Vaccination against RhoC induces long-lasting immune responses in patients with prostate cancer: results from a phase I/II clinical trial

Abstract

Background: Peptide-based vaccination is a rational option for immunotherapy of prostate cancer. In this first-in-man phase I/II study, we assessed the safety, tolerability and immunological impact of a synthetic long peptide vaccine targeting Ras homolog gene family member C (RhoC) in patients with prostate cancer. RhoC is a small GTPase overexpressed in advanced solid cancers, metastases and cancer stem cells.

Methods: Twenty-two patients who had previously undergone radical prostatectomy received subcutaneous injections of 0.1 mg of a single RhoC-derived 20mer peptide emulsified in Montanide ISA-51 every 2 weeks for the first six times, then five times every 4 weeks for a total treatment time of 30 weeks. The drug safety and vaccine-specific immune responses were assessed during treatment and thereafter within a 13-month follow-up period. Serum level of prostate-specific antigen was measured up to 26 months postvaccination.

Results: Most patients (18 of 21 evaluable) developed a strong CD4 T cell response against the vaccine, which lasted at least 10 months following the last vaccination. Three promiscuouslypresented HLA-class II epitopes were identified. Vaccine-specific CD4 T cells were polyfunctional and effector memory T cells that stably expressed PD-1 (CD279) and OX-40 (CD134), but not LAG-3 (CD223). One CD8 T cell response was detected in addition. The vaccine was well tolerated and no treatment-related adverse events of grade ≥3 were observed.

Conclusion: Targeting of RhoC induced a potent and long-lasting T cell immunity in the majority of the patients. The study demonstrates an excellent safety and tolerability profile. Vaccination against RhoC could potentially delay or prevent tumor recurrence and metastasis formation.

Trial registration number: NCT03199872.

Keywords: T-Lymphocytes; immunotherapy; prostatic neoplasms; translational medical research; vaccination.

Conflict of interest statement

Competing interests: CG and H-GR receive a research grant from RhoVac ApS. PTS is consultant at RhoVac ApS. CS is employee of DanTrials. TB is owner and CEO of DanTrials.

© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

RV001-specific T cells are induced after RhoC vaccination. (A) Vaccination and monitoring schedule. Patients were vaccinated 11 times. For immunoassays, blood was taken prevaccination, three times during the vaccination phase (vaccination) and four times postvaccination (follow-up 1 and follow-up 2) (blood drops). PBMCs were prestimulated with the RV001 and expanded for 12 days before IFN-γ ELISpot testing (0.2×106 cells/well, except for Patient 21 visits 2–13, and Patient 012 visits 16+17: 0.17×106 cells/well). (B) Exemplary result of an ELISpot (Patient 011). ddH2O and PHA were used as negative and positive control, respectively. (C–E) RV001-specific mean spot counts per analyzed time and normalized to 0.2×106 cells/well. Three independent ELISpot experiments were performed (indicated by the gaps). The sums of RV001-specific mean spot numbers (V6+8+13) are shown for strong- (C; n=7; ≥2500 spots), intermediate- (D; n=7; ≥1500–2500 spots), and weak/non- (E; n=7; 0–1500 spots) responders. (F) RV001-specific mean spot counts per patient and visit normalized to 0.2×106 cells/well. Light green indicates a statistical significance according to the DFR(2x) permutation test. n=21 patients. ddH2O, bidistilled water; IFN-γ, interferon-γ; na, cells not available; PHA, phytohemagglutinin; RhoC, Ras homolog gene family member C.

Figure 2

RV001-responding cells are multifunctional. ddH2O stimulated cells harvested from the ELISpot were restimulated with RV001 for 12 hours. Expression of CD107a, CD154, IL-2, TNF, and IFN-γ was examined by ICS on live CD4 and CD8 lymphocytes and the % of RV001-specific cells calculated for each of the 5 markers within CD4 or CD8 cell subsets. (A) Overview of CD4 T cell responses during vaccination (n=18 patients). (B) Mean+95% CI of cumulative marker expression on RV001-specific CD4 T cells for strong- (n=7), intermediate- (n=7) and weak- (n=4) responders. Kruskal-Wallis test with Dunn’s post-test. (C) Min to max percentages of RV001-specific CD4 T cells expressing one to five markers simultaneously, classified per strong- (n=7), intermediate- (n=7), and weak- (n=4) responders. Median values are indicated. Two-way ANOVA with Tukey’s post-test. (D) Mean+95% CI of RV001 specific CD4 T cells expressing each of the five activation markers or combinations thereof (all patients, n=18). (E) 12 day-cultured PBMCs from Patient 004 at visit 14 were restimulated with ddH2O (upper dot-plot panel) or RV001 (lower dot-plot panel). The activation marker expression was examined on living CD4 (upper rows) and CD8 (lower rows) lymphocytes. Percentages of marker+ cells within CD4 or CD8 cells are given. *P≤0.05, **p≤0.01, ***p≤0.001. Responder groups are defined based on the ELISpot results. ANOVA, analysis of variance; ddH2O, deionized water; IFN-γ, interferon-γ; IL-2, interleukin-2; TNF, tumor necrosis factor.

Figure 3

RV001-specific T cells are effector memory T cells. PBMCs from Patient 005 (visits 6–15) and Patients 009 and 018 (visits 6–17) were thawed, rested and stimulated either with RV001 or ddH2O for 12 hours. Live RV001-specific CD4 lymphocytes were identified by TNF expression and examined for the expression of CD45RA, CCR7, PD-1, OX-40, and LAG-3. (A) Exemplary results (Patient 009): CD4+TNF+ cells (black) are overlaid on the whole CD4 cell population (gray). Numbers indicate CD4+TNF+ cell counts. (B) Expression profile of PD-1 (upper row) and OX-40 (lower row) for n=3 patients at visit 6 and visit 15. Numbers indicate MFI ratios of the two receptors between CD4+TNF+CD45RA-CCR7- cells (dark gray) and CD4+TNF-CD45RA-CCR7- cells (light gray). Histograms show event counts normalized to mode. (C–E) Assessment of Tregs (CD3+CD8-CD4+CD127-CD25+Foxp3+) from 5 patients (4 visits). (C) Mean+95% CI of total CD4 cells (left-axis, black curves) and Tregs (right axis, blue curves) within lymphocytes. (D) Kinetics of Treg percentages within CD4 T lymphocytes for each of the 4 patients. (E) Exemplary dot-plots for Patient 003, percentages of CD3+CD8-CD4+CD127-CD25+Foxp3+ cells are indicated. ddH2O, deionized water; MFI, median fluorescence intensity; TNF, tumor necrosis factor.

Figure 4

The RV001 sequence comprises several promiscuous HLA-class II epitopes and one HLA-B*27:05 restricted epitope. The expression of CD107a, CD154, IL-2, TNF and IFN-γ was examined on live CD4 or CD8 cells. (A) Cells were restimulated with RV001 or with RV001-derived 15mer peptides (ATR15, AGL15, LQV15) for 12 hours. Shown are the percentage or mean+SD percentage (n=2 repeated measurements) of peptide-specific CD4 cells expressing each activation marker for three patients at visit 16. (B) LCLs were preloaded with RV001, ATR15, or AGL15 and incubated with HLA-matched patient cells at 1:2 ratio for 12 hours. Shown are the specific percentages of CD4 cells expressing the indicated activation markers. (C) Cells were restimulated either with the RV001 peptide alone or with RV001-preloaded C1R or C1R-HLA-B*27:05 cells for 12 hours in the ICS. Shown are the percentages of specific marker expression on CD8 cells. IFN-γ, interferon-γ; IL-2, interleukin 2; LCLs, lymphoblastic cell lines; TNF, tumor necrosis factor.