Dendritic cells are professional antigen presenting cells (APC) and are key regulators of the immune system. Located at the boundary between the inside and the outside world, dendritic cells provide a bridge between the innate and adaptive immune system. As sentinels throughout the body, dendritic cells capture and process antigen, undergo maturation, and subsequently, migrate to the secondary lymphoid organs, where they present the processed antigens to naive T cells and initiate primary T cell responses. In vivo, dendritic cells originate from bone marrow-derived CD34-positive hematopoietic progenitor cells (HPC), which can give rise to two classic myeloid dendritic cell subsets,

1. e., Langerhans cells (LC), which line the epithelial layers of the skin, and interstitial dendritic cells (IDC), which can be found in the dermis and throughout the body (Banchereau et al. 2000, Annu Rev. Immunol 18, 767; Figdor et al. 2004, Nat Med 10, 475). Besides that, CD34-positive HPC can also give rise to a nonclassic, plasmacytoid dendritic cell (PDC) subset, which has been described to secrete large amounts of interferon (IFN)-a/3 upon viral encounter, thereby representing the first barrier to intruding viruses (Siegal et al. 1999, Science 284, 1835; Shortman and Liu 2002, Nat Rev Immunol

2, 151 ). Several protocols have been developed to generate human dendritic cells in vitro. Dendritic cells can be generated from blood or bone marrow-derived CD34-positive hematopoietic progenitor cells or peripheral blood-derived CD14-positive monocytes (Romani et al. 1994, J Exp Med 180, 83; Caux et al. 1996, J Exp Med 184, 695; Sallusto and Lanzavecchia 1994, J Exp Med 179, 1 109). Upon culture in the presence of GM-CSF, TNF-a, and TGF-β or IL-4, CD34-positive hematopoietic progenitor cells can be differentiated into Langerhans cells or interstitial dendritic cells, respectively (Caux et al., loc. cit; Romani et al. 1996, J Immunol Methods 196, 137; Strobl et al. 1996, J Immunol 157, 1499). Besides that, interstitial dendritic cells can also be generated from CD14- positive monocytes. In the presence of different cytokine combinations, CD14-positive monocytes can develop into dendritic cells with distinct phenotype and function. As described, upon culture with GM-CSF and IL-4, IFN-α/β, or IL-15, monocytes are able to develop into IL-4-dendritic cells, IFN-dendritic cells, or IL-15-dendritic cells, respectively (Zhou and Tedder 1996, PNAS 93, 2588; Luft et al. 1998, J Immunol 161 , 1947; Mohamadzadeh et al. 2001 , J Exp Med 194, 1013). The development of such dendritic cell differentiation protocols has been of great importance for the study of dendritic cell biology and the subsequent implementation in clinical dendritic cell vaccination studies.

Usually, when dendritic cells are generated in vitro from progenitor cells, culture medium contains serum from (non-human) animals, in most cases from fetal bovines, i.e. Fetal Bovine Serum (FBS), sometimes also referred to Fetal Calf Serum (FCS). While in some cases the differentiation of progenitor cells into dendritic cells has been performed without serum, the progenitor cell lines, before differentiation, were nevertheless cultured in serum-containing media (see Ning et al. 201 1 , J. Biomed. and Biotechnol. 201 1 ). This has the unfortunate side effect that there are still serum components present in the dendritic cell lines after differentiation despite serum-free differentiation. In order to obtain large amounts of dendritic cells, progenitor cells such as leukemic cells or blast cells, e.g. AML blast cells have to be cultured and expanded and for these purposes culture medium containing (non-human) animal serum is used. However, there are concerns with using animal serum, such as the risk of contamination of such progenitor cells that may be differentiated in dendritic cells by animal proteins or pathogens for the manufacture of dendritic cells for human therapies.

Accordingly, the technical problem may thus be seen to comply with the aforementioned concerns to ensure that the dendritic progenitor cell lines are adapted to a serum-free environment so that they can be differentiated into dendritic cells without residual serum components.

The present invention provides as a solution to the technical problem means and methods for serum-free cultivation of progenitor cells for dendritic cells.

Specifically, the present invention provides a method for the serum-free cultivation of progenitor cells for dendritic cells comprising culturing said cells in serum-free cell culture medium containing GM-CSF as well as progenitor cells for dendritic cells obtainable by the method of the present invention, said progenitor cells are contained in serum-free medium or in a human blood-derived product as described herein.

Indeed, although serum-free media and methods for cultivating various cell lines without animal serum are known in the art, to the inventors' best knowledge, no such serum-free cultivation has been described or done for progenitor cells for dendritic cells up to the present invention. A reason may be that serum-free cultivation for one cell line cannot be readily transferred to another cell line, since each of them may have specific demands. The present inventors found that animal serum-free cultivation of progenitor cell lines for dendritic cells requires in particular the presence of GM-CSF in serum-free culture medium.

The term "cultivation of cells" or "culturing of cells" in medium (either with serum or serum free) in the context of the present invention refers to the seeding of the cells into the culture vessel, to the growing of the cells in medium in the logarithmic phase until, in case of adherent culturing, a monolayer is formed, or, in case of a suspension culture, a sufficient cell density is established and/or to the maintenance of the cells in medium as soon as the monolayer is formed or to the maintenance of the cells in suspension, respectively. The term "cultivation of cells" or "culturing of cells" in medium also includes that all of the above mentioned steps are performed with serum free medium, so that no animal serum products are present during the whole cultivation process of the cells. Yet, as described herein, the above mentioned steps may also be performed with serum containing medium in case progenitor cells are cultured for a certain period of time in order to, e.g. expand them prior to being cultured without serum.

Culturing of progenitor cells can be done in any container suitable for culturing cells, for instance in dishes, roller bottles or in bioreactors such as the WAVE™ bioreactor system. Suitable conditions for culturing cells are known (see e.g. Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and R.I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition, Wiley-Liss Inc., 2000, ISBN 0-471 - 34889-9. The term "serum-free" medium refers to any cell culture medium that does not contain sera from non-human animal origin. Suitable serum-free cell culture media are known to the person skilled in the art.

The serum-free culture medium applied in the method of the present invention is preferably a chemically defined serum-free medium or is a human blood-derived product.

A chemically defined serum-free medium may comprise salts, vitamins, buffers, energy sources, amino acids and other substances.

A preferred chemically defined serum-free medium contains X-VIVO 10, X-VIVO 15 or X- VIVO 20 medium, with X-VIVO 15 medium being preferred. These media can be purchased from Lonza. However, apart from these preferred chemically defined serum- free media also other such chemically defined serum-free media are known in the art and may also be applied in the method of the present invention. A preferred human blood-derived product is a human platelet lysate or human serum albumin. Preferably, serum-free cell culture medium contains between 5 and 100, optionally at least 5, 10, 15 or 20 units/ml GM-CSF, with at least 20 units/ml GM-CSF being preferred. As amounts of around 1000 units/ml GM-CSF are generally used to differentiate progenitor cell lines into dendritic cells, the amount of GM-CSF should be low to ensure that the progenitor cell line does not differentiate.

It is preferred that GM-CSF is the only cytokine present in said serum-free cell culture medium. Of note, GM-CSF alone is not able to differentiate progenitor dendritic cells into dendritic cells. As mentioned above, upon culture in the presence of GM-CSF, TNF-a, and TGF-β or IL-4, CD34-positive hematopoietic progenitor cells can be differentiated into Langerhans cells or interstitial dendritic cells, respectively (Caux et al., loc. cit; Romani et al. 1996, J Immunol Methods 196, 137; StrobI et al. 1996, J Immunol 157, 1499). Other reagents for maturing dendritic progenitor cells into dendritic cells are well known (for example IFN-γ, CD40 ligand (L), LPS and DNA (Banchereau et al. 2000 Annu Rev Immunol 18, 767-81 1 ). Besides that, interstitial dendritic cells can also be generated from CD14-positive monocytes. In the presence of different cytokine combinations, CD14- positive monocytes can develop into dendritic cells with distinct phenotype and function. Also, upon culture with GM-CSF and IL-4, IFN-α/β, or IL-15, monocytes are able to develop into IL-4-dendritic cells, IFN-dendritic cells, or IL-15-dendritic cells, respectively (Zhou and Tedder 1996, PNAS 93, 2588; Luft et al. 1998, J Immunol 161 , 1947; Mohamadzadeh et al. 2001 , J Exp Med 194, 1013). However, it was surprisingly found that the dendritic progenitor cell lines could be cultivated in the absence of serum if low amounts of GM-CSF were added to the serum-free media using the methods of the invention to generate serum-free dendritic cell progenitor lines. For this reason, the present invention also encompasses the use of a serum-free medium containing GM-CSF as the only cytokine present in the serum-free medium for use in the cultivation of dendritic progenitor cells. These cells can be kept stably in culture and then differentiated into dendritic cells by the addition of further cytokines as known in the art and summarized above.

The terms "cell" and "cell line" can be used interchangeably herein. The term "cell line" means a cell line or cells which can be grown under in vitro culture conditions as indicated, e.g., in the appended examples. Additionally, said term also embraces cells of a single type that have been grown in the laboratory for several generations. Moreover, said term encompasses subclones, in particular subclones of the specific progenitor cells as described herein, i.e., MUTZ-3 (DSMZ ACC 295), DCOne (DSMZ ACC 3189), THP-1 (ATCC Accession No. TIB-202), HL-60 (ATCC Accession No. CCL-240), or KG-1 (ATCC Accession No. CCL-246), with MUTZ-3 (DSMZ ACC 295) or DCOne (DSMZ ACC 3189) being preferred.

The term "subclones" when used in accordance with the present invention means cells or cells of a cell line which occur due to naturally occurring alterations, e.g., mutations or loss of genetic material, but still exhibit those characteristics as the source progenitor cell or cell line, e.g. MUTZ-3 (DSMZ ACC 295) or DCOne (DSMZ ACC 3189).

The term "progenitor cell of the invention", "progenitor cell for a dendritic cell" or "progenitor dendritic cell" as well grammatical variants thereof as used herein means cells that can be differentiated or transformed by means and methods known in the art into dendritic cells.

Preferably, at least 40% of the progenitor cells are CD34-positive cells. It is further preferred that the CD34-positive cells are in addition CD1 a- and CD83-.

It is yet further preferred that the CD34-positive progenitor cells are in addition CD3-, CD4+, CD5-, CD7-, CD8-, CD13+, CD14+, CD15+, CD19-, and/or HLA-DR+. The expression level of the cell surface markers, such as CD34, CD1 a, or CD83 and those further described herein are preferably determined by ELISA assay or flow cytometry.

The term "progenitor cell line" refers to a cell line of dendritic progenitor cells. Examples of such cell lines are THP-1 (ATCC TIB-202), HL-60 (ATCC CCL-240), KG-1 (ATCC CCL- 246), MUTZ-3 cells (DSMZ Accession No. ACC 295) and DCOne cells (DSMZ Accession No. ACC 3189).

In a preferred embodiment, the progenitor cell lines of the invention are obtained from a patient suffering from leukemia, in particular from acute myeloid leukemia (AML), more particularly AML of type FAB M4 (acute myelomonocytic leukemia). Preferred examples of such AML cells are those of the cell line MUTZ-3 (DSMZ ACC 295) or DCOne (DSMZ ACC 3189). Other examples of progenitor cells of the invention are cells from the cell line THP-1 (ATCC Accession No. TIB-202), HL-60 (ATCC Accession No. CCL-240), or KG-1 (ATCC Accession No. CCL-246).

In general, such serum-free cultivation of a cell line requires approximately 50 generations starting from known cell lines in cultivation medium including serum and stepwise reduction of the amount of serum until the cell lines are cultivated in serum-free medium. However, in the present case it was found that the cultivation of the dendritic cell progenitor cell lines in serum-free medium was only possible in the presence of low amounts of GM-CSF (5-100 units/ml, preferably 20 units/ml).

It is preferred that prior to culturing progenitor cell lines under (non-human animal) serum- free conditions, said progenitor cells are cultured in conditioned medium in order to step- wise adapt them to (non-human animal) serum-free conditions. This can be achieved, e.g. by reducing the amount of conditioned medium over the course of time.

Accordingly, the present invention provides in a preferred embodiment that progenitor cells have been cultivated prior to cultivation in serum-free cell culture medium in cell culture medium containing conditioned medium derived from 5637 cells (DSMZ Accession No. ACC 35) or GM-CSF and serum, and/or in serum-free cell culture medium containing conditioned medium derived from 5637 cells (ATCC HTB-9).

The cell culture medium used for culturing progenitor cells prior to culturing them in serum-free medium may be minimal essential medium (MEM):

The serum that may be contained in a cell culture medium for culturing progenitor cells prior to culture them in serum-free medium may be fetal bovine/calf serum. Said fetal bovine/calf serum may be present in the cell culture medium in an amount of 20% (v/v).

The conditioned medium derived from 5637 cells that may be contained in a cell culture medium for culturing progenitor cells prior to culture them in serum-free medium may be present in the cell culture medium in an amount of 10% (v/v). The present invention further provides a method for differentiating progenitor cells as described herein which are (or were) cultured in a serum-free medium into dendritic cells. Thus, the method of the present invention for the serum-free cultivation of progenitor cells for dendritic cells comprising culturing said cells in serum-free cell culture medium containing GM-CSF and then further comprises a step of differentiating said progenitor cells into dendritic cells.

The present invention also provides dendritic cells obtainable by (or which are obtained by) the afore-described method, which dendritic cells are contained in serum free medium. Methods for differentiating or transforming progenitor cells for dendritic cells into dendritic cells are known in the art; e.g. from WO 03/023023, WO 2009/019320, Bontkes et al. (2006), J. Immunother 29(2):188-200; or Ning et al. (201 1 ), J Biomed Biotechnol.172965. The term "dendritic cell" as used herein refers to antigen-presenting cells which play a critical role in the regulation of the adaptive immune response. Dendritic cells are unique antigen-presenting cells and have been referred to as "professional" antigen presenting cells since the principal function of dendritic cells is to present antigens, and because only dendritic cells have the ability to induce a primary immune response in resting naive T lymphocytes. To perform these functions, dendritic cells are capable of capturing antigens, processing them, and presenting them on the cell surface along with appropriate co-stimulation molecules. Dendritic cells also play a role in the maintenance of B cell function and recall responses. Thus, dendritic cells are critical in the establishment of immunological memory. A dendritic cell may preferably be obtainable (or is obtained) by the methods as described herein.

Said term further encompasses "immature dendritic cells" and "mature dendritic cells". Immature dendritic cells may be mature into mature dendritic cells by means and methods known in the art. Accordingly, a dendritic cell as referred to herein also encompasses "dendritic cells of type 1 (DC type 1 )" and "dendritic cells of type 2 (DC type 2)". While DC type 1 mainly secret IL-12, DC type 2 mainly secrete IL-10.

Said term also includes "interstitial-like dendritic cells stage/phenotype" as well as Langerhans-like dendritic cells. In a further preferred embodiment, the method further comprises introducing additional genes into the dendritic cells of the invention which encode and/or express receptors for or inhibitors of stimulatory molecules. Said stimulatory molecules are preferably selected from the group consisting of GM-CSF, TNF alpha, LPS, PGE2, CD40 ligand, polylC, calcium, PMA, TGF beta 1 , IL-7, IL-13 and/or IL-4.

In a still preferred embodiment of the method, at least one immunotherapeutic agent gene is introduced into the dendritic cells of the invention, wherein said immunotherapeutic agent is a gene which encodes a tumor antigen, a viral antigen, an antigen of a parasite, bacteria or microorganism.

In another preferred embodiment of the method, the dendritic cells of the invention are fused with other cells or cell lines.

In a further preferred embodiment of the method, the dendritic cells of the invention having different activation and/or effector stages are obtained as a result of different stimulatory molecules, the dosage thereof and/or the order of contacting, as described in the following examples.

Furthermore, the present invention relates to a vaccine composition comprising the dendritic cells of the present invention. It also relates to a pharmaceutical composition comprising dendritic cells of the present invention and optionally a pharmaceutically acceptable carrier or excipient. Dendritic cells are preferably present in the vaccine composition or pharmaceutical composition in a therapeutically effective cell number. A therapeutically effective cell number refers to a cell number which prevents, ameliorates or treats the symptoms of cancer referred to in this specification. Dendritic cells of the present invention are preferably loaded with an antigen, e.g. a tumor antigen. Such loaded dendritic cells may also be comprised by a vaccine composition of the present invention.

Accordingly, in one embodiment of the vaccine composition of the invention, immature dendritic cells of the present invention are loaded with tumor antigens by culturing said cells with tumor cell lysates.

In another embodiment of the vaccine composition of the invention, mature dendritic cells of the present invention are loaded with tumor antigens and/or tumor antigenic peptides by culturing said cells with the tumor antigen and/or tumor antigenic peptides.

In case the vaccine is being prepared by the method for the production of a vaccine of the invention, the resulting mature dendritic cells are loaded by both (i) tumor cell lysate and (ii) tumor antigen and/or peptides derived from the tumor antigen.

In another preferred embodiment of the vaccine the dendritic cells of the invention, are caused to undergo apoptosis or necrosis, e.g., by irradiating the cells or by containing at least one suicide gene. A tumor antigen as used herein means an antigenic substance produced in tumor cells, i.e., it triggers an immune response in the host. Tumor antigens are well known in the art. The identification and generation of antigenic peptides derived from a tumor antigen is known in the art. The production of said peptides can be achieved, e.g., by peptide synthesis. Loading of the dendritic cells of the invention with a tumor antigen and/or peptides of said tumor antigen can be carried out by cultivating said cells in the presence of the tumor antigen and/or peptides derived from said tumor antigen. It is also encompassed by the scope of the invention that not only one tumor antigen is used for loading of the dendritic cells of the invention, but also two, three or even more different tumor antigens. In addition, it is evident to those skilled in the art that not only one peptide of a specific tumor antigen is used for loading, but also a pool of at least two, three, four or even more different peptides. Also in this case, ELISA assays can be used in order to analyze the efficiency of the loading procedure.

As evident to those skilled in the art, the term "vaccination" as used herein means antigen- specific active immunotherapy.

Advantageously, the vaccine composition is for the amelioration, treatment, or prevention of cancer. Accordingly, the vaccine of the invention is preferably a cancer vaccine.

The term "treatment" as used herein denotes the improvement or even elimination of one or more symptoms associated with a cancer referred to herein, by the administration of the dendritic cells or cell lines or vaccine of the invention. An improvement may also be seen as amelioration of said symptom(s).

The term "prevention" as used herein means the avoidance of the occurrence or reoccurrence of a cancer, as referred to herein, by the administration of the dendritic cells or cell lines of the invention or a vaccine of the invention.

The dendritic cells or the vaccine of the invention can be administered either by intradermal or subcutaneous injection. However, it is not excluded that other routes of administration are being used for the administration of the dendritic cells or cell lines or the vaccine of the invention.

An issue concerning the use of the dendritic cells of the invention as a vaccine is the quantity of cells necessary to achieve an optimal anti-cancer or anti-tumor effect. The dosage for administration may be variable, may include an initial administration followed by subsequent administrations, and can be ascertained by the skilled artisan armed with the present disclosure. Typically, the administered dose or dosage will provide for a therapeutically effective amount of the cells, i.e., one achieving the desired local or systemic effect and performance. The number of dendritic cells of the invention to be administered to a patient in need thereof is in the range of between 10 ² to 10 ⁹ , or between 10 ³ to 10 ⁹ , or between 10 ⁴ to 10 ⁹ , such as between 10 ⁴ and 10 ⁸ , or between 10 ⁵ and 10 ⁷ , e.g. about 1 x 10 ⁵ , about 5 x 10 ⁵ , about 1 x 10 ⁶ , about 2 x 10 ⁶ , about 5 x 10 ⁶ , about 1 x 10 ⁷ , or about 2 x 10 ⁷ , about 3 x 10 ⁷ , about 4 x 10 ⁷ , about 5 x 10 ⁷ , about 6 x 10 ⁷ , about 7 x 10 ⁷ , about 8 x 10 ⁷ , about 9 x 10 ⁷ , or about 1 x 10 ⁸ cells can be administered to a human subject. However, the precise determination of a therapeutically effective dose, i.e. cell number, may be based on factors individual to each patient, including their size, age, size of tumor, and amount of time since the tumor occurred, and can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.

The cells can be administered one time or, preferably, more times, for example two, three, four, five, six or even more times, depending on the need and outcome of the vaccination cancer therapy. For example, the cells can be administered once a week, in another embodiment at week 0, then at week 2, followed by week 4, 6, 8 and so on.

The clinical outcome of the vaccination can be analyzed by means and methods known in the medical art, such as PCR analysis or ELISA assays which can be used for analyzing the presence or absence of specific tumor markers, magnetic resonance tomography, computer tomography, ultrasound, X ray, etc.

For the generation of the vaccine composition of the invention, the serum-free dendritic cells of the present invention are particularly advantageous, for the reasons communicated elsewhere herein.

The invention further pertains to a kit comprising the progenitor cells, dendritic cells or the vaccine composition of the invention and an instruction sheet. The term "kit" as used herein refers to a collection of means comprising the progenitor cells, dendritic cells of the invention or the vaccine composition of the invention and an instruction sheet which are provided in separate or common vials in a ready-to-use manner for carrying out the treatment or prevention of cancer as set forth herein. In an aspect, the kit comprises additional means for treating or preventing of cancer as set forth herein, such as means for administration, for instance, syringes and injection needles, etc. Furthermore, in an aspect, the kit comprises instructions for carrying out the vaccination for the prevention, amelioration or treatment of cancer as set forth herein. These instructions can be provided as a manual or can be in the form of a computer implementable algorithm on a data storage medium which upon implementation is capable of governing one or more steps of the treatment, amelioration or prevention of cancer. The instructions comprise information with respect to the cell number to be administered, the route of administration, time schedule for administration and the like.

The invention further pertains to a test system comprising the progenitor cells for dendritic cells and/or dendritic cells of the invention.

For instance, the test system can be used for testing vaccines and immunotherapeutics development, e.g. for immunomonitoring, T cell priming and restimulation, antigen processing and epitope characterization, evaluation of antigen immunogenicity and/or dendritic cell functional studies. The test system can also be used for the development of pharmaceuticals, foodstuffs or cosmetics, in particular for investigating of immunostimulatory or immunosuppressive effects (e.g. on maturation) of foodstuffs and ingredients (e.g. bacteria, food additives, trace contaminants), of cosmetics and their ingredients (e.g. allergy-inducing substances) and of novel pharmaceuticals and biologicals. The advantages of the test system of the invention are evident to the person skilled in the art: It is easy and fast to use. It further contains standardized quality controlled cells.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein the terms "about" and "approximately" means within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The terms "a," "an," "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term "consisting of" excludes any element, step, or ingredient not specified in the claims. The transition term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

FIGURES

Figure 1 shows a growth curve of MUTZ-3 cells grown for 1 10 days in conditioned medium further comprising animal serum.

Figure 2 shows a growth curve of MUTZ-3 cells grown for > 100 days in serum-free medium containing GM-CSF.

EXAMPLES

The invention will now be illustrated by examples which shall however, not be construed as limiting the scope of the invention.

Generation of serum-free MUTZ-3 cells of the present invention

MUTZ-3 cells were cultured under standard conditions with culture medium that contains serum (about 20% v/v) and a conditioned medium of 5637 cells (CM5637, about 10% v/v).

In order to change to a serum-free medium, a serum-free replacement for the serum itself and the conditioned medium of CM5637 (also containing serum) would have to be found.

Accordingly, MUTZ-3 cells were cultivated in a chemically defined medium, i.e. X-VIV015 (Lonza Ltd Switzerland) without addition of serum, but in presence of conditioned medium from CM5637 cells for up to 14 months. To save the cells at this stage of medium exchange, cells were cryo-preserved. In a next step, conditioned medium from CM5637 cells was replaced by 20 Units/mL Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) in slow process. The slow process involved a stepwise reduction of the amount of CM5637 conditioned medium by dilution with the new medium. Finally, the cells were cryo-preserved in serum-free medium containing 12.5% Dimethyl sulfoxide (DMSO) and 20 U/ml GM-CSF.