Bacillus Calmette-Guérin (BCG) is the gold standard immunologic agent for patients with high-grade non-muscle invasive bladder cancer (NMIBC). Nevertheless, responses to BCG are heterogeneous and limited options exist when BCG therapy fails. Preclinical data in animal models of bladder cancer (BC) suggests that priming with percutaneous BCG vaccine prior to intravesical BCG instillation can enhance BCG-specific immunity and improve outcome. To study the safety, immunogenicity and preliminary efficacy of this approach, we administered percutaneous BCG 21 days prior to intravesical BCG instillation in “prime patients” with NMIBC (n=13) in a prospective single-arm clinical trial. Immune responses and clinical outcomes were monitored and compared to a contemporaneous cohort of “control patients” (n=9) receiving intravesical BCG without prior percutaneous BCG. Priming was well tolerated and no grade ≥3 adverse events were observed. Compared to control, prime patients had improvement in both local and systemic measures of BCG-specific immunity, scored by increased post-BCG urinary IL-8 and IL-17A and increased circulating CD4, CD8, and γδ T cell proliferation and effector function in response to BCG. Furthermore, ex vivo cytotoxicity of circulating NK and γδ T cells against RT4 BC was significantly increased in prime patient after BCG treatment and was mediated in part by NKG2D, suggesting an important role of innate effector immune mechanisms underlying BCG’s antitumor activity. Remarkably, no prime patients progressed whereas 3 out of 9 control patients progressed to muscle-invasive disease and underwent cystectomy. Thus, BCG priming safely enhances BCG-specific immunity and could improve outcome for patients with NMIBC.

αPD-L1 bladder cancer (BC) immunotherapy is effective in <30% of cases. To address the large αPD-L1-unresponsive subset of patients, we tested αIL-2/IL-2 complexes (IL-2c) that block IL-2 from binding high-affinity IL-2Rα (CD25) for preferential IL-2Rβ (CD122) binding. Regulatory T cells (Tregs) capture IL-2 by CD25 whereas CD8+T, γδ T, and NK cells use CD122. We hypothesized that the tumor microenvironment, including local immune cells in primary versus metastatic BC, differentially affects immunotherapy responses. We used PD-L1+ mouse BC cell lines MB49 and MBT-2, for intravesical ([IVe] in bladder) and intravenous (IV) challenge studies of local versus metastatic BC. αPD-L1 or IL-2c alone reduced tumor burden and extended survival in IVe MB49 and MBT-2. Treg depletion using FOXP3DTR mice further enhanced IVe IL-2c effects, consistent with the known tumor-promoting role of Tregs in human BC. Using in vivo cell depletion approaches, we found that γδ T cells and NK cells, but not CD8+ T cells, were necessary for IL-2c efficacy in bladder. γδ T cells also reduced intratumoral Th17 cells that promote MB49 growth and are elevated in human BC. We confirmed γδ T cell effects in δ TCR KO mice, which abrogated IL-2c efficacy but not αPD-L1 efficacy. Neither αPD-L1 nor IL-2c alone treated metastatic MB49 and MBT-2 BC but the combination improved survival in both. These data are consistent with our recent findings in human BC patients in whom γδ T cell and NK cell cytotoxicity improved BCG immunotherapy. Thus, IL-2c is a promising novel BC immunotherapy that can improve bladder-specific immunity in primary BC. In metastatic BC, combination with αPD-L1 may also be a successful BC treatment strategy due to engagement of innate and adaptive immune responses.

The mechanistic target of rapamycin (mTOR) integrates environmental inputs to regulate cellular growth and metabolism in tumors. However, mTOR also regulates T cell differentiation and activation, rendering applications of mTOR inhibitors towards treating cancer complex. Recent preclinical data supports distinct biphasic effects of rapamycin with higher doses directly suppressing tumor cell growth and lower doses enhancing T cell immunity. We conducted a randomized controlled pre-surgical clinical trial to study the tumor and immune effects of low dose mTOR complex 1 inhibition with rapamycin for patients with invasive, non-metastatic bladder cancer. At 3 mg daily for four weeks, rapamycin significantly inhibited tumor mTORC1 (ribosomal protein S6 phosphorylation), and achieved high bladder tissue levels. Rapamycin also reduced surgery-mediated T cell exhaustion, evidenced by a significant decrease in the prevalence of dysfunctional programmed death-1 (PD-1) expressing circulating T cells without decreasing T cell proliferation or cytokine production. In mice challenged with MB49 orthotopic bladder tumors, a 2 cm surgical laparotomy reduces the efficacy of α-PD-L1 immunotherapy but efficacy can be partly restored with presurgical administration of rapamycin. These findings support favorable delivery, pharmacodynamic activity, and immune modulatory profile of low dose rapamycin in bladder cancer and identifies a novel role for mTOR inhibition in modulating surgery-induced immune dysfunction.

αPD-L1 is an FDA-approved bladder cancer (BC) immunotherapy but is effective in ≤30% of cases. We tested conjugates of αIL-2 antibody + IL-2 (IL-2c) that block IL-2 from binding high-affinity IL-2Rα (CD25) for preferential IL-2Rβ(CD122) binding. CD25 and CD122 are preferred for IL-2 capture by regulatory T cells and anti-tumor effector T cells (Teff), respectively, allowing IL-2c to target Teff preferentially. Orthotopic, intravesical (in bladder) MB49 BC produces PD-L1+BC tumors in syngeneic BL6 mice. αPD-L1 or IL-2c treated intravesical, but not lung metastatic MB49 produced by intravenous injection. Still, in metastatic BC, αPD-L1 + IL-2c (combo) reduced lung metastases and extended survival. Preliminary data found combo treatment efficacy was better in lung versus bladder. In subcutaneous (SQ) B16 melanoma, combo was better than single agents, and increased CD8+CXCR5+TCF-1+Tim-3−PD-1+T stem cells (CXCR5+SC, see X. Zhang poster) vs. single agents. Neither αPD-L1 nor combo increased CXCR5+SC in MB49 in bladder or lung metastases, suggesting a novel treatment mechanism. To test the impact of tumor PD-L1 on treatment efficacy, we made PD-L1KOMB49, but it did not grow in bladder or SQ in wild type mice, but grew similar to control MB49 in immunodeficient NSG mice SQ, suggesting tumor PD-L1 microenvironment-specific effects. PD-L1KOB16 grew well SQ in wild type mice, excluding a PD-L1-specific defect. Thus, tumor PD-L1 differs in immune evasion in a tumor-dependent manner. Selective IL-2 targeting to CD122 improves αPD-L1 treatment of metastatic BC. Mechanisms differ from melanoma, which could be due to tumor, PD-L1, or microenvironment effects. We are assessing mechanisms and αPD-1 treatment effects (also FDA-approved for BC).