Development of PCVs and FAST vaccines
In order to develop both the PCV and FAST vaccines we synthesized 788 peptides (20mers) representing 200 different Fs neoantigens. These peptides were spotted on glass slides and used to screen mouse sera. The basic procedure to make the vaccines and the challenge protocol is depicted in Fig. 1a. To produce PCVs, mice were injected with 500 4T1 tumor cells and 7 days later, blood from each mouse was screened on the peptide microarrays for antigen discovery. For each mouse the 10 most reactive FSPs, relative to non-cancer controls, were chosen for the vaccine. As many FSPs were represented by more than 1 peptide on the microarray, each mouse received between 13 – 23 individual peptides (Fig. 1c, Supplementary Table 1).
While most FSP reactivities were personal to a single mouse, others were reactive in multiple mice, not surprisingly given that the mice are an inbred strain that received the same cell line. To develop the breast cancer FAST (BC-FAST) vaccine, the same data was analyzed and the 10 FS peptides that were the most frequently positive across all 24 samples were chosen as the shared antigens (Fig. 1b, Supplementary Table 2). The same process was applied using the mouse pancreatic cell line, KPC, to produce the PC-FAST vaccine. The frequency of occurrence of the reactive peptides chosen for 4T1 and KPC is shown in Table 1 and Fig. 1b.
BC-FAST and FS PCVs significantly reduce primary tumor growth as monotherapies
Each vaccine was tested against 4T1 primary tumors according to the procedure outlined in Fig. 1a. BALB/c mice were injected with 500 4T1 cells and were treated with either BC-PCV on day 12 and 19 or BC-FAST on day 7 and 14. We chose to vaccinate the FAST vaccine 5 days earlier than the PCV to reflect the advantage of a premade vaccine over a personal one. Human PCVs generally take months to produce. The low injection number of 4T1 cells was required to have sufficient time to prepare the PCV, simulating the time lag to produce PCVs for human use. Each vaccine was administered with poly (I:C) as adjuvant. The control set (Mock) consisted of mice injected with PBS. As another negative control, a non-reactive vaccine (NR) was prepared from FS peptides that were not reactive with sera from 4T1 tumor bearing mice (Supplementary Fig. 1). An additional cohort of mice were vaccinated with the PC-FAST vaccine on the same schedule as the BC-FAST vaccine.
Tumor growth was monitored for each group. Seven out 10 BC-FAST vaccinated mice were tumor free versus 2 out of 10 mock vaccinated mice (p = 0.0074) (Fig. 2a, Table 2). There were 5 tumor-free mice in the BC-PCV vaccinated group (n.s) while the PC-FAST vaccinated group had similar tumor-free curves (3/10) as the control arm . All mice vaccinated with NR developed tumors. For the tumor-bearing mice, the PCV and BC-FAST vaccines significantly delayed tumor growth and reduced the tumor volume at the endpoint (Fig. 2b, Supplementary Fig. 2). However, in contrast to the BC-FAST and PCV vaccinees, the tumors in the NR and PC-FAST groups grew as fast as the control group (Fig. 2b, bottom). Additionally, BC-FAST vaccination prolonged survival in comparison to all the other vaccine groups and mock (p = 0.00167)(Fig. 2c).
Tumor-free mice in the mock, BC-FAST and PC-FAST groups were re-challenged with twice as many 4T1 (1,000) cells on day 43 after the initial inoculation (Supplementary Fig. 3, 5) and received a vaccine boost 21 days post re-challenge (day 64). Although a majority of mice developed tumors, tumors in the BC-FAST group grew at a slower rate than the controls (p < 0.01) while the delayed tumor growth of PC-FAST vaccinated mice was not statistically significant. Intriguingly, 2 mice vaccinated with BC-FAST, with and without ICI, had a small nodule that remained the same size during the entire re-challenge experiment (35 days). Remarkably, BC-FAST vaccinated mice that received prior ICI treatment (BC-FAST/ICI) when re-challenged showed an increased tumor latency (24 days versus 19 days) and had even smaller tumors (average 307 mm3) than control (average 1100 mm3) and vaccine alone (average 607 mm3) (Supplementary Fig. 3), suggesting a long-term immune-stimulation effect by the combined immunotherapy. These results indicate that the BC-FAST vaccine may be as effective as the BC-PCV in this model.
PCVs and FAST vaccines in combination with ICIs
As many current clinical trials of PCVs include ICI treatment, we tested the effect of anti-PD-1 plus anti-CTLA-4 ICI treatment on FAST and PCV efficacy. In these studies, PCV treated mice received 4 ICI injections after the peptide vaccinations while FAST vaccinated mice received 3 ICI injections. We reduced the number of ICI doses due to the rapid and fatal reactions in controls groups after repeated administration of the ICI (4-5 doses) observed in our study and this adverse event was also observed and investigate by Mall et. al. 2016 . Interestingly, while the BC-FAST/ICI and BC-PCV/ICI groups did not show statistically significantly improved tumor-free curves relative to the vaccine alone or the Mock group, the PC-FAST vaccine did (p =0.0370) (Fig. 3a). In fact, we observed an earlier tumor onset in the presence of ICI for BC-FAST group, probably due to the immune cell infiltration causing the phenomenon called “pseudoprogression” [22, 23]. Similar to the monotherapy results, those mice that developed tumors had significantly slower growth in the PC-FAST/ICI, BC-FAST/ICI and PCV/ICI groups relative to controls (Fig. 3b). However, the BC-FAST/ICI group showed slightly larger tumors than the vaccine alone, indicating again possible pseudoprogression. Importantly, the addition of ICI to the BC-PCV group extended their survival window by 10 days. Morbidity for NR-PCV/ICI vaccinees exceeded that of the control mice and this group had to be sacrificed. As observed for the vaccine therapy alone, the number of tumor free mice and survival rates were improved compared to the control group (Fig 3c,d and Table 2). We conclude that including ICI did not improve the BC-FAST vaccine performance, but did potentiate the BC-PCV and pancreatic cancer (PC-FAST) vaccines efficacies.
PCVs and FAST vaccines reduce lung metastases
The effect on the incidence of secondary spontaneous tumors was also assessed. We noted significant reductions in lung metastases for the BC-FAST vaccination (with and without re-challenge (circles)), BC-PCV and PC-FAST (with and without re-challenge) vaccination versus the control mice (Fig. 4a). The average number of metastases observed in the PCV group was less than the FAST vaccinated groups, though this result was non-significant. Additionally, vaccinated tumor-free mice did not have measurable lung metastases. Similar to the monotherapy results, there was a significant reduction in lung metastases for the BC-FAST/ICI, PCV/ICI and PC-FAST/ICI groups versus control but non-significant differences between groups (Fig. 4b). Surprisingly, association between tumor size (weight) and the number of pulmonary metastases revealed an enhanced efficacy of both the tumor specific (BC-FAST) and non-specific (PC-FAST), FAST vaccines in controlling of tumor dissemination (fig. 4c). While mock and negative control (NR-PCV) groups demonstrated that larger primaries tumors also have larger number of metastases, FAST vaccinated animals with large tumors had fewer pulmonary tumors. This was not observed in mice vaccinated with PCV as monotherapy. By adding ICI to the PCVs or NR vaccine (negative control), produced a similar pattern observed for the FAST vaccines, but did not improved FAST anti-metastasis capacity (fig. 4d). Altogether, these data suggest that the anti-metastatic mechanism of the PCV and FAST, as monotherapies, are different; while PCV reduces tumor dissemination by controlling primary tumor, FAST vaccines control pulmonary metastases regardless its efficacy against the primary tumors.
BC-FAST and PCVs elicit robust neoantigen specific T-cell responses.
Peptides for the PCVs and the BC-FAST vaccines were selected based upon antibody reactivity on the microarrays so we expected them to also be reactive by ELISA. The antibody response from pooled sera (collected at the endpoint) to each peptide in the BC-FAST vaccine demonstrated that, as expected, the injection of the tumor alone in the Mock mice elicited an antibody response to most peptides (Supplementary Fig. 6). Interestingly, vaccination with BC-FAST or PC- FAST alone or with ICI treatment did not generally increase the antibody responses (Supplementary Fig. 6). For the PCV group, each FS antigen was assessed in each mouse and it was found that most peptides were reactive to varying degrees in each mouse (Supplementary Fig. 6).
We also evaluated the T-cell responses to the FS antigens and found that the Mock vaccinated group had a moderate T-cell response to the shared antigens in the BC-FAST vaccine but did not react with the PCV antigen peptides, as measured by ELISpot of mouse splenocytes (Fig. 5a,c). However, vaccination with the BC-FAST (n.s.) vaccine or PCVs (p = 0.01) did produce a T-cell response to pooled peptides in most of the mice (Fig. 5a,c, Supplementary Fig. 7). However, addition of ICI to either group did not significantly affect the number of reactive T-cells (Fig. 5b,d). The T-cell response against the 4T1 tumor cells was measured and as expected, there was little reactivity in the Mock group. However, the BC-FAST, BC-PCV and PC-FAST vaccinees had significant T-cell responses against the tumor in most of the vaccinated mice (Fig. 6).
BC-FAST evokes a robust cellular immune response
To better understand the phenotype of the T-cell populations produced by the BC-FAST vaccine, we used FACS to evaluate the cytokine-producing T cell populations in naïve BALB/c mice and vaccinated tumor-bearing mice with splenocytes stimulated in vitro by BC-FAST peptides (Fig. 7). In naïve mice, we observed the presence of CD4+ T-cells producing IFN-γ, TNF-α, or IL-2 cytokine in response to ex vivo stimulation by the shared FS peptides. This indicates a Th1 subtype profile, but no polyfunctional T-cells were observed. Naïve CD8+ T cells showed a high frequency of single-positive cells for cytokines (IFN-γ, TNF-α or IL-2) and granzyme B, with low frequency of triple-positive effector cells (IFN-γ, TNF-α and Granzyme B). Interestingly, we observed a small frequency of IFN-γ and PD-1 double positive cells in both CD4+ and CD8+ T-cells. PD-1 expression in effector T cells has a complex significance, suggesting both T cell dysfunction as well as T cell activation/high avidity. These data indicate the immunogenicity of these peptides and their ability to induce antigen-specific effector cells.
Mock vaccinated mice had a predominantly Th1 CD4+ T-cell response with production of IFN- γ and CD8+ T-cells that were positive for IFN-γ or granzyme B. In contrast, BC-FAST vaccinated mice, both with and without ICI treatment, had T-cells that could produce effector cytokines (IFN-γ, TNF-α or IL-2 ) and degranulate (Granzyme B). Furthermore, we detected an increased frequency of CD4+ T cells producing granzyme B indicating their cytotoxic activity (CTL), possibly as a result of chronic exposure to the tumor . Similar to naïve mice, we detected an increase in the frequency of cells single and double-positive (with IFN-γ) for PD-1, indicating a exhaustion/activation phenotype. Altogether, these data demonstrate that FAST vaccination induces T-cells with potent ability to produce both effector cytokines and degranulate, despite PD-1 expression.