Cancer of Colon

Example 2

For Western blot analysis 20 μg of total protein extracted from cells lyzed with Laemmli-lysis buffer was used. Extracts were diluted in reducing sample buffer (Roth), subjected to SDS-PAGE and subsequently electrotransferred onto PVDF membrane (Pall). Immunostaining was performed with polyclonal antibodies reactive to CLDN6 (ARP) and beta-Actin (Abcam) followed by detection of primary antibodies with horseradish-peroxidase conjugated goat anti-mouse and goat anti-rabbit secondary antibodies (Dako).

Tissue lysates from up to five individuals were tested for each normal tissue type. No CLDN6 protein expression was detected in any of the normal tissues analyzed. In contrast to normal tissues, high expression of CLDN6 protein was detected in samples from ovarian cancer and lung cancer. CLDN6 expression was detected in NIH-OVCAR3 (ovarian cancer), MKN7 (gastric cancer), AGS (gastric cancer), CPC-N (SCLC), HCT-116 (colon cancer), FU97 (gastric cancer), NEC8 (testicular embryonal carcinoma), JAR (placental choriocarcinoma), JEG3 (placental choriocarcinoma), BEWO (placental choriocarcinoma), and PA-1 (ovarian teratocarcinoma).

Example 11

MPV.10.34.d IRC Effectiveness in Human Assays

While the in vitro functional test results of the above experiments were promising, the next desired step in the analysis was to perform similar experiments in human-based assays. To this end, the response of mock human cellular immune system components to tumor cells exposed to MPV.10.34.d IRC was examined in vitro. Human CMV (HCMV) was selected for this study since human CMV is highly prevalent (infecting 50-90% of the human population) and mostly asymptomatic in healthy individuals. (See, Longmate et al., Immunogenetics, 52(3-4):165-73, 2001; Pardieck et al., F1000Res, 7, 2018; and van den Berg et al., Med. Microbiol. Immunol., 208(3-4):365-373, 2019). Importantly, HCMV establishes a life-long persistent infection that requires long-lived cellular immunity to prevent disease. Hence, it is rational to hypothesize that a complex adaptive cell-mediated anti-viral immunity developed over many years to strongly control a viral infection in an aging person can be repurposed and harnessed to treat cancer.

In these experiments, CD8+ T cell responses to CMV peptides were tested in three different human tumor cell lines, including HCT116, OVCAR3, and MCF7. All three of these human tumor cell lines are HLA-A*0201 positive.

In vitro cytotoxicity assays. HTC112, human colon cancer cells, MCF7, human breast cancer cells, and OVCAR3, human ovarian cancer cells (all from ATCC, Manassas, VA, US) were seeded overnight at 0.01 to 0.2×10⁶ per well per 100 μL per 96 well plate. The next day (about 20 to 22 hrs later), each cell line was incubated for one hour at 37° C. under the following conditions: (1) CMV peptide at a final concentration of 1 μg/mL (positive control), (2) MPV.10.34.d at a final concentration of 2.5 μg/mL (negative control), (3) CMV-conjugated MPV.10.34.d IRC at a final concentration of 2.5 μg/mL, (4) CMV-conjugated HPV16 IRC at a final concentration of 2.5 μg/mL, and (5) no antigen (negative control). After 1 hour, the cells were washed vigorously with 200 μL of media for three times to remove non-specific binding. Human patient donor CMV T cells (ASTARTE Biologics, Seattle, WA, US) were added at the E:T (effector cell:target cell) ratio of 10:1 and incubated in a tissue culture incubator for 24 hrs at 37 C, 5% CO₂. The total final volume of each sample after co-culture was 200 μL. Cell viability was measured after co-culturing. Cell viability was measured with CELLTITER-GLO® (Promega, Madison, WI, US). This assay provides a luciferase-expressing chemical probe that detects and binds to ATP, a marker of cell viability. The amount of ATP generated from tumor cells was quantified according to manufacturer protocols. In these assays, reduced luciferase activity indicates cell death and suggests greater immune redirection and greater cytotoxicity.

The results are provided in FIG. 25. CMV-conjugated MPV.10.34.d IRC (“VERI-101” in FIGS. 25A, 25B, and 25C) was equally effective as CMV-conjugated HPV16 IRC (“CMV AIR-VLP” in FIGS. 25A, 25B, and 25C) in redirecting human healthy donor CMV pp65-specific CD8+ T-cells (Astarte Biologics, Inc., Bothell, WA, US) to kill immortalized HLA.A2 positive human colon cancer cells (HCT116), human ovarian cancer cells (OVCAR3), and human breast cancer cells (MCF7). The control samples (“No Ag” or “VERI-000” in FIGS. 25A, 25B, and 25C) showed no background tumor killing. Together, these data demonstrate that MPV.10.34.d IRC redirects mouse and human immune responses against tumor cells in vitro.

Example 4

Cells were plated at 6,000 cells/well in a 96-well plate and treated with the indicated concentration of zafirlukast 24 hours later. Cells were allowed to grow for an additional 24 hours before alterations in growth were measured by the PrestoBlue cell proliferation assay in triplicate. Fluorescence readings with an excitation at 570 nm and emission of 600 nm were collected and treated samples were converted to a percentage of the untreated controls.

OVCar8 (human ovarian cancer cell line), HCT116 (human colon cancer), HeLa (cervical cancer cell line) and MDA-MB-231 (breast cancer cell line) cell lines were examined for their ability to inhibit cell proliferation. OVCar8 and HCT116 cells exhibited an IC50 in the 5-10 μM range. HeLa cervical cancer cells and MDA-MB-231 breast cancer lines were less sensitive. Table 1 illustrates the results of a cell proliferation assay testing zafirlukast against OVCar8 cells, HCT116 cells, HeLa cells and MDA-MB-231 cells.