Rapamycin enhances BCG-specific γδ T cells during intravesical BCG therapy for non-muscle invasive bladder cancer: a randomized, double-blind study

Abstract

Background: Although intravesical BCG is the standard treatment of high-grade non-muscle invasive bladder cancer (NMIBC), response rates remain unsatisfactory. In preclinical models, rapamycin enhances BCG vaccine efficacy against tuberculosis and the killing capacity of γδ T cells, which are critical for BCG's antitumor effects. Here, we monitored immunity, safety, and tolerability of rapamycin combined with BCG in patients with NMIBC.

Methods: A randomized double-blind trial of oral rapamycin (0.5 or 2.0 mg daily) versus placebo for 1 month was conducted in patients with NMIBC concurrently receiving 3 weekly BCG instillations (NCT02753309). The primary outcome was induction of BCG-specific γδ T cells, measured as a percentage change from baseline. Post-BCG urinary cytokines and immune cells were examined as surrogates for local immune response in the bladder. Secondary outcomes measured were adverse events (AEs) and tolerability using validated patient-reported questionnaires.

Results: Thirty-one patients were randomized (11 placebo, 8 rapamycin 2.0 mg, and 12 rapamycin 0.5 mg). AEs were similar across groups and most were grade 1-2. One (12.5%) patient randomized to 2.0 mg rapamycin was taken off treatment due to stomatitis. No significant differences in urinary symptoms, bowel function, or bother were observed between groups. The median (IQR) percentage change in BCG-specific γδ T cells from baseline per group was as follows: -26% (-51% to 24%) for placebo, 9.6% (-59% to 117%) for rapamycin 0.5 mg (versus placebo, p=0.18), and 78.8% (-31% to 115%) for rapamycin 2.0 mg (versus placebo, p=0.03). BCG-induced cytokines showed a progressive increase in IL-8 (p=0.02) and TNF-α (p=0.04) over time for patients on rapamycin 2.0 mg, whereas patients receiving placebo had no significant change in urinary cytokines. Compared with placebo, patients receiving 2.0 mg rapamycin had increased urinary γδ T cells at the first week of BCG (p=0.02).

Conclusions: Four weeks of 0.5 and 2.0 mg oral rapamycin daily is safe and tolerable in combination with BCG for patients with NMIBC. Rapamycin enhances BCG-specific γδ T cell immunity and boosts urinary cytokines during BCG treatment. Further study is needed to determine long-term rapamycin safety, tolerability and effects on BCG efficacy.

Keywords: immunity; immunotherapy; innate; urinary bladder neoplasms.

Figure 1

Clinical trial schema. AE, adverse event; DHT, delayed hypersensitivity test.

Figure 2

Rapamycin is tolerated during intravesical BCG treatment. (A) AUA symptom scores tracked over the first 4 weeks of treatment. Mean±SEM. (B) BCI QoL questionnaire scores assessing urinary (B) function and (C) bother (inconvenience) and bowel habit (D) function and (E) bother (inconvenience). BCI scores were calculated by standardizing Likert scale question responses to a 0 to 100 scale, where a score of 100 indicated the patient answered ‘no problem/bother’ to all questions. Mean±SEM. AUA, American Urologic Association; BCI, Bladder Cancer Index; QoL, quality of life.

Figure 3

Rapamycin (2.0 mg daily) increases urinary IL-8 and TNF-α during intravesical BCG. Cytokines in post-BCG urine weeks 1–3 of BCG maintenance treatment detected by Luminex. P value, linear regression on slope over time for each group. Mean±SEM.

Figure 4

Increased urinary γδ T cells in response to BCG treatment in patients treated with rapamycin. (A) Percentage of each T cell subset and (B) activated or degranulating γδ T cells were analyzed among cells collected from week 1 post-BCG urine pellets by flow cytometry. Expression level of (C, D) CD107a and CD44 on γδ T cells and (E, F) CD44 and CD56 on NK cells from week 1 post-BCG urine pellets were also shown as MFI. γδ T cell, CD4⁺ and CD8⁺ T cell populations were gated under live CD45⁺ and CD3⁺ cells in urine, and NK cells were gated as CD45⁺CD3⁺CD56⁺ cells. P value indicates t-test or Mann-Whitney test, based on normality, for placebo versus rapamycin-treated group. Mean±SEM. MFI, mean fluorescence intensity.

Figure 5

Increased proliferation and function of circulating γδ T cells by combination treatment of BCG and rapamycin. PBMCs from both baseline and day 28 were cocultured with BCG for 7 days. Proliferation and cytokines were measured by flow cytometry. Shown are (A) a representative example of visit 6 (day 28) response of one patient from each group (with highest response in γδ T cells) and (B) AN of proliferated IFNγ producing T cells were calculated based on day 7 live cell count multiplied by the percentage of the population. Then, changes of day 28 response over baseline were calculated and compared between Placebo group and each rapamycin group (bottom graph). P value indicates t-test or Mann-Whitney test, based on normality, for placebo versus each rapamycin-treated group. Mean±SEM. PBMC, peripheral blood mononuclear cells.