An engineered oncolytic virus expressing PD-L1 inhibitors activates tumor neoantigen-specific T cell responses

Oncolytic viruses offer an in situ vaccination approach to activate tumor-specific T cell responses. However, the upregulation of PD-L1 expression on tumor cells and immune cells leads to tumor resistance to oncolytic immunotherapy. In this study, we generate an engineered oncolytic virus that coexpresses a PD-L1 inhibitor and GM-CSF. We find that the oncolytic virus is able to secrete the PD-L1 inhibitor that systemically binds and inhibits PD-L1 on tumor cells and immune cells. Importantly, the intratumoral injection with the oncolytic virus overcomes PD-L1-mediated immunosuppression during both the priming and effector phases, provokes systemic T cell responses against dominant and subdominant neoantigen epitopes derived from mutations, and leads to an effective rejection of both virus-injected and distant tumors. In summary, this engineered oncolytic virus is able to activate tumor neoantigen-specific T cell responses, providing a potent, individual tumor-specific oncolytic immunotherapy for cancer patients, especially those resistant to PD-1/PD-L1 blockade therapy.


Statistics
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Software and code
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Data analysis
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Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. And sample size of at least 5 mice in each group was widely accepted in life science experiment to support the consistency of the data and conclusion. No statistical method was used to predetermine sample size No data was excluded. In Figure 3, when tumors reach >1000 mm^3, the mice were euthanized in accordance with the guidelines of the Institutional Animal Care and Use Committee of USC.
All attempts at replication were successful, and standard deviations were within expected ranges. Unless otherwise noted, each experiment was repeated three or more times.
After tumor injection when tumor size reached about 100mm^3, all mice were allocated randomly for each group. And within animal controls were performed wherever possible. And the possible confounders 'experimenter' and 'day of experiment' were equally matched between groups. For experiments other than mice studies, randomization is irrelevant to our study, because different groups were defined by either different cell lines or different conditions. For all in vitro or vivo experiments whose data were generated by machine reading, blinding or not won't affect the results. For caliper measurement of tumor size, different people were involved in measuring even they were aware of group labeling which was conducted partially blinded. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
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Methodology
Sample preparation Instrument Software other antibodies were validated by flow cytometry.
Human embryonic kidney cell line 293T, osteosarcoma HUTK-143B, monkey kidney fibroblasts CV1, murine adenocarcinoma Py230, murine melanoma B16-F10, Murine lymphoma EL4 were purchased from the American Type Culture Collection (ATCC). Murine colon adenocarcinoma cells MC38 was purchased from Kerafast Certificates of analysis were provided with cell line by ATCC. ATCC authenticates cell lines routinely with the following tests: Short Tandem Repeat (STR) Profiling Cellular Morphology Karyotyping Cytochrome C Oxidase I (COI) Assay Testing All cell lines were routinely tested for mycoplasma contamination and found negative.
the study didn't involve wile animals The study didn't involve samples collected from the field Institutional Animal Care and Use Committee of USC which IACUC approved For Figure 1d. Isolation of bone marrow cells -Age-matched mice were sacrificed by cervical dislocation and bones from hind legs, forelegs were isolated. Bone marrow (BM) was extracted by crushing the bones using mortar and pestle in RPMI + 2% FBS. The suspension was filtered through a 40 #m mesh and centrifuged for 5 min at 1,500 rpm. Freshly isolated bone marrow cells from mice were cultured in complete RPMI1640 media supplemented with 10% FBS, 20 ng/mL GM-CSF, and 40 ng/mL IL-4 for dendritic cell differentiation. For Figure 4. Mice with tumors are euthanized by CO2 asphyxiation. Using autoclaved surgical instruments rinsed with 70% ETOH, cut tumor into small (< 3mm) pieces and incubate in 5 ml dissociation solution (RPMI medium supplemented with 5% FBS, Collagenase type I (200 U/ml) and DNase I (100 #g/ml)) for 30 min at 37°C, Pipetting (using a 1,000 #l pipet tip) and vortexing every 10 minutes during the incubation. If myeloid cells will be subsequently isolated, substitute 5% FBS and Collagenase type I with 10% FBS and Collagenase type IV (200 U/ml), respectively. Splenic cells were obtained by physical dissociation of the spleen on a cell strainer.
After incubation, pass cell suspension through a 70 #m cell strainer and wash twice with 10 ml PBS buffer for staining. Cells were blocked with Fc blocking reagent, and then stained with antibody cocktail for 30 min on ice. If intracellular staining was performed, BD Cytofix/Cytoperm Fixation and Permeabilization Solution was used according to the manufacturer's manual. Counting beads (San diego, biolegend) were added in the final cell suspension to quantify cells when necessary.