Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

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This method can help address key challenges in the field of cancer immunotherapy by facilitating the development of oncolyctic vectors for targeted immunomodulation. The main advantage of the applied reverse genetic system is its versatility;therefore, vectors can be adapted to address various research questions and target tumors with diverse immune signatures. The virus propagation steps especially determining the optimal time point to harvest virus are difficult to learn from a text protocol because syncytia formation must be assessed visually over time.

To begin design and clone recombinant immunomodulatory vectors as described in the manuscript. This protocol describes specific steps for the development of an oncolytic measles vaccine vector, encoding a bispecific T cell engager. To rescue measles virus from cDNA, 24 hours before transfection plate measles virus producer cells evenly on a 6 wall plate.

Seed 200, 000 cells in 2 milliliters DMEM containing 10 percent FBS per well to achieve 65 to 75 percent confluency at the time of transfection. Mix 5 micrograms of recombinant DNA encoding the measles virus antigenome that will be used to transfect the cells. Appropriate plasmids and a fluorescent reporter in a total volume of 200 microliters of DMEM.

Add 18.6 microliters of liposomal transfection reagent to the mixture and immediately flick the tube to mix. Incubate this transfection mix for 25 minutes at room temperature. To transfect the MV producer cells remove the medium from the 6 well plate grown to 65 to 75 percent confluency and add 1.8 milliliters of DMEM with 2 percent FBS and 50 micrograms per milliliter Kanamycin per well.

Then add the transfection mix dropwise to each well and swirl carefully. Incubate the cells overnight at 37 degrees Celsius and 5 percent carbon dioxide. On the following day replace the medium with 2 milliliters of fresh DMEM with 2 percent FBS and 50 micrograms per milliliter Kanamycin and repeat this when medium becomes acidic recognized by the yellow color.

Use a microscope to observe cells daily for reporter gene expression and syncytia formation. When large syncytia consisting of 20 or more cells are visible or when cells become too dense harvest the virus. 24 hours before the anticipated harvest seed approximately 1.5 million MV producer cells in 12 milliliters of DMEM with 10 percent FBS on 10 centimeter dishes to achieve 65 to 75 percent confluency at the time of virus inoculation.

To collect the virus progyny use a cell scraper to carefully scrape adherent producer cells from the 6 wall plate with transfected cells. Transfer the medium containing the scraped cells into a centrifuge tube. Centrifuge for at least 2500 times G and 4 degrees Celsius for 5 minutes to remove cell debris.

After centrifugation mix the cell-free supernatant with serum free medium to a final volume of 4 milliliters to prepare inoculum. Remove the medium from the 10 centimeter dish with MV producer cells and add the inoculum to the cells. Incubate for at least 2 hours at 37 degrees Celsius and 5 percent carbon dioxide.

After incubation add 6 milliliters of DMEM with 10 percent FBS and incubate cells overnight. Then replace the medium with 12 milliliters of fresh DMEM with 10 percent FBS and incubate at 37 degrees Celsius and 5 percent carbon dioxide until harvesting. Observe the cells at least twice daily.

Before syncytia burst when membrane disruption become visible harvest the virus. To harvest the first passage remove supernatant from the plate and add 600 microliters of serum free medium. Then use a cell lifter to scrape the cells and transfer to a clean tube.

Immediately after that freeze the virus suspension in liquid nitrogen and store at 80 degrees Celsius for at least 24 hours to ensure thorough freezing. To determine titers of virus stocks in octuplicates per aliquot using 96 wall plates. First pull the producer cells from T75 cell culture flasks into 50 milliliter conical tube.

Count the cells and adjust the cell suspension to 150, 000 cells per milliliter in DMEM with 10 percent FBS. Add 90 microliters of DMEM with 10 percent FBS per well of a 96 wall plate. Then add 10 microliters from one aliquot of the virus stock to all 8 wells in the first column of the plate.

Mix thoroughly by pipetting up and down at least 10 times. Use a multichannel pipette to transfer 10 microliters of each well, from the first to the second column, and mix thoroughly by pipetting up and down. Repeat this for each column to obtain serial tenfold dilutions of the virus using fresh pipette tips for each dilution step.

Discard 10 microliters from each well of the last column. Add 100 microliters of cell suspension with 150, 000 cells per milliliter to each well making sure there are no cell clumps. Incubate at 37 degrees Celsius, 5 percent carbon dioxide for 48 hours.

Check for syncytia using a light microscope. Count syncytia in each well of the column with the highest dilution factor containing visible syncytia. For viruses encoding a fluorescent reporter, syncytia can be counted using fluorescence microscopy.

For analysis of measles virus replication kinetics one day prior to infection seed 100, 000 vero cells in 1 milliliter of DMEM with 10 percent FBS per well on 12 wall plates with at least 2 wells for each time point of interest. To infect the cells replace the medium with 300 microliters of serum free medium containing the respective amount of the virus. Incubate at 37 degrees Celsius and 5 percent carbon dioxide for at least 2 hours.

Remove inoculum and add 1 milliliter of DMEM with 10 percent FBS and continue incubation. At relevant time points after infection harvest viral progyny by directly scraping cells into the medium. Transfer contents from each well to an individual tube.

Snap freeze in liquid nitrogen and store at 80 degrees Celsius until titration and then proceed as described in the manuscript. To begin evaluation of BTE induced cell mediated cytotoxicity by LDH release, first isolate secreted transgene products expressed by virus infected target cells and isolate immune effector cells as described in the manuscript. Then seed target cells in a U-bottom 96 wall plate and include samples as described in the manuscript.

Add previously isolated immunomodulators at desired concentrations to the respective samples. Add immune defector cells at desired ratios and add medium to a total volume of 100 microliters per well. Incubate for the desired previously determined time frames at 37 degrees Celsius and 5 percent carbon dioxide.

45 minutes before sample collection add 10 microliters of 10X Lysis Solution to wells containing T max samples and the corresponding medium controls and continue incubation. Centrifuge the cells at 250 times G for 4 minutes. Transfer 50 microliters of supernatant from each well to wells of a flat bottom 96 wall plate making sure not to transfer the cells.

After preparing substrate solution according the manufacturer add 50 microliters to each well. Incubate at room temperature in the dark for 30 minutes or until T max samples turn deep red. After incubation add 50 microliters of stop solution to each well.

Centrifuge for 1 minute at 4000 times G and then use a hollowed needle to remove air bubbles. Measure optical absorbance at 490 nanometers and calculate as described in the manuscript. After inoculation with unmodified and BTE encoding oncolytic measles viruses growth curves of the compared vectors on vero cells appear similar.

Cell viability curves on mc38 murine colorectal carcinoma cells stably expressing human carcinoembryonic antigen and the MV receptors CD46 after inoculation show that lytic activity of the transgene encoding virus lags behind in the murine tumor cell line. Flow cytometry of target antigen expressing cells incubated with BTE's at five different dilutions showed BTE binding by cells in a concentration dependent manner. In immunoblot after magnetic pull down of BTE associated cells showed that when non targeting BTE's were used there were no detectable amounts of cells;whereas when targeting BTE samples were used bound cells were present in the elution fraction.

BTE mediated target antigen specific and concentration dependent cytotoxicity of murine T cells is indicated by representative LDH release assay and shows that in the present example 15 percent specific cell killing was achieved at a relatively high BTE concentration of 1 microgram per milliliter compared to reference cells. While attempting this procedure, it is important to remember to regularly monitor cells to check progression of infection. Don't forget that recombinant measles vaccine strains though attenuated may be hazardous especially to immuno-suppressed individuals.

Avoid exposure by following the appropriate bio safety procedures. Individuals handling viruses should be vaccinated before performing these methods. Following this procedure viral expression of further transgenes can be achieved in order to analyze virus hosts interactions and therapeutic effects.

The development of these techniques has paved the way for researchers in the field of biotherapy to explore the effects of locally expressed immunomodulators in numerous tumorous entities;In Vitro, In Vivo, and also in clinical trials.

Journal (Cancer Research)

Journal (Cancer Research)

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

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