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  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
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Definitions

• B A comprises-E2B-Ll-L2-L3-E2A-L4;
• transgene cassette encodes IFNa shown in SEQ ID NO: 98 or a sequence at least 95% identical thereto, such as SEQ ID NO: 98.
• the adenovirus contains two BiTEs.
• virus genome contains coding sequences of DNA. Endogenous (naturally occurring genes) in the genomic sequence of the virus are not considered a transgene, within the context of the present specification unless then have been modified by recombinant techniques such that they are in a non-natural location or in a non-natural environment.
• the transgene or transgene cassette encodes a monocistronic or polycistronic mRNA, and for example the cassette is suitable for insertion into the adenovirus genome at a location under the control of an endogenous promoter or exogenous promoter or a combination thereof.
• the transgene cassette encodes monocistronic mRNA.
• the oncolytic virus is EnAd or an active derivate thereof which retains the essential beneficial properties of the virus.
• EnAd is disclosed in WO2005/118825 (incorporated herein by reference) and the full sequence for the virus is provided herein SEQ ID NO: 28.
• the chimeric E2B region is disclosed herein as SEQ ID NO: 60.
• the given element is a full-length sequence i.e. the full-length gene. In one embodiment the given element is less than a full-length and retains the same or corresponding function as the full-length sequence.
• E1A and/or E1B The skilled person can easily identify whether E1A and/or E1B are present or
• the E4 region has the sequence from 32188bp to 29380bp of SEQ ID NO: 28.
• parenteral formulation is in the form of an infusion.
• the formulation is provided as a formulation for topical administrations including inhalation.
• Suitable inhalable preparations include inhalable powders, metering aerosols containing propellant gases or inhalable solutions free from propellant gases.
• Inhalable powders according to the disclosure will generally contain a virus as described herein with a physiologically acceptable excipient.
• the therapeutic suspensions or solution formulations can also contain one or more excipients.
• Excipients are well known in the art and include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensions can be encapsulated in liposomes or biodegradable microspheres.
• the formulation will generally be provided in a substantially sterile form employing sterile manufacture processes.
• Nebulisable formulation according to the present disclosure may be provided, for example, as single dose units (e.g., sealed plastic containers or vials) packed in foil envelopes. Each vial contains a unit dose in a volume, e.g., 2 mL, of solvent/solution buffer. Treatment
• the administration of the additional cancer treatment at the same time or approximately the same time as the oncolytic adenovirus formulation.
• the treatment may be contained within the same formulation or administered as a separate formulation.
• Figure 1 Shows schematics of the NG-615, NG-640 and NG-641 transgene cassettes
• Figure 34 shows a graph indicating the CD25 expression levels on CD3+ T cells in ascites samples obtained from a cancer patient and infected with viruses of the present disclosure.
• Figure 35 shows a graph indicating the number of FAP+ cells in ascites samples obtained from a cancer patient and infected with viruses of the present disclosure.
• SEQ ID NO: 60 DNA sequence corresponding to E2B region of the EnAd genome (bp
• SEQ ID NO: 96 FLT3L amino acid sequence
• IFNa ELISA was carried out using the Verikine Human IFN alpha Kit (Pbl assay science), MlPla ELISA was carried out using the Human CCL3 Quantikine ELISA kit (R & D systems), Flt3L ELISA was carried out using the Flt3L human ELISA kit (Abeam), CXCL9 ELISA was carried out using the CXCL9 human ELISA kit (Abeam) and CXCLIO ELISA was carried out using the CXCLIO human ELISA kit (Abeam). All assays were carried out according to the manufacturers' protocol.
• CCR5 reporter cells were seeded (5xl0 3 cells/well) and incubated for 20- 24 hours. 5 ⁇ _, of supernatant from the treated tumour cells was then added to each well and incubated for 90 minutes. Luciferase reporter activity was then detected using a detection solution and quantification on a luminescence plate reader. Responses demonstrating the presence of functional ⁇ were detected in supernatants from NG-615 treated carcinoma cells and supernatants from cells treated with a positive control virus known to express MlPla, NG-347 ( Figure 6B).
• FAP BiTE-mediated target cell lysis is specific to FAP-expressing cells
• NG-601, NG-602, NG-603, NG-604, NG-605 and NG-606 viruses were characterised using the methods described below.
• NG-603, NG-604, NG-605, NG-606 and EnAd were assessed in co-culture with SKOV tumour cells and CD3 + T-cells using xCELLigence.
• NHDF cells and SKOV cells were seeded in a 48-well E-plate at 4xl0 3 and lxlO 3 cells/well, respectively. Plates were incubated for 18 hrs, 37°C, 5% CO2, before cells were either infected with 100 ppc of EnAd, of NG-603, NG-604, NG-605 or NG-606 or were left uninfected. After 2 hour incubation, 37,500 CD3 + T-cells were added to each well.
• EnAd-SA-EpCAMBiTE and EnAd-SA-ControlBiTE were assessed using xCELLigence technology.
• SKOV cells were plated in 48-well E-plate at le4 cells/well respectively. Plates were incubated for 18 hrs, 37°C, 5% C02, before cells were either infected with 100 virus particles per cell (ppc) or were left uninfected. After two hours, PBMC T-cells (5:1) in normal serum or patient exudate fluid (final, 50%) were added.
• xCELLigence was used to measure target cell cytotoxicity every 10 minutes.
• Digested DNA was purified by phenol/chloroform extraction and precipitated for 16hrs, -20°C in 300 ⁇ 1 >95% molecular biology grade ethanol and ⁇ 3M Sodium Acetate.
• the precipitated DNA was pelleted by centrifuging at 14000rpm, 5 mins and was washed in 500 ⁇ 1 70% ethanol, before centrifuging again, 14000rpm, 5mins.
• the clean DNA pellet was air dried, resuspended in 500 ⁇ 1 OptiMEM containing 15 ⁇ lipofectamine transfection reagent and incubated for 30 mins, RT. The transfection mixture was then added drop wise to a T-25 flask containing 293 cells grown to 70% confluency.

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