcutaneous, intramuscular, local or intradermal, with intranodal, intradermal, subcu-taneous, intrarectal, intravenous or local being preferred.
A suitable route of application may exhibit varying suit-ability, depending on the particular disease.
For example, application of effective dendritic cells for the therapy of autoimmune diseases is directed to tolerance of the immune system, whereas a suitable route of applica-tion for the treatment or prophylaxis of tumor or infec-tious diseases is intended to support activation of the im-mune system. Those skilled in the art will be able to de-termine suitable routes of administration by means of per se known methods. The drugs can be administered to an indi-vidual in a suitable dose, one dose comprising from 100 to 1012 effective dendritic cells, preferably from 105 to 1010 .
The effective dendritic cells are loaded with a suitable form and quantity of antigens which also may vary depending on the type of use. A single dose is preferably adminis-tered once a week and up to greater intervals of e.g. one month, 3 months, one year or even longer intervals. Shorter intervals may also be suitable, e.g. once per day. Those skilled in the art will be able to determine suitable time intervals and doses, preferably using methods of immuno-monitoring and adjusting the doses correspondingly. Suit-able methods are well-known to those skilled in the art, and some of them will be described in the examples.
The kind of dosage will be determined by the attending phy-sician according to the clinical factors. As is familiar to those skilled in the art, the kind of dosage will depend on various factors, such as size, body surface, age, sex, or general health condition of the patient, but also on the particular agent being administered, the time period and

- 20 -type of administration, and on other medications possibly administered in parallel.
In a preferred embodiment the effective dendritic cells are loaded with a number of antigens. In another preferred em-bodiment doses of effective dendritic cells loaded with suitable antigens are combined with doses directly compris-ing the antigens or single antigens or portions thereof in suitable formulations, with no ex vivo loading of dendritic cells. This is advantageous in that semi-allogenic ex vivo loaded inventive dendritic cells strongly induce immune re-sponse, being supported by the alloresponse as a kind of danger signal and associated with partial specific immuni-zation by presentation of overlapping MHC molecules, and combined with an immune response directed to the dendritic cells in vivo. Such a combination is particularly suitable in breaking up tolerances and anergies.
In a preferred embodiment inventive immature effective den-dritic cells (iDC form) loaded with a corresponding antigen are used for the treatment of autoimmune diseases. In an-other preferred embodiment the cells are locked in the iDC
form in a transient or stable fashion, for which purpose methods are used that are well-known to those skilled in the art, e.g. locking by genetically engineered modifica-tions. In another preferred embodiment, following loading in the immature form (iDC) or mature form (mDC), the cells are further matured and used as loaded effective dendritic cells (mDC) in the treatment or prophylaxis of tumor or in-fectious diseases.
A drug according to the invention comprises a pharmacologi-cal substance which contains the dendritic cells in a suit-able solution or administration form. Administration thereof can be effected either alone or in combination with one or more adjuvants or other suitable material enhancing

- 21 -the drug effect. QS-21, GPI-0100 or other saponins, water-oil emulsions such as Montanide adjuvants, polylysine, polyarginine compounds, DNA compounds such as CpG, Detox, bacterial vaccines such as typhoid vaccine or BCG vaccines are used as preferred adjuvants and mixed with the den-dritic cells of the invention in a suitable manner accord-ing to per se known methods.
Preferred forms of adjuvants are co-stimulatory factors, cytokines and/or growth factors such as GM-CSF or IL-2 or IL-12. They can also be incorporated in a genetic form in the cells of the cell lines according to the invention, preferably in a stable fashion.
The inventive use of the drug is in the prophylaxis and/or treatment of cancerous diseases, tumors, infections and/or autoimmune diseases. In a preferred embodiment the cancer-ous disease or the tumor to be treated or prevented is se-lected from the group of cancerous diseases or tumor dis-eases of head and nape, lungs, mediastinum, gastrointesti-nal tract, sexual apparatus/urinary system, gynecological system, breast, endocrine system, skin, cancerous diseases or tumor diseases during childhood, primary tumors, metas-tasizing cancer, soft-tissue sarcoma or osteosarcoma, mela-noma, neoplasms of the central nervous system, lymphoma, leukemias, paraneoplastic syndrome, peritoneal carcinomato-sis and/or malignancy related to immunosuppressed malig-nancy.
The infection to be treated or prevented with the drug of the invention is selected from bacterial infections, viral infections, fungous infections, infections with protozoa and/or infections with helminths. In a preferred fashion, the bacterial, viral, fungous infection, infection with protozoa and/or infection with helminths, which is to be treated or prevented, is selected from infections such as

- 22 -sepsis or septic shock, fever of unknown origin, infectious endocarditis, intra-abdominal infections and abscesses, acute infections, diarrhea diseases, bacterial food poison-ing, sexually transmittable infections, inflammatory pelvis infections, urinary tract infections, pyelonephritis, os-teomyelitis, infections of the skin, muscles or soft tis-sue, infections by injection of drugs, infections by bites, scratches or burns, infections in graft recipients, hospi-talism infections and/or intravascular infections caused by equipment. In a more preferred embodiment the infection to be prevented or treated is selected from bacterial infec-tions such as pneumococcal infections, staphylococcal in-fections, streptococcal infections, enterococcal infec-tions, diphtheria, various corynebacterial infections, an-thrax, Listeria monocytogenes infections, tetanus, botu-lism, gas gangrene, antibiotics-associated colitis, various clostridial infections, meningococcal infections, gonococ-cal infections, Moraxella (branhamella) catarrhalis infec-tions, infections with other Moraxella species, Klingella infections, hemophilus influenza infections, infections with other hemophilus species, infections with the HACEK
group, infections by other gram-negative bacilli, Legion-ella infections, pertussis, infections by gram-negative en-terobacteria, helicobacterial infections, infections by pseudomonades and related organisms, salmonellosis, shigel-losis, infections by campylobacteria and related species, cholera, vibrio, brucellosis, tularemia, plague, various yersinia infections, Bartonella infections, including in-fections by cat scratches, Donovania (Granuloma inguinale), nocardiosis, actinomycosis, infections by multiple anaero-bic organisms, tuberculosis, leprosy, infections by non-tubercle bacteria, syphilis, endemic treponematosis, lepto-spirosis, relapsing fever, Lyme borreliosis, infections by rickettsia, mycoplasmas or chlamydia, viral infections such as Herpes simplex virus infections, Varicella zoster infec-tions, Epstein-Barr virus infections, including mononucleo-

- 23 -sis, cytomegalovirus infections, human Herpes virus type 6,7 or 8 infections, smallpox virus infections, Vaccinia infections, various poxvirus infections, parvovirus infec-tions, human papillomavirus infections, viral respiratory tract infections, influenza, viral gastroenteritis, entero-virus infections, reovirus infections, measles, rubella, mumps, rabies virus infections, other rhabdovirus infec-tions, infections caused by rodent and/or arthropod vi-ruses, infections with Marburg and/or Ebola viruses, fun-gous infections such as histoplasmosis, coccidioidomycosis, blastomycosis, cryptococcosis, candidiasis, aspergillosis, mucormycosis, miscellaneous mycoses, prototheca infections, Pneumocystis carinii infections, infections with protozoa such as ameba infestation, infections with free-living ameba, malaria, infections by parasites of red blood cells, Leishmaniosis, trypanosomiasis, toxoplasma infections, in-testinal infections by protozoa, trichomonad colpitis, in-fections with helminths such as trichinosis, infections with other tissue nematoda, infections with intestinal nematoda, filariosis, infections such as loiasis, onchocer-cosis or dracontiasis, schistosoma, trematoda infections or cestoda infections.
The autoimmune disease to be treated or prevented by means of the drug according to the invention is selected from autoimmune diseases such as allergic encephalomyelitis, autoimmune hemolytic anemia, autoimmune thyroiditis (Hashi-moto syndrome), autoimmune male sterility, pemphigoid, ab-dominal cave disease, Basedow disease, Goodpasture syn-drome, idiopathic thrombocytopenic purpura, insulin-resistant diabetes mellitus, myasthenia gravis, pernicious anemia, pemphigus vulgaris, polyarteritis nodosa, primary bile cirrhosis, Reiter syndrome, rheumatic fever, sarcoido-sis, Sjogren syndrome, systemic lupus erythematodes, sympa-thetic ophthalmia, multiple sclerosis, and/or viral myocar-ditis by Cocksakie B virus response.

- 24 -Another preferred embodiment of the invention is a method for the production of a drug, comprising the procedures ac-cording to the invention, said drug including dendritic cells loaded with antigens according to per se known meth-ods or fused with corresponding cells. The dendritic cells of the drug are formulated with a suitable pharmaceutical carrier according to methods per se known in autologous dendritic cell therapy. The drug thus obtained can be ad-ministered according to per se known methods. The dendritic cells of the drug take up antigens, process them, and pres-ent fragments thereof on their surface in the context with MHC molecules and co-stimulatory molecules. Following fur-ther maturing according to per se known methods, the cells in suitable formulation are used in humans. Another example is loading of mature dendritic cells according to per se known pulsing methods. The dendritic cells are autologous, allogenic or semi-allogenic dendritic cells or precursor cells thereof, or cells from cell lines having the func-tional properties of dendritic cells, which cells are suitably treated ex vivo for development and maturing ac-cording to per se known methods.
If necessary, the precursor cells are preferably matured by adding suitable factors, e.g. co-stimulatory factors, cyto-kines and/or growth factors such as IL-4 and GM-CSF, to form cells which are similar to iDCs in terms of function and phenotype. These cells are loaded with suitable anti-gens and matured further, if required. The resulting loaded effective dendritic cells (mDC) are cells which are similar to loaded dendritic cells in terms of function and pheno-type and are preferably used in the prophylaxis or therapy of tumor or infectious diseases. Alternatively, it is also possible to load the effective dendritic cells at a later stage as mDCs, e.g. in case of well-defined MHC class pep-tides as antigens. Alternatively, cells at varying precur-sor, differentiation and/or maturing stages can be trans-

- 25 -fected with DNA or RNA of antigens, co-stimulatory mole-cules and/or immunogens according to per se known methods of genetic engineering. Preferably, these are stable trans-formations of those cells undergoing division in the best way possible, preferably prior to the precursor stage and prior to differentiation. Alternatively, suitable stages of the dendritic cells according to the invention are fused as precursor cell, as immature cell or as mature cell with other cells according to per se known methods and option-ally further differentiated and/or matured. For treatment or prophylaxis of autoimmune diseases it is preferred to use the loaded cells at an immature stage as set forth in more detail above.
Without intending to be limiting, the invention will be ex-plained in more detail with reference to the following ex-ample.
Example 1:
MUTZ-3, a human CD34+, CD124+, CD116+ cell line for the production of effective DCs by cytokine-induced differen-tiation of dendritic cells from CD34+ precursor cells, and use of the effective DCs to induce functional T cell sub-sets for the production of immunotherapeutic agents Materials and methods Antibodies and reagents The following was used in the investigations:
PE-labelled monoclonal antibodies (mAbs) against CD40, CD34 and TCR Valpha 24 from Coulter Immunotech (Marseilles, France), against CD1a, CD54, CD83, and CD86 from Pharmingen

- 26 -(San Diego, CA), and against CD80 from Becton-Dickinson (San Jose, CA).
FITC-labelled mAb against HLA-DR, TCR VD 11 and CD14 from Becton-Dickinson, against CD116 (GM-CSF receptor) from Pharmingen.
CD1d expression was assessed using a murine mAb against CD1d (mAb CD1d27) (Spada et al. 1998, J. Exp. Med. 188(8), 1529-1534), followed by a FITC-labelled anti-mouse IgG1 mAb (Pharmingen). The isotype control mouse IgG1 is from Orga-non Technika-Cappel (Malvern, PA), FITC- and PE-labelled Simultest isotype controls from Becton-Dickinson. Langerin expression was detected by means of staining with the mAb DDCM4, followed by a FITC-labelled anti-mouse mAb. Antigen presentation by CD1d was blocked using Ab CD1d51 (Spada et al. 1998, J. Exp. Med. 188(8), 1529-1534).
Cell cultures The cytokine-dependent, human myelomonocytic leukemia cell line MUTZ-3 was cultured in MEM-alpha with ribonucleosides and deoxyribonucleosides (Gibco, Paisley, UK), heat-inactivated FCS, penicillin/streptomycin, and 10% condi-tioned medium of the human bladder carcinoma cell line 5637 (Quentmeier 1996, Leuk. Res. (4), 343-350). The cells were cultured in 6-well plates (Costar, Cambridge, MA) at 37 C
and 5% CO2 and passaged twice a week. The cell line THP-1 derived from an acute monocytic leukemia, the cell line KG-1 derived from an acute myelogenic leukemia, the chronic myeloid leukemia line K562, the cell line HL-60 derived from a promyelocytic leukemia, and the macrophage-like his-tiocytic lymphoma line U937 were obtained from the American Type Culture Collection (ATCC, Rockville, MD). These cell lines were cultured in IMDM or RPMI-1640 with heat-inactivated FCS, penicillin/streptomycin, 2M L-glutamine

- 27 -and P-mercaptoethanol and passaged twice a week in 80 cm2 tissue culture flasks (Costar).
Generation of immature (iDC) and mature DC-like (mDC) cells from leukemia cell lines The induction of a DC-like phenotype in leukemia cell lines was accomplished as follows:
The cells were washed and seeded at a cell density of 1x105/m1 (in a volume of 3 ml) in 24-well plates and incu-bated for 7 days with GM-CSF (100 ng/ml, Novartis/Schering-Plough, Arnhem, NL), IL-4 (1000 U/ml CLB) and low-dosed TNFalpha (2.5 ng/ml, CLB, Amsterdam, NL). On day 7 matura-tion was induced by adding either TNFalpha (75 ng/ml) or LPS (100 ng/ml, Sigma). To produce LC-like cells, the MUTZ-3 cells were cultured for 9 days in GM-CSF and low-dosed TNFa. The cells were then incubated in the presence or absence of TGF31 (1 ng/ml, R&D Systems, Abingdon, Oxon UK) and low-dosed TNFa for another 7 days, and the culture medium was renewed on the second day. The immature DCs (iDC) thus obtained were examined for expression of CD1a and langerin.
Flow cytometry The cultured cells were washed and resuspended at a cell number of 5x104 to 1x105 in 25 1 of ice-cold FACS buffer (PBS pH 7.5, 0.1% BSA, 0.2% sodium azide). The specific and fluorescence-labelled mAbs or the corresponding isotype controls were added, and the cells were incubated for 30 minutes at 4 C. The cells were washed once and resuspended in 250 1 FACS buffer. The labelled cells were analyzed on a FACStar (Becton-Dickinson) using the CellQuest software.

- 28 -Allogenic mixed lymphocyte reaction (MLR) Allogenic, non-adherent PBL were isolated from the periph-eral blood of healthy donors by means of gradient centrifu-gation on Hypaque Lymphoprep (Nycomed, Oslo, Sweden). The cells were seeded in round-bottom microtiter plates at a concentration of 5x104 cells/well and incubated with a dilu-tion series of MUTZ-3 DCs in 200 1 of culture medium for 5 days. The T cell proliferation was determined following a h pulse with 3H-thymidine (0.4 Ci/well, Amersham, Ayles-bury, UK) (standard methods).
Induction of IL-12/p70 and IL-10 secretion by mature MUTZ-3 DCs (MUTZ-3 mDC) The MUTZ-3 iDCs were washed and seeded in 48-well plates at a cell number of lx108 in MEM alpha (additives see above).
Immature MUTZ-3 .DCs (MUTZ-3 iDC) were matured by a treat-ment with TNFa in combination with either IFNy (1000 U/ml, Biosource, Camarillo, CA) or dexamethasone (1 mo1/1, Sigma) (incubation period 48 h) and subsequent stimulation with irradiated cells of a CD40 ligand-transfected J558 cell line (J558-CD4OL, 1x108 cells/well). The concentrations of the secreted cytokines IL-10 and IL-12 (p70 subunit) were determined using ELISA.
Induction of CD8+ T cells having specificity for influenza matrix proteins MUTZ-3 DCs were infected with 100 pfu/cell of a recombinant adenovirus. This adenovirus encodes the M1 matrix protein gene of the heminfluenza virus (RAd128). For RAd128 infec-tion, the DCs were washed with serum-free medium and incu-bated with lipofectamine (100 pfu/cell, 1.7 g/1 x 108 pfu).
After 2 hours the cells were washed with complete medium and incubated at 37 C and 5% CO2 overnight. Other MUTZ-3 DCs

- 29 -were loaded with the HLA-A2.1-binding Ml-derived peptide M158_66 (50 g/ml) together with beta-microglobulin (2.5 g/ml) in serum-free medium at 37 C overnight. CD8' T cells (responder) were isolated from HLA-A2+ PBMC using a CD8 T cell MACS isolation kit (Miltenyi Biotec). Antigen-loaded (virus or peptide) MUTZ-3 DCs (stimulator) at a re-sponder/stimulator ratio of 5:1 were used in complete IMDM
medium with 10% pooled human serum (CLB) and 5 ng/ml IL-7 (R&D Systems). After one week the T cells were examined for specificity in an 1FNy ELISPOT assay. To this end, irradi-ated T2 cells were used, loaded either with the Ml-derived peptide M15866 or, as a control, with the HLA-A2.1-binding HPV16-E7-derived peptide (E711.20).
The cells were loaded with the peptide (50 g/ml) and beta-microglobulin (2.5 g/ml) in serum-free medium at 37 C overnight.
Induction of CM* T cells having specificity for the melanoma-associated antigen MART-1 MUTZ-3 DCs were loaded with the HLA-A2.1-binding MART-1-derived peptide (ELAGIGILTV) (10 g/ml) for 4 hours at 37 C
in serum-free AIM-V medium (Gibco). CD8 + T cells (responder) were isolated from HLA-A2+ PBMC using a CD8 T cell MACS
isolation kit (Miltenyi Biotec). MART-1 peptide-loaded MUTZ-3 DCs (stimulator) at a responder/stimulator ratio of 10:1 were used in serum-free AIM-V medium (Gibco). After one week the T cells were examined for specificity in an IFNy ELISPOT assay. To this end, irradiated T2 cells were used, loaded either with the HLA-A2.1-binding MART-1 pep-tide (ELAGIGILTV) or, as a control, with the HLA-A2.1-binding CEA-derived peptide CEA.78 (IMIGVLVGV). The cells were loaded with the peptide (1 g/ml) in serum-free AIM-V
medium (Gibco) at 37 C overnight.

- 30 -Induction of CD8+ T cells with specificity for the tumor antigens MUC-1 and asialoglycophorin by stimulation with tumor cell lysates The tumor cell lysates were produced either from tumor cell lines or from primary material:
a) Cell lysates from tumor cell lines were produced using 4 cycles of alternating freezing in liquid nitrogen and sub-sequent thawing according to per se known methods.
b) Cell lysates from solid tumor primary material were pro-duced as follows: Solid tumors were treated using the tri-ple enzyme method, thereby producing single-cell suspen-sions. This method is well-known to those skilled in the art and is frequently used in various variants in most tu-mor-pathological/immunological laboratories. Following sur-gical removal of the tumor, all further steps are carried out under aseptic conditions. The tumor was dissected into pieces about 5 mm3 in size and placed in a vessel with ster-ile triple enzyme medium (0.1% collagenase, 0.002% deoxyri-bonuclease, 0.01% hyaluronidase in Hank's buffered saline, HBSS). This was stirred with a magnetic stirrer at room temperature overnight until the solid pieces of tissue had dissolved. Thereafter, the undigested pieces of tissue were removed using a coarse wire grid, and following careful washing in HBSS, the remaining cells were centrifuged with a Ficoll gradient so as to separate monocytes and lympho-cytes from the tumor cell suspension. The tumor cells were subsequently lysed using 4 cycles of alternating freezing in liquid nitrogen and subsequent thawing.
MUTZ-3 DC were loaded with a tumor cell lysate in serum-free AIM-V medium (Gibco) at 37 C overnight. CD8+ T cells (responder) were isolated from HLA-A2+ PBMC using a CD8 T cell MACS isolation kit (Miltenyi Biotec). MUTZ-3 DCs loaded with tumor cell lysate (stimulator) at a re-sponder/stimulator ratio of 10:1 were used in serum-free

- 31 -AIM-V medium (Gibco). After one week the T cells were exam-ined for specificity in an IFNy ELISPOT assay. To this end, antigen-loaded MUTZ-3 DCs or peptide-loaded T2 cells were used. The MUTZ-3 DCs were loaded with a tumor cell lysate or with asialoglycophorin (protein) in serum-free AIM-V me-dium (Gibco) at 37 C overnight. T2 cells were loaded with the HLA-A2.1-binding MUC1 peptide MUC-1.2 (LLLLTVLTV) (1 g/ml) in serum-free AIM-V medium (Gibco) for 4 hours.
IFDly ELISPOT assay Multiscreen 96-well filtration plates (Millipore, Molsheim, France) were coated for 3 h at room temperature (RT) or overnight at 4 C with the mAb 1-D1K (50 g/ml, 15 g/ml) in filtrated PBS (Mabtech, Nacka, Sweden). The plates were washed 6 times with serum-free medium and subsequently blocked with filtrated complete medium with 10% FCS for 0.5-1 h at RT. Subsequently, 7.5x103 to 1x105 effector cells/well were incubated with 1x104 target cells at 37 C
and 5% CO2 overnight. The cells were discarded and the plates were washed 6 times with filtrated PBS/0.05% Tween 20. Each well was added with 50 1 mAb 7-86-1 (1 g/ml in filtrated PBS), and the plates were allowed to stand for 2-4 h at RT. Following 6 wash steps with filtrated PBS/0.05%
Tween 20, 50 l/well streptavidin-coupled alkaline phospha-tase (diluted 1:1000 in PBS) was added, and the plates were incubated for 1-2 h at RT. After 6 additional wash steps with filtrated PBS/0.05% Tween 20, 50 1 of alkaline phos-phatase reagent (AP conjugate substrate kit, Biorad, Hercu-les, CA) was added, and this was allowed to stand for 15 min to 1 h, until stain dots had developed. The reaction was quenched with tap water, and the stain dots were counted by two independent persons.

=

- 32 -Activation of tetanus toxoid (TT)-specific T cells PBMC of donors with partial HLA matching (expressing HLA-DR11, HLA-DQ7, HLA-B44 and HLA-A2) were selected, and the CD4+-PBL were isolated using MiniMACS separation columns (Miltenyi Biotec). Following a 1.5 h adherence to the plas-tic surface to remove contaminating APC, the cells were in-cubated with a dilution series of TT-pulsed, immature MUTZ-3 DCs (50 mg/ml, Bilthoven, NL, 12 h in serum-free me-dium) in 200 1 of medium for 7 d at 37 C and 5% CO2. T cell proliferation was assessed following a 5 h pulse with 3H-thymidine (0.4 Ci/well, Amersham, Aylesbury, UK) (standard methods).
Presentation of a-galactosylceramide to va244114311+ NKT cells Va24+ T cells, including va24-7411+ NKT cells, were ob-tained from PBL by positive selection using autoMACS
(Miltenyi Biotec). The purified NKT cells were then co-cultured for 7 days with immature or mature MUTZ-3 DCs pulsed with DMSO (vehicle control) or 100 ng/ml a-galactosylceramide (alpha-GalCer, Pharmaceutical Research Laboratory, Kirin Brewery, Japan), with addition of ng/ml recombinant human IL-7 (R&D Systems) and 10 ng/ml recombinant human IL-15 (R&D Systems), in the presence or absence of blocking anti-CD1d antibodies (CD1d51, 10 g/ml). The absolute number of NKT cells and the expan-sion factor were determined using FACS analyses.

- 33 -Results Differentiation of MUTZ-3 cells into effective DCs of vary-ing differentiation stages and effector stages MUTZ-3 cells acquire the phenotype of immature DCs upon cy-tokine administration Initially, we determined the potential of leukemic cell lines of differentiating in the presence of cytokines rou-tinely used to induce DCs. More specifically, we investi-gated the cell lines treated with cytokines for induced ex-pression of CD1a, a major characteristic of immature den-dritic cells (iDC), on the surface of the cells. Three of six tested cell lines (MUTZ-3, KG-1, THP-1) responded to the cytokine cocktail GM-CSF, IL-4 and low-dosed TNFa. The amount of CD1a-positive cells after 7 days in culture was highest in the cell line MUTZ-3 (20%), while the cell lines KG-1 and THP-1 showed 10% and 5% CD1a-positive cells, re-spectively (Table 1). In the latter two cell lines differ-entiation was accompanied by marked expression of the DC
maturing marker CD83, thus confirming earlier results (Hulette et al. 2001, Arch. Dermatol. Res. 293(3), 147-158;
St. Louis et al. 1999, J. Immunol. 162(6), 3237-3248).
KG-1 and THP-1 did not respond to further cytokine stimuli, and also, no further modification of the CD1a/CD83 pheno-type was observed. Neither CD1a nor CD83 were detected in the remaining 3 investigated cell lines. All of the tested cell lines were expressing the GM-CSF receptor (CD116), but only the cell line MUTZ-3 was also expressing the IL-4 re-ceptor (CD124). This demonstrates the unique ability of MUTZ-3 cells to become CD1a-positive without simultaneously expressing CD83, i.e., acquiring the iDC phenotype.

- 34 -MIITZ-3 is a CD34-positive DC differentiation model derived from precursor cells In addition to neo-expression of CD1a, further morphologi-cal and phenotypical changes were observed following cyto-kine stimulation of MUTZ-3 cells. Typically, the MUTZ-3 cells were non-adherent, round or somewhat lobular cells.
Subsequent to differentiation, the MUTZ-3 iDCs were no more than loosely adherent, forming lumps of large cells and de-veloping hair-like, cytoplasmatic projections - a morpho-logical characteristic of DCs (Fig. la, b). Analysis of the cell surface markers showed sparse expression of CD14, CD86, CD54 and CD40, and strong expression of CD34 and HLA-DR by non-stimulated MUTZ-3 cells (Figs. 2 and 3). Follow-ing induction of CD1a expression on the cell surface, a down-regulated expression of CD14 (monocyte marker) and CD34 (marker of hematopoietic precursor cells) was ob-served. Expression of the co-stimulatory and adhesion mole-cules CD80, CD86, CD40, CD54, and HLA-DR was strongly up-regulated on MUTZ-3 iDCs compared to the non-stimulated cell population (Fig.3). Stimulation of the MUTZ-3 iDCs with TNFa induced expression of the DC maturing marker CD83 with a further increase of CD1a expression and all co-stimulatory molecules. Similar observations were made when the MUTZ-3 iDCs had been matured with LPS, CD40 ligand-transfected J558 cells or polyIC (results not shown). No further proliferation was observed upon addition of cytoki-nes to the MUTZ-3 iDCs or mature DCs (mDC).
Accordingly, MUTZ-3 cells are capable of differentiating into DCs (MUTZ-3 DC) under the influence of GM-CSF, IL-4 and low-dosed TNFa, passing through two different stages of differentiation - an immature (MUTZ-3 iDC) and a mature phenotype (MUTZ-3 mDC).

- 35 -Such down-regulation of CD34 and CD14 suggests that MUTZ-3 cells represent a population of precursor cells in the dif-ferentiation of the CD34-positive stem cells. Differentia-tion of CD34-positive stem cells gives rise to formation of at least two types of precursor cells which ultimately ma-ture into interstitial and Langerhans cells (LC). To deter-mine whether MUTZ-3 cells would develop into LC-like cells, we have cultured MUTZ-3 cells in the presence or absence of TGFP1. TGFP1 is known to induce a LC phenotype in DCs de-rived from CD34-positive cells (Caux et al. 1997, Blood 90(4), 1458-1470). We observed not only an increase of the amount of CD1-positive MUTZ-3 cells from 20% to 80%, but also strong langerin/CD1a double staining under the influ-ence of TGF31, the latter indicating that these cells ex-hibit specific characteristics of LC cells.
MUTZ-3 DCs induce proliferation of allogenic lymphocytes In mixed lymphocyte reactions, MUTZ-3 mDCs were capable of stimulating proliferation of allogenic T cells, and indeed, to a higher degree compared to MUTZ-3 iDCs or non-stimulated MUTZ-3 cells. Incorporation of 3H-thymidine (lym-phocyte proliferation) increased by 6-10 times compared to non-stimulated MUTZ-3 cells and incorporation of 3H-thymidine increased by 2-3 times compared to MUTZ-3 iDCs was measured at a MUTZ-3/PBL ratio of 40:1 (Fig. 5). Such enhanced stimulatory properties of MUTZ-3 mDCs compared to MUTZ-3 iDCs probably reflect the observed increase of ex-pression of the co-stimulatory and adhesion markers CD80, CD86, CD40 and CD54 (as shown in Fig.3).
MUTZ-3 DCs respond to Th-polarizing stimuli and assume a DC1 or DC2 phenotype during maturing DCs can secrete IL-12, a potent type 1 T cell-inducing cy-tokine (Kalinski et al. 1998, J. Immunol. 161(6), 2804-

- 36 -2809). Furthermore, it has been demonstrated that non-preprogrammed iDCs under the influence of particular stim-uli assume the capability of secreting mainly IL-12 (DC2 phenotype) or the type 2-inducing cytokine IL-10 (DC2 phe-notype) (Vieira et al. 2000, J. Immunol. 164(9), 4507-4512;
Langenkamp et al. 2000, Nat. Immunol. 1(4), 311-316). To investigate whether MUTZ-3 iDCs would develop either the DC1 or DC2 phenotype, maturing of the MUTZ-3 iDCs was in-duced in the presence of IFNy or dexamethasone. When stimu-lating MUTZ-3 mDCs (after maturing in the presence of TNFa) in the presence or absence of CD40 ligand-transfected J558 cells, small amounts of IL-10 and IL-12 were produced (Fig.
6). On the other hand, maturing of MUTZ-3 iDCs in the pres-ence of IFNy gave IL-12 production which, in addition, mas-sively increased when stimulation of the cells by said transfected J558 cells continued (post-maturation). In marked contrast, no IL-12 at all was produced by MUTZ-3 mDCs when maturing thereof was effected in the presence of dexamethasone. However, increased IL-10 production was de-tected in these cell cultures. These results show that non-preprogrammed MUTZ-3 DCs can be modified into the DC1 or DC2 phenotype under suitable conditions.
MUTZ-3 cells as effective DCs having the ability to process and present antigens and to induce an immune response One central function of DCs as professional antigen-presenting cells (APC) is their ability to stimulate CD4-and CD8-positive T cells and (as recently shown) present lipids and hydrophobic antigens to NKT cells. We therefore investigated whether MUTZ-3 DCs would be capable of spe-cifically processing and presenting antigens in this way.

- 37 -MUTZ-3 DCs activate influenza-specific, cytotoxic T lympho-cytes via class I MHC
Molecular typification indicated that MUTZ-3 cells were positive to the HLA antigens HLA-A2, HLA-A3, HLA-B44, HLA-DR10, ILA-DR11, HLA-DR52, HLA-DQ5, and HLA-DQ7. HLA-A2 ex-pression was confirmed by FACS analysis using the mono-clonal antibodies MA2.1 and BB 7.1 (results not shown). We then investigated whether MUTZ-3 DCs would be capable of processing and presenting antigens via the HLA-A2 class I
molecule. MUTZ3 DCs were loaded with the immunodominant A2-binding Ni heminfluenza (flu) peptide, or the cells were infected with adenoviruses encoding the entire M1 sequence (to test the capability of HLA class I processing). In both cases, T2 cells loaded either with the MI flu peptide or, as a control, with the HPV-derived E7 peptide were used as stimulator cells in the IFNy ELISPOT assay for cytotoxic T
lymphocytes (CTL) which might have formed during co-culturing of MUTZ-3 DCs and T cells. Unstimulated T cells were added to determine the base line of the flu-specific CTL reaction. No specific CTL response was observed under these conditions (results not shown). An HLA-A2-restricted, flu-specific CTL expansion was detected upon co-culturing of the CTLs with MUTZ-3 DCs which were either loaded with the flu peptide or infected with the Ml-encoding adenovirus (Fig. 7a 1,2). These results demonstrate that MUTZ-3 DCs are capable of processing and presenting flu peptides, re-sulting in a stimulation of flu-specific, class I MHC-restricted CTLs.
MUTZ-3 DCs induce MART-1-specific, cytotoxic T lymphocytes via class I MHC
HLA-A2-dependent, MART-1-specific CTL expansion and activa-tion (IFNy secretion) was detected upon co-culturing of the CTL with MUTZ-3 DCs loaded with the modified MART-1 peptide

- 38 -ELAGIGILTV (Fig. 9). These results show that MUTZ-3 DCs are capable of sensitizing naive CTLs via class I MHC.
Tumor cell lysate-loaded MUTZ-3 DCs induce cytotoxic T lym-phocytes specific for different tumor antigens HLA-A2-dependent, tumor cell lysate-specific CTL expansion and activation (IFNy secretion) was detected upon co-culturing of the CTLs with MUTZ-3 DCs loaded with tumor cell lysate (Fig. 10). Activation of these CTLs was also possible by restimulation with the MUC1 peptide LLLLTVLTV
and by restimulation with the protein asialoglycophorin.
These results show that MUTZ-3 DCs are capable of inducing a polyspecific cellular anti-tumor immune response.
Generation of immature MUTZ-3 (iDC) from precursor cells using GM-CSF, TNFa and various IL-4 concentrations or IL-13 MUTZ-3 cells from the current culture were washed twice with PBS and seeded at a cell density of lx105 cells/ml into a volume of 5 ml of culture medium in a 6-well plate and incubated for 7 days with GM-CSF (1000 U/m, Leukomax, No-vartis), low-dosed TNFa (2.5 ng/ml, Peprotech) and various concentrations of IL-4 (between 0.1 U/ml and 1000 U/ml, Peprotech). In another test IL-4 was replaced with IL-13 (100 ng/ml). This concentration corresponds to approxi-mately the 40fold concentration of the IL-4 concentration used (100 U/ml). Cytokine was added on each second to third day. After 7 days of incubation the cells were character-ized by flow cytometry (see Figs. 11 and 12).
Stimulation of TT-specific, CD4-positive T cells by TT-pulsed MUTZ-3 iDCs The capability of peptide processing via the class II MHC
pathway was investigated by pulsed loading of MUTZ-3 iDCs

- 39 -with peptides derived from the "common recall" TT antigen and subsequent co-culturing with allogenic CD4-positive T cells partially matching with respect to the HLA type.
Strong stimulation of the TT-specific CD4-positive T cells was observed when MUTZ-3 iDCs were loaded with TT peptides in a pulsed fashion, as compared to the vehicle as a con-trol, and the control values were similarly low as in the case of CD4-positive cells alone (Fig. 7b). These results show that MUTZ-3 cells are capable of processing and pre-senting antigens via the class II MHC pathway.
Glycolipid presentation by MUTZ-3 DCs to Va24-positive/
14611-positive NKT cells CD1 molecules represent a specialized class of antigen-presenting molecules capable of presenting lipids, glyco-lipids and hydrophobic peptides. It has been demonstrated that the glycolipid a-GalCer can be presented to Va24-positive/V311-positive NKT cells (Brossay et al. 1998, J.
Exp. Med. 188(8), 1521-1528). To investigate whether MUTZ-3 DCs would be capable of presenting a-GalCer, we initially demonstrated that MUTZ-3 DCs express the CD1d molecule (re-sults not shown). MUTZ-3 iDCs and mDCs were then loaded with a-GalCer or vehicle and co-cultured with purified NKT
cells for 7 days in the presence of 10 ng/ml IL-7 and IL-15 (van der Vliet et al. 2001, J. Immunol. Methods 247(1-2), 61-72). a-GalCer-loaded MUTZ-3 mDCs were superior in induc-ing NKT cells compared to MUTZ-3 iDCs (loaded both with a-GalCer and vehicle) and vehicle-loaded MUTZ-3 mDCs. The termination of antigen presentation by CD1d blocking con-firmed the conclusion that MUTZ-3 mDCs are capable of pre-senting glycolipid antigens via the non-classical antigen-presenting CD1d molecules (Fig. 8).

- 40 -Key to the drawings Table 1. FACS analysis of CD1a and CD83 expression in leu-kemia cell lines. CD1a and CD83 expression was investigated using flow cytometry following 7 days of incubation with cytokines. With MUTZ-3 cells a neo-expression of CD1a but not of C1D83 was observed. Minor induction of CD1a expres-sion with associated CD83 expression was measured for KG-1 and to an even lesser extent also for THP-1 cells. a % posi-tive cells represents the total number of cells with posi-tive staining with a particular CD marker within a gated cell population. b Cells stained by PE-conjugated anti-CD1a and FITC-conjugated anti-CD83 monoclonal antibodies repre-sent double-positive cells. C Cells were stained with FITC-conjugated anti-CD116 monoclonal antibodies. d Published in Drexler, H.G. 2001, The Leukemia-Lymphoma Cell Line Facts Book, Academic Press.
Figure 1. Microscopic images of differentiated MUTZ-3 cells following addition of cytokines. a) Unstimulated MUTZ-3 cells, b) MUTZ-3 iDCs after culturing for 7 days in the presence of GM-CSF, IL-4 and low concentrations of TNFa.
The cells are no more than loosely adherent, showing a den-dritic morphology (enlarged 40fold).
Figure 2. MUTZ-3 DCs show characteristics of immature and mature DCs in the presence of cytokines. The scatter plot representation illustrates the phenotype a) of unstimulated MUTZ-3 cells, b) of immature MUTZ-3 iDCs and c) of TNFa-induced mature MUTZ-3 mDCs. The numbers relate to the per-centage of cells positive to the respective CD marker. All cells were stained with PE- or FITC-conjugated, antigen-specific, monoclonal antibodies. The data are derived from one experiment which is representative of five experiments.

- 41 -Figure 3. The differentiation of MUTZ-3 cells is associated with the induction of expression of co-stimulatory mole-cules. FACS analysis indicates induction of the co-stimulatory molecules CD86 and CD40, of adhesion molecule CD54 and class II HLA molecule HLA-DR during MUTZ-3 differ-entiation; unstimulated MUTZ-3 (dotted line), immature MUTZ-3 iDCs (solid line) and mature MUTZ-3 mDCs (fat solid line). The data are derived from one experiment which is representative of five experiments.
Figure 4. TGF01 induces expression of the LC-associated surface molecule langerin on MUTZ-3 cells. CD34-positive MUTZ-3 cells were initially cultured in the presence of GM-CSF/TNFa and subsequently in the presence or absence of TGF01. The numbers in the left upper corner relate to the percentage of CD1a/langerin double-positive cells within a gated cell population, or to the percentage of cells stained with an isotypic antibody as a control. The data are derived from one experiment which is representative of three experiments.
Figure 5. The ability of MUTZ-3 cells to stimulate lympho-cytes. Unstimulated MUTZ-3, immature MUTZ-3 iDCs and mature MUTZ-3 mDCs were co-cultured with lymphocytes non-matching in MHC in an allogenic mixed lymphocyte reaction. MUTZ-3 mDCs had a strong stimulatory capacity compared to unstimu-lated MUTZ-3 cells (by 6.3 higher difference in 3H-thymidine incorporation compared to unstimulated cells, and by 2.3 higher difference compared to MUTZ-3 iDCs). The data are derived from one experiment which is representative of four experiments.
Figure 6. Non-preprogrammed MUTZ-3 iDCs can be modified into the DC1 or DC2 phenotype during maturing under the in-fluence of IFNy or dexamethasone. MUTZ-3 iDCs cultured in the presence of IFNy secrete IL-12. No IL-12 production is

- 42 -observed when culturing the cells with dexamethasone. Simi-larly, the cells do not secrete any IL-10 when treated with IFNy. IL-12 and IL-10 concentrations were determined using ELISA. The cytokine concentrations are given in pg/ml per 105 cells. The data are representative of four individual experiments.
Figure 7. MUTZ-3 cells have the capability of processing and presenting antigens. (a) Class I MHC presentation.
MUTZ-3 iDCs stimulate a flu-specific CTL reaction by pre-senting the flu peptide restricted to HLA-A2.1. (1) MUTZ-3 DCs were loaded with the HLA-A2.1-binding heminfluenza-derived matrix protein M158-66 and co-cultured with CD8-positive T cells. To detect CTL proliferation the produc-tion of IFNy by the CTLs was measured which were co-cultured with T2 cells as target cells. The T2 cells were either loaded with the M1 flu peptide (black squares) or with the HPV16-derived peptide E7 as control (white squares). (2) MUTZ-3 DCs were infected with recombinant adenoviruses including the M1 matrix protein gene and sub-sequently co-cultured as described above. Again, CTLs were stimulated with T2 cells loaded either with the M1 flu pep-tide (black circles) or with the E7 peptide (white cir-cles). The data are derived from one experiment which is representative of three experiments. (b) Class II MHC anti-gen presentation. MUTZ-3 mDCs process and present peptides derived from the common recall TT antigen and stimulate TT-specific CD4-positive T cells. The data are derived from one experiment which is representative of three experi-ments.
Figure 8. Presentation of a-GalCer via CD1d. MUTZ-3 iDCs were loaded either with a-GalCer or with vehicle (DMSO) as control and subsequently cultured for 48 h in the presence or absence of higher-dosed TNFa. Thereafter, mDCs were co-cultured for 9 days in the presence or IL-7 And IL-15 and

- 43 -in the presence or absence of CD1d-blocking antibodies with NKT cells isolated from healthy donors. The results show the relative yield of NKT cells following co-culturing with MUTZ-3 iDCs and mDCs previously loaded with vehicle and a-GalCer, with or without blocking of a-GalCer presentation by the CD1d-blocking antibody. The data are derived from one experiment which is representative of three experi-ments.
Figure 9. MUTZ-3 DCs are capable of sensitizing naive CTLs.
CTLs were stimulated with MART-1 ELAGIGILTV peptide-loaded MUTZ-3 DCs (prime) and after one week restimulated over-night with MART-1 ELAGIGILTV or CEA IMIGVLVGV peptide-loaded T2 cells. The IFNy ELISPOT showed strong antigen-specific (MART-1) activation of the CTLs; restimulation with an irrelevant antigen (CEA) gave only sparse activa-tion of the cells.
Figure 10. MUTZ-3 DCs are capable of inducing a polyspeci-fic anti-tumor CTL response.
CTLs were stimulated with tumor cell lysate-loaded MUTZ-3 DCs (prime) and after one week restimulated with tumor cell lysate-loaded MUTZ-3 DCs, with asialoglycophorin-loaded MUTZ-3 DCs, or with MUC1 LLLLTVLTV peptide-loaded T2 cells.
The IFNy ELISPOT showed strong activation of the CTLs by restimulation of the cells with tumor cell lysate, with the MUC I peptide and with asialoglycophorin.
Figure 11: MUTZ-3 cells from the current culture were incu-bated for 7 days with GM-CSF (1000 U/ml), low-dosed TNFa (2.5 ng/ml), and varying concentrations of IL-4 (between 0.1 U/ml and 1000 U/ml). The results show that a reduction of CD124 expression can be observed with increasing IL-4 concentration.

- 44 -Figure 12: MUTZ-3 cells from the current culture were incu-bated for 7 days with GM-CSF (1000 U/ml), low-dosed TNFa (2.5 ng/ml), and in a comparative fashion with IL-4 (100 U/ml) or IL-13 (100 ng/ml). The concentration of IL-13 corresponds to approximately the 40fold concentration of the IL-4 used. Characterization of the surface molecules by means of flow cytometry shows that comparable expression of surface molecules can be observed in a 7-day incubation with IL-13 instead of IL-4, in addition to GM-CSF and low-dosed TNFa.
In the context with the invention the term "sensitize"
means transferring T lymphocytes into a state of suscepti-bility to an antigen-specific stimulus.
Cell line % positive Cytokine receptor cellsa expression CD1ab CD83b CD116 CD124 (GM-CSF (IL-4 receptor)c receptor)d Table 1: FACS analysis of CD1a and CD83 expression on leu-kemia cell lines. CD1a and CD83 expression was investigated using flow cytometry on day 7 after cytokine addition. CD1a but not CD83 expression can be induced in MUTZ-3 cells.

- 45 -KG-1 and, to a minor extent, TH-1 give CD1a (low) expres-sion in connection with CD83 expression.
a %
positive cells represents the total number of cells with positive staining with a particular CD marker within a gated cell population.
= Cells were stained with PE-labelled anti-CD1a and FITC-labelled anti-CD83 monoclonal antibodies; this result rep-resents double-positive cells.
= Stained with anti-CD116 FITC-labelled monoclonal anti-bodies.
= By Drexler, H.G. 2001, The Leukemia-Lymphoma Cell Line Facts Book, Academic Press.

Claims (53)

Claims:

1. A method for producing immature dendritic cells or cell lines, said method comprising culturing an MUTZ-3 cell line with stimulatory molecules: (a)GM-CSF, TNF.alpha. and IL-4;
(b) GM-CSF, TNF.alpha. and IL-13; or (c) GM-CSF, TNF.alpha., and TGF.beta.1; until said immature dendritic cells or cell lines are obtained.

2. The method of claim 1, wherein said immature dendritic cells or cell lines produced have a phenotype corresponding to at least one of: interstitial dendritic cells, Langerhans dendritic cells, and CD1a-positive cells.

3. A method for producing mature dendritic cells or a mature dendritic cell line from MUTZ-3-derived immature dendritic cells or an MUTZ-3-derived immature dendritic cell line, said method comprising culturing said MUTZ-3-derived immature dendritic cells or said MUTZ-3-derived immature dendritic cell line with stimulatory molecules: IFN.gamma., dexamethasone, TNF.alpha., LPS, CD40 ligand, polyinsosinic-polycytidylic acid (polyIC), or any combination thereof, until said mature dendritic cells or said mature dendritic cell line are obtained.

4. The method of claim 3, wherein said mature dendritic cells or cell lines produced have a phenotype corresponding to at least one of: CD83-positive cells, DC type 1 cells, and DC type 2 cells.

5. The method of any one of claims 1-4, wherein said MUTZ-3 cell line is CD34-positive.

6. The method of any one of claims 1-5, wherein a CD34 gene is introduced into said MUTZ-3 cell line.

7. The method of any one of claims 1-6, wherein additional genes are introduced into said MUTZ-3 cell line, and wherein said additional genes encode and/or express receptors for or inhibitors of said stimulatory molecules.

8. The method of any one of claims 1-7, wherein at least one immunotherapeutic agent-encoding gene is introduced into said MUTZ-3 cell line, wherein said at least one immunotherapeutic agent-encoding gene is a gene which encodes: a tumor antigen; a viral antigen; or an antigen of a parasite, bacteria or microorganism.

9. The method of any one of claims 1-8, further comprising fusing said dendritic cell or cell line produced by said method with other cells or cell lines.

10. The method of any one of claims 1-9, further comprising inducing apoptosis or necrosis in the immature or mature dendritic cell or cell line produced by said method.

11. The method of claim 10, wherein said apoptosis or said necrosis is caused by irradiation.

12. The method of any one of claims 1-10, further comprising incorporating at least one suicide gene into said MUTZ-3 cell line.

13. The method of any one of claims 1-12, further comprising loading the immature or mature dendritic cells or cell lines produced by said method with a further antigen.

14. An MUTZ-3-derived immature dendritic cell or cell line, or an MUTZ-3-derived mature dendritic cell or cell line produced by the method of any one of claims 1-13.

15. The dendritic cell or cell line of claim 14 loaded with at least one antigen.

16. The dendritic cell or cell line of claim 15, wherein said antigen is a tumor antigen or an infection antigen or an antigenic portion thereof.

17. The dendritic cell or cell line of claim 16, wherein said tumor antigen or said infection antigen is: a peptide, a protein, a lipid, a lipopeptide, a lipoprotein, a carbohydrate, a glycolipid, a glycopeptide, a glycoprotein, a phosphorylated protein, a phosphorylated peptide, a post-translationally modified protein or peptide, or is encoded by DNA or RNA which has been transfected into said MUTZ-3 cell line.

18. A pharmaceutical composition for immunotherapy comprising the dendritic cells or cell line of any one of claims 14-17, and a pharmaceutically acceptable carrier.

19. The dendritic cell or cell line of any one of claims 14-17 for use as an immunotherapeutic agent.

20. Use of the dendritic cell or cell line of any one of claims 14-17 for immunotherapy or for the manufacture of a medicament for immunotherapy.

21. The dendritic cell or cell line of any one of claims 14-17 for use in the prophylaxis or treatment of infectious, tumor and/or autoimmune diseases.

22. Use of the dendritic cell or cell line of any one of claims 14-17 for the prophylaxis or treatment of infectious, tumor and/or autoimmune diseases, or for the manufacture of a medicament for same.

23. The dendritic cell or cell line of any one of claims 14-17 for use in the processing and/or presentation of antigens.

24. The dendritic cell or cell line of claim 23, wherein said antigens to be processed and/or presented comprise peptides, proteins, lipids, lipopeptides, lipoproteins, carbohydrates, glycolipids, glycopeptides, glycoproteins, phosphorylated proteins or phosphorylated peptides.

25. Use of the dendritic cell or cell line of any one of claims 14-17 for the processing and/or the presentation of antigens.

26. The use of claim 25, wherein said antigens to be processed and/or presented comprise peptides, proteins, lipids, lipopeptides, lipoproteins, carbohydrates, glycolipids, glycopeptides, glycoproteins, phosphorylated proteins or phosphorylated peptides.

27. A test system comprising the dendritic cell or cell line of any one of claims 14-17.

28. The test system of claim 27 for testing immunoactivity-inhibiting and/or -modulating substances.

29. The test system of claim 27 for testing tumor vaccines or for testing the influence of substances, pharmacological agents, cosmetics or foodstuffs on the immune system.

30. Use of the test system of claim 27 for testing immunoactivity-inhibiting and/or -modulating substances.

31. Use of the test system of claim 27 for testing tumor vaccines or for testing the influence of substances, pharmacological agents, cosmetics or foodstuffs on the immune system.

32. The dendritic cell or cell line of any one of claims 14-17 for the manufacture of a pharmaceutical composition for immunotherapy.

33. Use of the dendritic cell or cell line of any one of claims 14-17 for the manufacture of a pharmaceutical composition for immunotherapy.

34. An MUTZ-3 cell line for use in producing dendritic cells or cell lines.

35. The MUTZ-3 cell line of claim 34, wherein said dendritic cells are immature dendritic cells or cell lines.

36. The MUTZ-3 cell line of claim 35, wherein said immature dendritic cells or cell lines have a phenotype corresponding to at least one of: interstitial dendritic cells, Langerhans dendritic cells, and CD1a-positive cells.

37. The MUTZ-3 cell line of claim 35, wherein said dendritic cells are mature dendritic cells.

38. The MUTZ-3 cell line of claim 37, wherein said mature dendritic cells or cell lines have a phenotype corresponding to at least one of: CD83-positive cells, DC
type 1 cells, and DC type 2 cells.

39. The MUTZ-3 cell line of any one of claims 34-38, wherein said dendritic cells or cell lines are CD34-positive.

40. The MUTZ-3 cell line of any one of claims 34-39, wherein said dendritic cells or cell lines are apoptotic or necrotic.

41. The MUTZ-3 cell line of any one of claims 34-40, wherein said dendritic cells or cell lines are loaded with an antigen or a polynucleotide molecule encoding same.

42. The MUTZ-3 cell line of any one of claims 34-40, wherein said antigen is: a tumor antigen; a viral antigen;
or an antigen of a parasite, bacteria or microorganism.

43. Use of an MUTZ-3 cell line for producing dendritic cells or cell lines.

44. The use of claim 43, wherein said dendritic cells are immature dendritic cells or cell lines.

45. The use of claim 43, wherein said immature dendritic cells or cell lines have a phenotype corresponding to at least one of: interstitial dendritic cells, Langerhans dendritic cells, and CD1a-positive cells.

46. The use of claim 43, wherein said dendritic cells are mature dendritic cells.

47. The use of claim 46, wherein said mature dendritic cells or cell lines have a phenotype corresponding to at least one of: CD83-positive cells, DC type 1 cells, and DC
type 2 cells.

48. The use of any one of claims 43-47, wherein said dendritic cells or cell lines are CD34-positive.

49. The use of any one of claims 43-48, wherein said dendritic cells or cell lines are apoptotic or necrotic.

50. The use of any one of claims 43-49, wherein said dendritic cells or cell lines are loaded with a further antigen or a gene encoding same.

51. The use of any one of claims 43-50, wherein said exogenous antigen is: a tumor antigen; a viral antigen; or an antigen of a parasite, bacteria or microorganism.

52. An MUTZ-3-derived immature dendritic cell or cell line.

53. A MUTZ-3-derived mature dendritic cell or cell line.