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A prime/boost vaccine platform efficiently identifies CD27 agonism and depletion of myeloid-derived suppressor cells as therapies that rationally combine with checkpoint blockade in ovarian cancer

Cancer immunotherapies have generated remarkable clinical responses for some patients with advanced/metastatic disease, prompting exploration of rational combination therapies to bolster anti-tumor immunity in patients with limited response or those who experience tumor progression following an initial response to immunotherapy. In contrast to other tumor indications, objective response rates to single-agent PD-1/PD-L1 blockade in ovarian cancer are limited, suggesting a need to identify combinatorial approaches that lead to tumor regression in a setting where checkpoint blockade alone is ineffective. Using a pre-clinical model of aggressive intraperitoneal ovarian cancer, we have previously reported on a heterologous prime/boost cancer vaccine that elicits robust anti-tumor immunity, prolongs survival of tumor-bearing mice, and which is further improved when combined with checkpoint blockade. As tumor control in this model is CD8 + T cell dependent, we reasoned that the prime/boost vaccine platform could be used to explore additional treatment combinations intended to bolster the effects of CD8 + T cells. Using whole tumor transcriptomic data, we identified candidate therapeutic targets anticipated to rationally combine with prime/boost vaccination. In the context of a highly effective cancer vaccine, CD27 agonism or antibody-mediated depletion of granulocytic cells each modestly increased tumor control following vaccination, with anti-PD-1 therapy further improving treatment efficacy. These findings support the use of immunotherapies with well-defined mechanisms(s) of action as a valuable platform for identifying candidate combination approaches for further therapeutic testing in ovarian cancer. Supplementary Information The online version contains supplementary material available at 10.1007/s00262-021-02936-1.

protocol or were purchased directly from Jackson Laboratory prior to experimental use. Experimental mice were 6-8 weeks of age at study onset. All performed experiments and procedures were reviewed and approved by the Roswell Park IACUC prior to conducting experiments.

Monitoring of T cell responses
Blood, spleen, and peritoneal wash (collected following IP injection of PBS to assess immunological changes in the peritoneal TME) were collected at indicated time points to monitor tumor-specific CD8+ T cell responses. Red blood cells were removed using ACK lysis buffer and CD8+ T cell responses to the immunodominant epitope of ovalbumin (OVA257-264; SIINFEKL) were measured using Tetramer staining. For direct ex vivo analysis of CD8+ T cell function based on IFN-γ production, cell suspensions were stimulated with SIINFEKL peptide (1μg/ml) for 5hrs in the presence of Brefeldin A (10μg/ml) (Sigma Aldrich, St Louis, MO). Data were acquired using a LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar, Ashland, OR).

Analysis of immune infiltrate in primary human ovarian tumors
Ovarian cancer patient tumors (FIGO stage IC-IV) were surgically removed at Roswell Park and received in the laboratory from tissue procurement under protocol I215512. Tumor samples were minced with surgical scissors and mechanically disrupted with a Miltenyi Gentlemacs tissue disruptor, followed by passage through a 300μm strainer and centrifugation at 400 x G for 5 min.
Supernatant was discarded and cells passed through a 100μm cell strainer.
Cells were washed, counted, and whole tumor single cell suspensions plated in 6 well plates (1-4x10 6 cells/well) in cRPMI and incubated in a 37 tissue culture incubator for 24-48hrs prior to staining. Cells were collected by gentle scraping/pipetting and stained for flow cytometric analysis as detailed. Note that Tumor speciments 3 and 4 were distinct tumors removed from the same patient (Stage IIIC).

Immunohistochemistry
The protocol used to assess CD3+ TIL by IHC in IE9-mp1 solid tumors has been previously described [McGray et al, JITC, 2019]. Briefly, solid tumor nodules from the omentum along with surrounding tissue were excised from mice and fixed in 10% Neutral Buffered Formalin for 3 days and were processed, sectioned, and stained at the Roswell Park Pathology Shared Resource. An independent Pathologist at Roswell Park conducted CD3+ TIL scoring on stained tumor sections.

CD70 Transcriptional Analysis
Nanostring analysis of solid IE9-mp1 tumor nodules following treatment using the mouse PanCancer Immune Profiling Panel has been described previously [McGray et al, JITC, 2019]. Normalized data from this study were used to assess CD70 expression in treated or untreated control tumors.

Statistical Analysis
Two-tailed, unpaired t tests were used to compare data from two treatment groups. One and two way Analysis of Variances (ANOVA) were used for data analysis of more than two groups and/or across multiple time points and a Bonferroni post test was utilized to determine significant differences between groups. Survival data was compared using a Logrank test. Linear regression and R 2 values were used to determine the degree of correlation between data sets.