Patent Application
20190203184
The present invention provides a process for parvovirus production using an essentially serum-free medium which is suitable to increase parvovirus production compared to a standard medium.
H-1 PV belongs to the genus Protoparvovirus within the Parvovirinae subfamily of Parvoviridae (Cotmore et al., 2014). It consists of a non-enveloped icosahedral capsid 25 nm in diameter and contains a single-stranded DNA genome about 5 kb long, encoding non-structural proteins—notably NS1 (83 kDa) and NS2 (25 kDa)—and the capsid proteins VP1 (81 kDa) and VP2 (65 kDa). Another capsid protein, VP3 (63 kDa), is generated by post-translational cleavage of VP2 (Faisst et al., 1995; Halder et al., 2012; Hanson and Rhode, 1991; Toolan et al., 1960). Protoparvoviruses replicate in a S-phase-dependent fashion and undergo a lytic cycle after infection of permissive cells (Burnett et al., 2006).
While the natural host of H-1 PV 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., 2012; Rommelaere et al., 2010). In xenograft models, H-1PV has been shown to suppress a number of human tumors, including cervical tumors (Faisst et al., 1998; Li et al., 2013), pancreatic tumors (Angelova et al., 2009b; Grekova et al., 2011), mammary carcinomas (Dupressoir et al., 1989), gliomas (Geletneky et al., 2010; Kiprianova et al., 2011), and lymphomas (Angelova et al., 2009a). In addition, H-1PV has been shown to be successful in eliminating cancer stem cells (EP 2 404 609 A1). On the basis of these preclinical proofs of concept, a first clinical trial (phase I/IIa) of H-1 PV was launched in 2011, for patients with recurrent glioblastoma multiforme (Geletneky et al., 2012).
To test and eventually exploit the therapeutic potential of H-1 PV, 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-1 PV production is routinely carried out in various cell cultures, such as human NB-324K 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., 1993). 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 10-20% 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 (WO 2011/157447 A2; Allaume et al., 2012) 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 (WO 2012/033328 A2). 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.K. Patent Application No. GB 901673; Keay, L., Biotechnol. Bioengin. 17:745-764 (1975)) to provide sources of nitrogen and other essential nutrients.
However, none of these approaches provided a culture medium that is optimal for the cultivation of non-commercial NB-324K cells for the large scale production of H1-PV for preclinical or clinical applications. Since NB-324K 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-324K cells to increase the productivity of H-1 PV. Such culture media should allow easier and more cost-effective production and purification of the parvovirus produced by cultured NB-324K 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.
Oncolytic protoparvovirus research has reached the stage of translation into clinical practice, with a first phase I/IIa study of H-1 PV in patients with recurrent resectable malignant glioma (Geletneky et al., 2012). 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://clinicaltrails.gov/ct2/show/NCT02653313) or neuroblastoma (Lacroix et al., 2010; Li et al., 2013). 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-1 PV for preclinical and clinical applications in the anticancer virotherapy.
The process of the present invention uses an essentially serum-free cell culture medium comprising the ingredients glucose, glutamine, glutamate, lactate, ammonium, proteins (e.g. growth factors, insulin, etc.), wherein the medium is capable of supporting the cultivation of epithelial cells in vitro, preferably NB-324K cells, for the production of H-1 PV.
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 cell, i.e. a cell which adheres to the culture vessel, preferably epithelial cells like NB-324K 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 epidermal 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 preferably 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 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-1 by growing H-1 infected producer cell line NB-324K (“the master cell bank” or “master cell line”) in a serum-free culture medium, wherein the medium comprises the below mentioned ingredients. 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 an serum-free cell culture medium comprising the ingredients glucose, glutamine, glutamate, lactate, ammonium, proteins (e.g. growth factors, insulin, etc.), wherein the medium is capable of supporting the cultivation of epithelial cells in vitro, preferably NB-324K cells, for the production of H-1 PV.
The preferred 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 10 μg/μl.
The medium of the present invention can be used to grow human epithelial cells, preferably NB-324K cells, to high density and/or to increase parvovirus production. In one preferred embodiment, the process of the present invention uses an essentially serum-free cell culture medium containing about 16-22 mM glucose, 3-5 mM glutamine, 0.1-0.6 mM glutamate, 0.5-1.0 mM lactate, less than 0.3 mM ammonium and 3-10 μg/μl proteins, wherein the medium is capable of supporting the cultivation of NB-324K cells for the production of H-1 PV.
Preferably, the culture medium contains 17-20 mM glucose, about 4 mM glutamine, about 0.15-0.5 mM glutamate, about 0.7 mM lactate, less than 0.2 mM ammonium and 4-8 μg/μl proteins. (e.g. supplements, epithelial growth factor and insulin).
In a particular preferred embodiment the culture medium contains 19.14 mM glucose, 4.25 mM glutamine, 0.174 mM glutamate, 0.669 mM lactate, less than 0.05 mM ammonium and about 5 μg/μl proteins. These proteins include EGF, insulin and proteineous supplements. This medium is called “VP-SFM”.
In another preferred embodiment, the culture medium contains 17.49 mM glucose, 4.25 mM glutamine, 0.486 mM glutamate, 0.641 mM lactate, 0.18 mM ammonium, 7.5 ng/μl rEGF and about 7.5 μg/μl proteins. These proteins include EGF and proteineous supplements. This medium is called “Opti-Pro SFM”.
Supplements which may be added to the present media include insulin, soy peptides (e.g. Hypep 1510), Sheff-Vax Plus ACF, Insulin-Transferrin-Selenium A, Insulin-Transferrin-Seelenium X and CD Lipid Concentrate. Insulin is available commercially, for example from Life Technologies, Inc. (Rockville, Md.). Soy peptides are commercially available, for example from Quest International (Norwich, N.Y.).
In a preferred embodiment, the medium of the present invention is combined with a supplement, preferably soy proteins. One suitable soy protein is ultrafiltered enzymatic digest of soy, e.g. Hypep 1510® (Kerry Ingredients & Favors, Norwich, N.Y., USA). Another suitable supplement is Sheff-Vax Plus ACF® (Kerry, Inc., Kildire, Ireland) which is a vegetable component-based hydrolysate that contains growth factors and trace elements.
In further preferred embodiment, the pH of the medium of the present invention should be adjusted to around 7.0-7.5, preferably about 7.2.
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-324K 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. While such examples are typical of those that might be used, other methods known to those skilled in the art may alternatively be utilized.
(A) Medium for Cell Cultivation and H-1 PV Production
The MEM medium was supplemented with 2 mM L-Glutamin. VP-SFM and OptiPro-SFM were each supplemented with 4 mM L-Glutamin.
(B) Cell Cultivation and H-1PV Production
NB-324K human newborn kidney cells (master cell bank) transformed with simian virus 40 (SV40) (Tattersall and Bratton, 1983) were cultured at 37° C. in different medium (see above).
After transferring MCB cells to each medium the cells have been adapted to the cells at least for 2 weeks. For production, NB-324K cells were seeded at 3.6×104 cells/cm2 and infected immediately with H-1 PV at a multiplicity of infection (MOI) of 0.01 plaque forming units (PFU) per cell. The infected cells were incubated for 4 days at 37° C. under 5% CO2 until the cytopathic effect (CPE), measured as the percentage of detached cells observed under a microscope, reached at least 30%. Cell density and viability were measured by staining living cells with 0.4% 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/1 mM EDTA. The medium supernatant and detached cells were centrifuged for 5 min at 5,000×g. The pellet was washed with PBS, resuspended in Virus Tris/EDTA buffer, pH 8.7 (VTE) containing 0.05 M Tris HCl, 0.5 mM EDTA, and subjected to three freeze/thaw cycles. After centrifugation for 5 min at 5,000×g, cell debris were discarded. The cell lysate was then sonicated at 48 W for 1 min in a Sonorex Super 10 P ultrasonic homogenizer (Bandelin, Germany) and treated with DNAse (50 U/ml, Sigma, Germany) for 30 min at 37° C.
(C) Plaque Formation Assay (PFU)
Plaque assays were done essentially as described by Tattersall and Bratton, 1983.
NB-324K cells were grown in monolayer cultures in MEM medium containing 5% FBS, 100 μg/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. They were infected at 60% confluence with serial dilutions of H-1PV and incubated for 1 h at 37° C. Then the inoculum was replaced with a bacto-agar overlay (1.7% in MEM containing 5% FBS). On day four post-infection, living cells were stained for 18-24 h by addition of 0.02% toluylene red staining solution (Sigma, Germany) containing bacto-agar (Becton Dickinson, Germany). The dishes were incubated at 37° C. under 5% CO2. Plaque-forming units were counted 5 days post-infection on a light box and their concentration expressed in PFU/ml.
(D) Immune Fluorescence Test
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 145 cm2 cell culture flasks (Greiner Bio-One). NB-324K 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 1.2 E6 cells/flask or 5E6 cells were seeded as controls. After 24-hour incubation time the cells on the slides were washed in PBS, fixed for 10 minutes in methanol and afterwards 5 minutes in acetone. Both liquids were precooled before in −20° C. For the Keratin detection the slides were pre-blocked with PBS with 0.05% BSA and afterwards incubated with the CH/HK-Keratin antibody for 1 hour. Afterwards slides were washed in PBS with 0.1% Triton, before the second antibody Cy3gp (Dianova) was incubated for 1 hour. The unbound second antibodies were removed by triple washing in PBS with 0.1% Triton. The slides were dipped in Ethanol and were dried. The slides were embedded with slide mounting medium (CELL LAB) including 14/ml DAPI, that stains the nucleus of the cells. The analysis occurred with the fluorescence microscope BZ-9000 (Keyence).
(E) Microscopy of Cells
With the light microscope (Axiovert 25) the cell morphology, density and shape is observed.
The inventors first determined the productivity of H-1 PV 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). As mentioned in the above Table VP-SFM* and Opti-Pro SFM* contain EGF as a proteineous ingredient. The cells were harvested as described above and the infectious particles have been analyzed via PFU Assay.
As shown in
These results demonstrate that the VP-SFM* and Opti-Pro SFM* media without FBS facilitate the rapid production of H-1 PV compared to standard medium MEM-Hepes without FBS (see
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., 1984) 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 5% FBS, NB-324K cells were harvested as described above and the infectious particles have been analyzed via PFU Assay.
As shown in
These results demonstrate that Opti-Pro SFM* has a better productivity without the addition of FBS in comparison with added 5% FBS (
Hypep 1510® and Sheff-Vax Plus ACF® were used as additives in VP-SFM to replace FBS. The cells were cultured with 5% FBS in VP-SFM and in serum-free VP-SFM supplemented with Hypep 1510® 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
These results demonstrate that the production of H1-PV in VP-SFM supplemented with Hypep 1510 showed the same yield compared to VP-SFM supplemented with 5% FBS. Thus, the VP-SFM medium of the present invention, when supplemented with Hypep 1510, can be used to culture H-1 PV-infected NB-324K cells in serum-free medium with comparable results as obtained with medium supplemented with 5% FBS.
An ideal pH for the H-1 PV production should be determined. Therefore the pH of 6.5, 7.0, 7.2, 7.5, 7.8, 8.0 and 8.5 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 4 hours. Because CO2-gasing is necessary for the cultivation of the cells, after 4 hours incubation lids with filter were used for 4 days incubation. The cells were harvest as described above and the infectious particles have been analyzed via PFU Assay.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 16189921.6 | Sep 2016 | EP | regional |
This application is a continuation of PCT/EP2017/072872, filed Sep. 12, 2017; which claims the priority of EP 16189921.6, filed Sep. 21, 2016. The contents of the above applications are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2017/072872 | Sep 2017 | US |
| Child | 16294223 | US |
