Patent Description:
H-1PV belongs to the genus Protoparvovirus within the Parvovirinae subfamily of Parvoviridae (Cotmore et al. It consists of a non-enveloped icosahedral capsid <NUM> in diameter and contains a single-stranded DNA genome about <NUM> kb long, encoding non-structural proteins - notably NS1 (<NUM> kDa) and NS2 (<NUM> kDa) - and the capsid proteins VP1 (<NUM> kDa) and VP2 (<NUM> kDa). Another capsid protein, VP3 (<NUM> kDa), is generated by post-translational cleavage of VP2 (Faisst et al. , <NUM>; Halder et al. , <NUM>; Hanson and Rhode, <NUM>; Toolan et al. Protoparvoviruses replicate in a S-phase-dependent fashion and undergo a lytic cycle after infection of permissive cells (Burnett et al.

While the natural host of H-1PV is the rat, this virus has recently raised much interest because it replicates preferentially in transformed cells, including a number of human tumor cells. The virus thus has oncolytic and oncosuppressive properties that have been demonstrated in various cell cultures and animal models (Nuesch et al. , <NUM>; Rommelaere et al. In xenograft models, H-1PV has been shown to suppress a number of human tumors, including cervical tumors (Faisst et al. , <NUM>; Li et al. , <NUM>), pancreatic tumors (Angelova et al. , 2009b; Grekova et al. , <NUM>), mammary carcinomas (Dupressoir et al. , <NUM>), gliomas (Geletneky et al. , <NUM>; Kiprianova et al. , <NUM>), and lymphomas (Angelova et al. In addition, H-1PV has been shown to be successful in eliminating cancer stem cells (<CIT>). On the basis of these preclinical proofs of concept, a first clinical trial (phase I/Ila) of H-1PV was launched in <NUM>, for patients with recurrent glioblastoma multiforme (Geletneky et al. Moreover, Allaume et al. , <NUM> already refers to the retargeting of H-1PV to cancer cells through genetic engineering of the viral capsid.

To test and eventually exploit the therapeutic potential of H-1PV, it is necessary to optimize the production of purified virus (vector) stocks in medium suitable for preclinical or clinical applications or basic research.

However, cell culture media formulations have been well documented in the literature and a number of media are commercially available. H-1PV production is routinely carried out in various cell cultures, such as human NB-<NUM> cells, using standard cell culture media.

The requirements of human cell culture in vitro comprise, in addition to basic nutritional substances, a complex series of growth factors (Werner et al. The supplementation of standard cell culture media with animal serum is essential for cell growth, metabolism, and to stimulate proliferation. The sera used most widely are bovine sera of adult or newborn animals, or of fetal origin in a range of <NUM>-<NUM>% v/v. Fetal bovine serum (FBS) is a cocktail of most of the factors required for cell attachment, growth and proliferation and is thus used as an almost universal growth supplement effective for most types of human and animal cells. However, the use of animal serum in cell culture also bears a number of disadvantages. These disadvantages can be seen either from a cell biological point of view, since serum in general is an ill-defined mixture of components in culture media. Also, for the biotechnological production of human therapeutics, animal-derived components cannot be applied in culture protocols. For example, there is a risk that the culture medium or products purified from it may be immunogenic, particularly if the supplements are derived from an animal different from the source of the cells to be cultured and induce an immunological reaction.

A number of serum-free media formulations are commercially available, such as those designed to support the culture of endothelial cells. Serum-free media are lower in protein content than medium supplemented with serum, which can simplify the purification process, increase the yield of the end-product, introduce improved control of cell culture conditions, increased lot-to-lot consistency, and optimized formulations for specific cell types. Further, characteristics and compositions of the cell culture media vary depending on the particular cellular requirements. Important parameters include osmolarity, pH, and nutrient formulations.

To overcome the limitations of the use of animal proteins in serum-free media several attempts have been made to construct animal cell culture media that are completely free of animal proteins. For example, some culture media have incorporated extracts of yeast cells into the basal medium (U. Patent Application No. <CIT>; <NPL>)) to provide sources of nitrogen and other essential nutrients. Moreover, the document <CIT> teaches a method of growing MDCK cell lines in serum-free medium, wherein these cell lines needed to be adapted to the serum-free media. In contrast, the prior art document <CIT> refers to the cultivation of infected NB-<NUM> cells in Minimum Essential Medium (MEM) supplemented with <NUM>% FBS.

However, none of these approaches provided a culture medium that is optimal for the cultivation of non-commercial NB-<NUM> cells for the large-scale production of H1-PV for preclinical or clinical applications. Since NB-<NUM> cells are generally difficult to grow, it is not possible to just adapt media known from the cultivation of other commercial organisms.

Thus, there remains a need for an essentially serum-free culture medium which facilitates the growth of NB-<NUM> cells to increase the productivity of H-1PV. Such culture media should allow easier and more cost-effective production and purification of the parvovirus produced by cultured NB-<NUM> cells and will provide more consistent results.

Thus, the technical problem underlying the present invention is to optimize the parvovirus large scale production in an essentially serum-free medium.

The solution of said technical problem is achieved by providing the embodiments characterized in the claims.

The present invention concerns a method for growing the producer cell line NB-<NUM> infected with parvovirus H-<NUM> as defined in independent claim <NUM>.

Oncolytic protoparvovirus research has reached the stage of translation into clinical practice, with a first phase I/lia study of H-1PV in patients with recurrent resectable malignant glioma (Geletneky et al. This trial is expected to be followed by further clinical studies aiming to assess efficacy and to extend the approach to other cancers such as pancreatic carcinoma (clinical trial: "ParvOryx02"; https://clinicaltrials. gov/ct2/show/NCT02653313) or neuroblastoma (Lacroix et al. , <NUM>; Li et al. These developments rely on the availability of robust procedures for protoparvovirus production in a large scale.

During the experiments resulting in the present invention major innovations for optimization of the H1-PV production, with elimination of unwanted contaminants in cell culture media through FBS were introduced. The inventors focused on developing an essentially serum-free medium as a basis for the production of H-1PV for preclinical and clinical applications in the anticancer virotherapy.

The process of the present invention uses a serum-free cell culture medium comprising the ingredients glucose, glutamine, glutamate, lactate, ammonium, proteins , wherein the medium is capable of supporting the cultivation of epithelial cells in vitro. In more detail, the present invention relates to a method for growing the producer cell line NB-<NUM> infected with parvovirus H-<NUM>, comprising culturing the infected NB-<NUM> cells in a serum-free medium comprising <NUM>-<NUM> glucose, <NUM>-<NUM> glutamine, <NUM>-<NUM> glutamate, <NUM>-<NUM> lactate, less than <NUM> ammonium and <NUM>-<NUM>µg/µl proteins comprising epithelial growth factor and Hypep <NUM>® (Kerry Ingredients & Favors, Norwich, NY, USA), wherein the medium has a pH of <NUM> - <NUM>.

The term "cell culture" means the maintenance of cells in an artificial, in-vitro environment. The media of the present invention can be used to culture adherent mammalian cells i.e., cells which adhere to the culture vessel. According to the present invention such cells are NB-<NUM> cells.

The term "cultivation" means the maintenance of cells in-vitro under conditions favoring growth, differentiation or continued viability, in an active or quiescent state, of the cells.

The phrase "cell culture medium" refers to a nutritive solution for cultivating cells.

The term "EGF" refers to an epithelial growth factor that stimulates proliferation and differentiation by binding to its receptor EGFR. Further, EGF regulates migration and cell growth of intestinal epithelial cells. The EGF is for example human EGF (hEGF).

The term "ingredient" refers to any compound, whether of chemical or biological origin, that can be used in cell culture media to maintain or promote the growth of proliferation of cells. The terms "component," "nutrient" and ingredient" can be used interchangeably and are all meant to refer to such compounds. Typical ingredients that are used in cell culture media include glucose, glutamine, glutamate, lactate, ammonium, growth factors, insulin and other proteins.

A "serum-free" medium is one which contains no serum. A serum-free medium is distinguished from low-serum and essentially serum-free media, both of which contain serum.

The invention relates to a process for producing parvovirus H-<NUM> by growing the H-<NUM> infected producer cell line NB-<NUM> ("the master cell bank" or "master cell line") in a serum-free culture medium, wherein the medium comprises the ingredients as defined in claim <NUM>. As shown in the Example part, the present invention produces a four-fold higher parvovirus titer than usually achieved with a standard FBS-containing medium.

The process of the present invention uses a serum-free cell culture medium comprising the ingredients glucose, glutamine, glutamate, lactate, ammonium, proteins comprising epithelial growth factor, Hypep <NUM>®, and, preferably insulin, wherein the medium is capable of supporting the cultivation of epithelial cells in vitro, preferably NB-<NUM> cells, for the production of H-1PV.

Thus, the growth factor used in the media of the invention is epithelial growth factor (EGF), preferably human EGF (hEGF), while the preferred insulin is human recombinant. The protein ingredients of the present media have no human or animal origin, and the protein concentration is ultra-low, preferably less than <NUM>µg/µl.

The medium of the present invention can be used to grow human epithelial cells i.e., NB-<NUM> cells, to high density and/or to increase parvovirus production. In summary, the process of the present invention uses an essentially serum-free cell culture medium containing about <NUM>-<NUM> glucose, <NUM>-<NUM> glutamine, <NUM>-<NUM> glutamate, <NUM>-<NUM> lactate, less than <NUM> ammonium and <NUM>-<NUM>µg/µl proteins comprising epithelial growth factor and Hypep <NUM>®, wherein the medium is capable of supporting the cultivation of NB-<NUM> cells for the production of H-1PV.

Preferably, the culture medium of the present invention contains <NUM>-<NUM> glucose, about <NUM> glutamine, about <NUM>-<NUM> glutamate, about <NUM> lactate, less than <NUM> ammonium and <NUM>-<NUM>µg/µl proteins. (i.e., the supplements epithelial growth factor, Hypep <NUM>®, and, preferably, insulin).

The culture medium "VP-SFM" that is referred to in the Examples of the present application contains <NUM> glucose, <NUM> glutamine, <NUM> glutamate, <NUM> lactate, less than <NUM> ammonium and about <NUM>µg/µl proteins. These proteins include EGF, insulin and proteineous supplements.

The culture medium "Opti-Pro SFM" that is referred to in the Examples of the present application contains <NUM> glucose, <NUM> glutamine, <NUM> glutamate, <NUM> lactate, <NUM> ammonium, <NUM> ng/µl rEGF and about <NUM>µg/µl proteins. These proteins include EGF and proteineous supplements.

Proteineous supplements which are added to the medium of the present invention are soy peptides i.e., Hypep <NUM>® (Kerry Ingredients & Favors, Norwich, NY, USA). Supplements which may be additionally added to the present media include insulin, Sheff-Vax Plus ACF, Insulin-Transferrin-Selenium A, Insulin-Transferrin-Selenium X and CD Lipid Concentrate. Insulin is available commercially, for example from Life Technologies, Inc. (Rockville, Md.

Thus, the medium of the present invention is combined with soy proteins, which are represented by Hypep <NUM>® (Kerry Ingredients & Favors, Norwich, NY, USA). An additional supplement, which may be suitable, is Sheff-Vax Plus ACF® (Kerry, Inc. , Kildire, Ireland), which is a vegetable component-based hydrolysate that contains growth factors and trace elements.

The pH of the medium of the present invention is adjusted to <NUM>-<NUM>, preferably about <NUM>.

The medium of the present invention may be used to facilitate cultivation of epithelial cells. In particular, these media may be used to cultivate the master cell line based on NB-<NUM> cells for which the inventors found out that it is an epithelial cell line.

While the present medium is particularly useful for culturing epithelial cells, it is understood that the medium may be used in any standard cell culture protocol whether the cells are grown in a method by which epithelial cells may be cultivated in vitro.

Furthermore, since the present medium is serum-free and has a low-protein concentration, the medium may be used for rapid production of a parvovirus e.g., H-1PV. In the present application it has been demonstrated that the production of H1-PV in VP-SFM supplemented with glutamine and soy peptides showed the same yield compared to a FBS-containing medium and are a suitable alternative for the serum-free production. Another suitable medium is Opti-Pro SFM supplemented with glutamine.

The following examples are intended to illustrate, but not to limit, the invention.

The MEM medium was supplemented with <NUM> L-Glutamin. VP-SFM and OptiPro-SFM were each supplemented with <NUM> L-Glutamin.

NB-<NUM> human newborn kidney cells (master cell bank) transformed with simian virus <NUM> (SV40) (Tattersall and Bratton, <NUM>) were cultured at <NUM> in different medium (see above).

After transferring MCB cells to each medium the cells have been adapted to the cells at least for <NUM> weeks. For production, NB-<NUM> cells were seeded at <NUM>. 6x10<NUM> cells/cm<NUM> and infected immediately with H-<NUM> PV at a multiplicity of infection (MOI) of <NUM> plaque forming units (PFU) per cell. The infected cells were incubated for <NUM> days at <NUM> under <NUM>% CO<NUM> until the cytopathic effect (CPE), measured as the percentage of detached cells observed under a microscope, reached at least <NUM>%. Cell density and viability were measured by staining living cells with <NUM>% trypan blue (Invitrogen™, Germany). Cells were counted with a Countess® Cell counter (Life Technologies, Germany) and morphology is microscopical observed.

For harvesting, the medium was aspirated ,and infected cells were treated with PBS/<NUM> EDTA. The medium supernatant and detached cells were centrifuged for <NUM> at <NUM>,000xg. The pellet was washed with PBS, resuspended in Virus Tris/EDTA buffer, pH <NUM> (VTE) containing <NUM> Tris HCl, <NUM> EDTA, and subjected to three freeze/thaw cycles. After centrifugation for <NUM> at <NUM>,000xg, cell debris were discarded. The cell lysate was then sonicated at <NUM> W for <NUM> in a Sonorex Super <NUM> P ultrasonic homogenizer (Bandelin, Germany) and treated with DNAse (<NUM> U/ml, Sigma, Germany) for <NUM> at <NUM>.

Plaque assays were done essentially as described by Tattersall and Bratton, <NUM>. NB-<NUM> cells were grown in monolayer cultures in MEM medium containing <NUM>% FBS, <NUM>µg/ml penicillin, <NUM>µg/ml streptomycin, and <NUM> L-glutamine. They were infected at <NUM>% confluence with serial dilutions of H-1PV and incubated for <NUM> at <NUM>. Then the inoculum was replaced with a bacto-agar overlay (<NUM>% in MEM containing <NUM>% FBS). On day four post-infection, living cells were stained for <NUM>-<NUM> by addition of <NUM>% toluylene red staining solution (Sigma, Germany) containing bacto-agar (Becton Dickinson, Germany). The dishes were incubated at <NUM> under <NUM>% CO<NUM>. Plaque-forming units were counted <NUM> days post-infection on a light box and their concentration expressed in PFU/ml.

The immune fluorescence test is based on the use of fluorescence-marked antibodies which bind to her specific antigen by which certain extra and intracellular structures can be proved. This principle is used for keratin staining with a CH/HK-Keratin antibody to for of epithelial cells evidence. Therefore the cells were cultivated on slides in <NUM> <NUM> cell culture flasks (Greiner Bio-One). NB-<NUM> human newborn kidney cells (master cell bank) were seeded with a density of 3E6 cells/flask. For the Keratin staining HEF (human embryonic fibroblasts) and HEK293T (human epithelial kidney cells) with a cell density of <NUM> E6 cells/flask or 5E6 cells were seeded as controls. After <NUM>-hour incubation time the cells on the slides were washed in PBS, fixed for <NUM> minutes in methanol and afterwards <NUM> minutes in acetone. Both liquids were precooled before in -<NUM>. For the Keratin detection the slides were pre-blocked with PBS with <NUM>. <NUM>% BSA and afterwards incubated with the CH/HK-Keratin antibody for <NUM> hour. Afterwards slides were washed in PBS with <NUM>. <NUM>% Triton, before the second antibody Cy3gp (Dianova) was incubated for <NUM> hour. The unbound second antibodies were removed by triple washing in PBS with <NUM>. <NUM>% Triton. The slides were dipped in Ethanol and were dried. The slides were embedded with slide mounting medium (CELL LAB) including 1µg/ml DAPI, that stains the nucleus of the cells. The analysis occurred with the fluorescence microscope BZ-<NUM> (Keyence).

With the light microscope (Axiovert <NUM>) the cell morphology, density and shape are observed.

The inventors first determined the productivity of H-1PV in different media including MEM-Hepes, VP-SFM supplemented with glutamine (VP-SFM*) and Opti-Pro SFM supplemented with glutamine (Opti-Pro SFM*) media, each without FBS (fetal bovine serum). The cells were harvested as described above and the infectious particles have been analyzed via PFU Assay.

As shown in <FIG>, H-1PV-infected NB-<NUM> cells cultured in the VP-SFM* and Opti-Pro SFM* media with <NUM>% FBS produced higher titers of H-1PV compared to standard medium MEM-Hepes with <NUM>% FBS.

These results demonstrate that the VP-SFM* and Opti-Pro SFM* media without FBS facilitate the rapid production of H-1PV compared to standard medium MEM-Hepes without FBS (see <FIG>). VP-SFM* with <NUM>% FBS (<NUM> ± <NUM>) and Opti-Pro SFM* (<NUM> ± <NUM>) with <NUM>% FCS showed the same or even an increase in productivity compared to MEM-Hepes <NUM>% FBS (comparison of <FIG> with <FIG>). Further, standard medium MEM-Hepes with <NUM>% FBS showed a substantial decrease in productivity or almost no productivity (<FIG>).

VP-SFM" and Opti-Pro SFM* differ by the presence of EGF from the standard medium MEM-Hepes. This shows that the growth factor EGF could stimulate cell proliferation (Fallon et al. , <NUM>) and this may facilitate the H1-PV productivity.

To examine the H-1PV productivity in MEM-Hepes, VP-SFM* and Opti-Pro SFM* media with each <NUM>% FBS, NB-<NUM> cells were harvested as described above and the infectious particles have been analyzed via PFU Assay.

As shown in <FIG>, VP-SFM* (+<NUM>% FBS) was about <NUM>. 0x ±<NUM> better in H-1PV productivity than the comparison-media MEM-Hepes (+<NUM>% FBS). Opti-Pro SFM* (+<NUM>% FBS) medium shows a lower PFU value than the comparison-medium MEM-Hepes (+<NUM>% FBS) (about <NUM>% of MEM-Hepes).

These results demonstrate that Opti-Pro SFM* has a better productivity without the addition of FBS in comparison with added <NUM>% FBS (<FIG> and <FIG>). In contrast, standard medium MEM-Hepes showed a much better productivity with <NUM>% FBS in comparison without FBS (<FIG> and <FIG>).

Hypep <NUM>® (Kerry Ingredients & Favors, Norwich, NY, USA) and Sheff-Vax Plus ACF® were used as additives in VP-SFM to replace FBS. The cells were cultured with <NUM> % FBS in VP-SFM and in serum-free VP- SFM supplemented with Hypep <NUM>® or Sheff -Vax Plus ACF ®. The cells were harvested as described above and the infectious particles have been analyzed via PFU Assay.

As shown in <FIG>, VP-SFM supplemented with Hypep <NUM>® (<NUM>*<NUM><NUM> PFU/cell) showed an increase in productivity in comparison to VP-SFM with <NUM>% FBS (<NUM>*<NUM><NUM> PFU/cell).

These results demonstrate that the production of H1-PV in VP-SFM supplemented with Hypep <NUM>® showed the same yield compared to VP-SFM supplemented with <NUM>% FBS. Thus, the VP-SFM medium of the present invention, when supplemented with Hypep <NUM>®, can be used to culture H-1PV-infected NB-<NUM> cells in serum-free medium with comparable results as obtained with medium supplemented with <NUM>% FBS.

An ideal pH for the H-<NUM> PV production should be determined. Therefore the pH of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> was adjusted in the MEM media either with HCl or NaOH. To ensure the stable pH during H-1PV virus infection cell culture bottles with closed lids were used to avoid gas exchange for <NUM> hours. Because CO<NUM>-gasing is necessary for the cultivation of the cells, after <NUM> hours incubation lids with filter were used for <NUM> days incubation. The cells were harvest as described above and the infectious particles have been analyzed via PFU Assay.

As shown in <FIG>, the pH should be adjusted in the range of <NUM> to <NUM>, preferably to about <NUM>, to achieve an optional yield of infectious particles.

As shown in <FIG> via light microscopical pictures, the morphology demonstrates the same polygonal shape with regular dimensions and size in all three mediums.

Claim 1:
A method for growing the producer cell line NB-<NUM> infected with parvovirus H-<NUM>, comprising
culturing the infected NB-<NUM> cells in a serum-free medium comprising <NUM>-<NUM> glucose, <NUM>-<NUM> glutamine, <NUM>-<NUM> glutamate, <NUM>-<NUM> lactate, less than <NUM> ammonium and <NUM>-<NUM>µg/µl proteins comprising epithelial growth factor and Hypep <NUM>®, wherein the medium has a pH of <NUM> - <NUM>.