Vaccination Against RhoC in Prostate Cancer Patients Induces Potent and Long-Lasting CD4+ T Cell Responses with Cytolytic Potential in the Absence of Clinical Efficacy: A Randomized Phase II Trial

Abstract

Background: A previous phase I/II study demonstrated potent and long-term immune responses in men with prostate cancer following vaccination with a 20mer synthetic peptide (RV001) derived from the Ras homolog gene family member C protein (RhoC). Moreover, a fraction of patients experienced prostate-specific antigen (PSA) responses, which prompted the initiation of a phase II double-blind randomized trial (NCT04114825). The primary endpoint was to study whether vaccination could postpone PSA progression. Furthermore, the study included an evaluation of vaccination-induced immune responses, and in-depth in vitro studies of RhoC-specific CD4⁺ T cell responses. Methods: Men with non-metastatic biochemical recurrence after either radical prostatectomy or radiation therapy were eligible for the study. Participants were randomized 1:1 to either subcutaneous injections of 0.1 mg/mL RV001 emulsified in Montanide ISA 51, or a placebo. Vaccinations were administered every 2 weeks for the first six times, then five times every 4 weeks for a total treatment time of 30 weeks. Blood samples were collected from a subset of patients (n = 38) over the course of vaccination, and peripheral blood mononuclear cells (PBMCs) isolated for immunological assessment of vaccine-induced immune responses. Experiments using PBMCs from a healthy donor and a patient were performed to study the phenotype and function of RV001-specific CD4⁺ T cells. Results: A total of 192 men entered the study. There was no difference in time to PSA doubling, with 7.5 versus 9.3 months, or in time to initiating further therapies, 11.2 versus 17.6 months for treatment and control groups, respectively. At long-term follow-up, 12.9% of the patients in the vaccination arm had developed metastasis compared to 12% in the placebo arm. No serious treatment-related side effects were observed, and treatment-related adverse events did not differ between groups. Immunological examinations in a subset of patients demonstrated that the vaccination induced potent, long-lasting CD4⁺ T cell responses capable of proliferation and cytokine production. RV001-specific CD4⁺ T cells were shown to mediate cytotoxicity against a RhoC-expressing cancer cell line in an HLA-class II-dependent manner. Conclusions: Men randomized to active treatment with RV001V demonstrated the induction of potent, functionally capable, anti RhoC-CD4⁺ T cell responses. However, there was no benefit in time to biochemical progression, and no difference in time to the initiation of second-line therapies.

Keywords: CD4+ T cells; RhoC; cancer vaccine.

Figure 1

Outline of the study flow. EoT, end of treatment; PSADT, PSA doubling time; RP, radical prostatectomy; RT, radiation therapy.

Figure 2

Time to PSA doubling compared to baseline PSA at study entry. p = 0.1327.

Figure 3

The majority of patients treated with RV001V develop an immunological response against the vaccine. T cell reactivity against the vaccine peptide (RV001) was tested in IFN-γ ELISpot ex vivo and after in vitro pre-stimulation (IVASS). (A) Exemplary ELISpot plate for patient 104-002 at visits 2 (V2, pre-vaccination) and 8 (V8, after the 6th vaccination) ex vivo (upper panel) or after IVASS (lower panel). (B,C) Heat-maps showing the specific mean spot counts (background subtracted) for each tested patient at visits 2 and 8 for the placebo treatment group ((B) saline + Montanide, n = 10) and the active treatment group ((C) RV001 + Montanide, n = 28). The scale represents the mean number of specific spots per well. Ex vivo and post-IVASS settings are shown in the left and right panels, respectively. The white rectangle indicates that the ELISpot test was not performed. Red frames indicate immunological responders according to the predefined criteria.

Figure 4

CD4⁺ RV001-specific T cell responses observed in HD2 upon stimulation with RV001. CD14⁻ cells from HD2 were stimulated with RV001-pulsed DCs or PBMCs for two and three rounds, respectively, and specific T cells were enriched based on TNFα secretion. Peptide-specific reactivity was assessed within the CD4⁺ subset using the ICS assay. (A) Flow cytometry dot plots demonstrating TNFα and IFNγ production in CD4⁺ T cells after the 4th and 5th stimulations and after enrichment. (B) Corresponding stacked bar charts illustrating the percentage of IFNγ⁺, TNFα⁺, and TNFα⁺/IFNγ⁺ CD4⁺ T cells when stimulated with RV001, after subtraction of the background. (C) Flow cytometry dot plots showing the expression of the cell surface degranulation marker CD107a on gated CD4⁺ T cells. FSC-A, forward scatter. (D) Corresponding bar plot illustrating the percentage of CD107a⁺ CD4⁺ T cells when stimulated with RV001 after subtraction of the background. (E) Stacked bars summarizing the percentage of IFNγ⁺, TNFα⁺, and TNFα⁺/IFNγ⁺ CD4⁺ T cells for each of the selected clones upon peptide restimulation. (F) Bar plot of the percentage of CD107a⁺ CD4⁺ T cells of the selected clones upon peptide restimulation. For all panels n = 1.

Figure 5

Primed T cells from a healthy donor can mediate cytotoxicity against RhoC- and HLA-II-expressing FM3 cancer cells. (A) Western blot of FM3 to demonstrate RhoC expression. Full blots in Figure S7. (B) Flow cytometry of HLA-II expression in FM3 cancer cells using a pan-HLA-DR antibody. FMO, fluorescence minus one; FSC-H, forward scatter height. (C) Clone 1B5 was co-cultured with FM3 (left) and peptide-pulsed FM3 (pFM3) (right). Cytokine production was examined in living CD4⁺ T cells. Percentages of TNFα⁺ and IFNγ⁺ are given. (D) For the same co-culture experiment, surface expression of the degranulation marker CD107a is shown. FSC-A, forward scatter. (E) Cytotoxicity of clone 1B5 against FM3 (green) and peptide-pulsed FM3 (pFM3) (blue) was assessed with the xCELLigence system at different effector-to-target (E:T) ratios (10:1; 3:1; 1:1) over a total period of 48 h. Statistical significance was determined by two-way analysis of variance (ANOVA). * indicates p < 0.05 and ** indicates p < 0.01.

Figure 6

Patient-derived CD4+ T cells can mediate cytotoxicity against FM3 cancer cells. (A) Twelve day-cultured PBMCs from patient 101-003 (003) were co-cultured with FM3 (left), FM3 with a blocking antibody against HLA-II (middle), and peptide-pulsed FM3 (right). Cytokine production was examined in living CD4+ T cells. (B) Stacked bar charts illustrate the percentage of IFNγ+, TNFα+, and TNFα+/IFNγ+ CD4+ T cells of the same co-culture experiment. For all conditions, n = 1. (C,D) Cytotoxicity of the CD4+ T cell line against FM3 (green) and peptide-loaded FM3 (pFM3, blue) was assessed with the xCELLigence system at different effector-to-target (E:T) ratios (10:1 (C) and 3:1 (D)) over 48 h. Killing of FM3 in the presence of the HLA-II blocking antibody was also assessed (grey). Statistical significance was determined by two-way ANOVA (C,D). ** indicates p < 0.01.

Figure 7

Preferential TCRBV usage in RV001-specific CD4⁺ T cells. (A) Dot plots gated on HD2 CD4⁺ T cells showing the population positive for BV20 in the lower right quadrant (single positives for fluorescein isothiocyanate, FITC), BV18 (single positives for phycoerythrin, PE), and BV5.1 (PE/FITC double positives). (B) Graphical representation of the percentage of CD4⁺ T cells positive for BV20 at baseline, after the 4th and 5th stimulations, and following TNFα enrichment. (C) PCR product from amplification of cDNA pooled from 9 clones electrophoresed in agarose gel and stained with SYBR green. Full blots in Figure S8. (D) Dot plots gated on live CD4⁺ T cells of patient 101-003 after IVASS and enrichment showing the population positive for BV20 in the lower right quadrant. (E) Graphical representation of the percentage of CD4⁺ T cells positive for BV20 in the same patient.