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JP-7856736-B2 - Optimized parvovirus H1 production

JP7856736B2JP 7856736 B2JP7856736 B2JP 7856736B2JP-7856736-B2

Inventors

  • ロイクス，バーバラ
  • フレートマン，ヴェロニカ
  • ミュラー，マルクス
  • ロメレーレ，ジャン
  • ダーム，ミヒャエル
  • クレブス，オッタインツ

Assignees

  • ドイチェス クレブスフォルシュンクスツェントルム スチフトゥング デス エッフェントリヒェン レヒツ

Dates

Publication Date
20260511
Application Date
20241212
Priority Date
20190617

Claims (4)

  1. A method for producing a master seed virus (MSV) composition, wherein the method is: (a) A process of providing a single-clone master cell bank (MCB) consisting of cells deposited in DSMZ (=German Collection of Microorganisms and Cell Cultures) under accession number DSM ACC3353 in accordance with the Budapest Convention. (b) A step of transfecting the MCB cells from step (a) with an H-1PV-producing plasmid clone , and (c) A method comprising the step of infecting the MCB cells of step (b) with an H-1PV-producing plasmid clone for at least two rounds to produce MSV.
  2. The method according to claim 1, wherein the transfection in step (b) of claim 1 is performed by calcium phosphate transfection.
  3. A method for producing parvovirus H-1 (H-1PV), wherein the method is: (a) The process of providing a single-clonal master cell bank (MCB) consisting of cells deposited in the DSMZ (=German Collection of Microorganisms and Cell Cultures) under accession number DSM ACC3353 in accordance with the Budapest Convention; (b) A step of pre-seeding the MCB cells of step (a) with a master seed virus (MSV) obtained by the method described in either claim 1 or 2. (c) A step of infecting cells with a cell density of 2.0–5.0 x 10⁴ cells/ cm² with MSV at an MOI of 0.5–5 x 10⁻² PFU/cell; (d) A step of growing the cells for 2 to 6 days, collecting the cells 2 to 6 days after infection, and obtaining a cell pellet by centrifugation; (e) The step of subjecting the resuspended cell pellet to a mechanical, physical or chemical cell lysis method in order to obtain a parvovirus-containing cell lysate; (f) A process of clarifying the parvovirus sample by filtration; and (g) The process of subjecting it to DNAse treatment; (h) Buffer exchange process for chromatography preparation; (i) A chromatography step to remove empty particles and most impurities; (j) Buffer exchange and concentration steps by desalting column or tangential flow filtration; and (k) A method comprising a final formulation step in iodixanol/Ringer or other formulation solution.
  4. The method according to claim 3, wherein the chromatography in steps (h) and (i) of claim 3 is anion exchange chromatography.

Description

DSMZ DSM ACC3353 Field of Invention The present invention provides a robust single-clonal master cell bank (MCB) for the optimized production of H-1 parvovirus (H-1 PV), which is suitable for increasing the production of infectious parvovirus compared to the standard producer, NB-324K mixed cells. Background of the Invention H-1PV belongs to the genus Protoparvovirus within the subfamily Parvovirinae of the family Parvoviridae (Cotmore et al., 2014). It consists of a 25 nm diameter non-enveloped icosahedral capsid and contains a single-stranded DNA genome approximately 5 kb long that encodes non-structural proteins—particularly NS1 (83 kDa) and NS2 (25 kDa)—as well as capsid proteins VP1 (81 kDa) and VP2 (65 kDa). Another capsid protein, VP3 (63 kDa), is produced 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 phase-dependent manner and undergo post-infection lysis cycles in tolerant cells (Burnett et al., 2006). While the natural host of H-1PV is rat, this virus has recently attracted considerable interest because it preferentially replicates in transformed cells, including some human tumor cells. The virus possesses tumor-disintegrating and tumor-suppressing properties, which 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 several human tumors, such as cervical tumors (Faisst et al., 1998; Li et al., 2013), pancreatic tumors (Angelova et al., 2009b; Grekova et al., 2011), breast cancer (Dupressoir et al., 1989), gliomas (Geletneky et al., 2010; Kiprianova et al., 2011), and lymphomas (Angelova et al., 2009a). Based on this preclinical evidence of the concept, the first clinical trial of H-1PV (Phase I/IIa) was initiated in 2011 in patients with relapsed glioblastoma multiforme (Geletneky et al., 2012). To test and ultimately utilize the therapeutic potential of H-1PV, it is necessary to develop efficient, simple, robust, and reproducible processes for virus production and purification. Purification methods have been published for small-scale production using cesium chloride (Halder et al., 2012; Paradiso, 1981) or iodixanol (Wrzesinski et al., 2003; Zolotukhin et al., 1999) density gradient centrifugation. Research into tumor-disintegrating protoparvoviruses has progressed to clinical implementation, including the first Phase I/IIa trial of H-1PV in patients with recurrent, resectable gliomas (Geletneky et al., 2012). Furthermore, a Phase I/II clinical trial has been initiated in patients with unresectable metastatic pancreatic cancer (ClinicalTrials.gov identifier: NCT02653313; manuscript in preparation). These developments depend on the availability of robust procedures for protoparvovirus production and characterization. Standardized procedures are necessary to generate preclinical data that can provide evidence of concept. Standard operating procedures are also necessary to transfer the technology and standards to accredited facilities responsible for producing clinical batches and establishing their specifications. However, the use of well-characterized virus preparation and analysis methods is truly essential, and required by regulatory authorities, to obtain valid and reproducible evidence of the therapeutic efficacy of tumor-disintegrating protoparvoviruses in oncology. H-1PV production is routinely performed in cell cultures of human neonatal kidney cells, such as NB-324K mixed cells (Patent Document 1). WO 2016/206807 A1 Brief explanation of the drawing Figure 1: Generation of master cell bank and master seed virus for H-1PV production. Cells of NB-324K mixed clones underwent two rounds of single colony selection in MEM, 5% FBS, 2% L-glutamine, and 0.2% gentamicin. The selected clone D8-G3 was first used to establish a research cell seed (RCS) and then to establish a master cell bank (MCB; passage 15 or longer) for H1-PV production. For master seed virus (MSV) generation, single-clonal MCB cells were transfected with sequenced pUC19ΔHindII/H1 plasmid DNA. After two rounds of MCB cell infection with H-1PV, the final passage was defined as MSV. For drug production, MCB working cell bank cells were infected with MSV (or the corresponding working seed virus (WSV)). Figure 2: Two rounds of single-cell clone selection for H-1PV GMP production. Selection in the first round (A). Clones D8 (4.07E+03 ± 2.88E+03 PFU/cell), E6 (4.86E3 ± 3.27E+02 PFU/cell), and C5 (5.0E+03 ± 1.05E+03 PFU/cell) (n=2) show the same productivity as the NB-324K mixed clone (6.9E3 PFU/cell). Furthermore, the production times of the three selected clones (approximately 33–35 h) are also similar to the production time of the mixed clone NB-324K (approximately 32 h; not shown), where clone D8 has the best production time of 32.9 h. As a result, it was decided to work with clone D8 in further e