IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

Abstract

CD40 is a central co-stimulatory receptor implicated in the development of productive anti-tumor immune responses across multiple cancers, including bladder cancer. Despite strong preclinical rationale, systemic administration of therapeutic agonistic antibodies targeting the CD40 pathway have demonstrated dose limiting toxicities with minimal clinical activity to date, emphasizing an important need for optimized CD40-targeted approaches, including rational combination therapy strategies. Here, we describe an important role for the endogenous IL-15 pathway in contributing to the therapeutic activity of CD40 agonism in orthotopic bladder tumors, with upregulation of trans-presented IL-15/IL-15Rα surface complexes, particularly by cross-presenting cDC1s, and associated enrichment of activated CD8 T cells within the bladder tumor microenvironment. In bladder cancer patient samples, we identify DCs as the primary source of IL-15, however, they lack high levels of IL-15Rα at baseline. Using humanized immunocompetent orthotopic bladder tumor models, we demonstrate the ability to therapeutically augment this interaction through combined treatment with anti-CD40 agonist antibodies and exogenous IL-15, including the fully-human Fc-optimized antibody 2141-V11 currently in clinical development for the treatment of bladder cancer. Combination therapy enhances the crosstalk between Batf3-dependent cDC1s and CD8 T cells, driving robust primary anti-tumor activity and further stimulating long-term systemic anti-tumor memory responses associated with circulating memory-phenotype T and NK cell populations. Collectively, these data reveal an important role for IL-15 in mediating anti-tumor CD40 agonist responses in bladder cancer and provide key proof-of-concept for combined use of Fc-optimized anti-CD40 agonist antibodies and agents targeting the IL-15 pathway. These data support expansion of ongoing clinical studies evaluating anti-CD40 agonist antibodies and IL-15-based approaches to evaluate combinations of these promising therapeutics for the treatment of patients with bladder cancer.

Figure 1.

Dendritic cells in the bladder microenvironment of mice responding to CD40 agonism have higher expression of IL-15Rα. (A) Schematic of the treatment of mice bearing orthotopic MB49 bladder tumors with anti-CD40 antibody. (B) Representative intravital luciferase imaging (left) and quantification of luminescence (center) and bladder weights (right) across mice at day 24 post-tumor implantation (n = 3–6 mice per group; bars represent SD). Responders (gray circles) and non-responders (blue circles) to anti-CD40 antibody therapy are indicated. (C) IL-15Rα mean fluorescence intensity across mice on type-1 conventional DCs (cDC1; defined as F4/80⁻Ly-6G⁻CD11c⁺MHCII⁺XCR1⁺), type-2 conventional DCs (cDC2; defined as F4/80⁻Ly-6G⁻CD11c⁺MHCII⁺SIRPα⁺), macrophages (defined as CD11b⁺F4/80⁺Ly-6G⁻), and neutrophils (defined as CD11b⁺Ly-6G⁺) in the bladder microenvironment as assessed by flow cytometry at day 24 post-tumor implantation (n = 3–6 mice per group). *p < 0.05, **p < 0.01.

Figure 2.

Endogenous IL-15 participates in the therapeutic activity of CD40 agonism. (A) Schematic of the treatment of mice bearing orthotopic MB49 bladder tumors with anti-CD40 antibody and/or anti-IL-15 blocking antibody or isotype-matched control antibody. (B) Representative intravital luciferase imaging (left) and quantification of luminescence (center) and bladder weights (right) across mice at day 12 post-tumor implantation (n = 5 mice per group; bars represent SD). (C) Representative flow cytometry plots and quantification across mice (n = 5 mice per group) of CD44^hiCD122⁺ CD8 T cells (top) and CXCR6⁺CD44^hiCD122⁺ CD8 T cells (bottom) in the bladders of mice treated as outlined in A. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3.

DCs in human non-muscle invasive bladder cancer express high levels of IL-15. Eight primary tumor samples from either untreated NMIBC patients (n=3) or from NMIBC patients that had progressed from NMIBC to MIBC following BCG therapy (n=5) were included in this single-cell RNA sequencing analysis. A) Phenograph clustering was performed and 29 clusters were identified. Canonical lineage markers for cell populations were utilized to assign cell identities to each cluster. B) tSNE plots were created for four representative markers: CD40, CD40L, IL15, and IL15RA. C) A matrix plot with column scaled expression of the same four markers of interest is shown to highlight the distribution and intensity of expression for each gene across each subset.

Figure 4.

CD40 agonism induces IL-15/IL-15Rα upregulation on dendritic cells in the bladder tumor microenvironment. Mice bearing orthotopic MB49 bladder tumors were treated intravesically with anti-CD40 antibody, BCG, or isotype-matched control antibody on days 6 and 9 post-tumor implantation. (A) Representative histogram and (B) quantification across mice of IL-15Rα mean fluorescence intensity on type-1 conventional DCs (cDC1; defined as F4/80⁻Ly-6G⁻CD11c⁺MHCII⁺XCR1⁺), type-2 conventional DCs (cDC2; defined as F4/80⁻Ly-6G⁻CD11c⁺MHCII⁺SIRPα⁺), macrophages (defined as CD11b⁺F4/80⁺Ly-6G⁻), and neutrophils (defined as CD11b⁺Ly-6G⁺) in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 5 mice per group). (C) Representative flow cytometry plots and (D) quantification across mice of IL-15 and IL-15Rα surface expression on cDC1s in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 5 mice per group). Left graph is gated on cDC1s and depicts proportions of cDC1s that are double-positive for surface IL-15 and IL-15Rα. Right graph is gated on IL-15Rα-expressing cDC1s and depicts proportions of cDC1s expressing IL-15Rα that is occupied by IL-15. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5.

Combination therapy with Fc-optimized anti-CD40 agonist antibody 2141-V11 and IL-15 enhances primary anti-tumor activity. (A) Schematic of the treatment of humanized hCD40/hFcyR mice bearing orthotopic MB49 bladder tumors with anti-CD40 antibody 2141-V11 and/or IL-15 or control (isotype-matched control antibody and/or vehicle). (B) Representative intravital luciferase imaging (left) and luminescence quantification (right) across mice at day 14 post-tumor implantation (n = 5 mice per group; bars represent SD). (C) Survival (top) and representative intravital luciferase imaging (bottom) of surviving mice at day 85 post-tumor implantation treated as outlined in A. (D) Representative intravital luciferase imaging (left) and quantification of luminescence (right, top) and bladder weights (right, bottom) at day 14 post-tumor implantation across mice treated with anti-CD40 antibody 2141-V11 and IL-15 or control in the absence or presence of additional anti-CD8 T cell depleting antibody (n = 5 mice per group; bars represent SD). (E) Representative intravital luciferase imaging (left) and quantification of luminescence (right, top) and bladder weights (right, bottom) at day 14 post-tumor implantation across WT or Batf3^−/− mice treated with anti-CD40 antibody 1C10 and IL-15 or control (n = 5 mice per group; bars represent SD). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6.

Combination therapy with Fc-optimized anti-CD40 agonist antibody 2141-V11 and IL-15 enhances anti-tumor memory responses. (A) Schematic of subcutaneous rechallenge (ten-fold tumor cell dose) of humanized hCD40/hFcyR mice surviving long-term (>90 days after initial orthotopic MB49 bladder tumor implantation) following initial therapy with anti-CD40 antibody 2141-V11 with or without IL-15. (B) Tumor growth (in the absence of any additional therapy) of long-term survivors compared with naïve mice receiving an equivalent MB49 tumor cell implant (n = 2–5 mice per group; bars represent SD). (C) Representative flow cytometry plots and quantification across mice (n = 2–5 mice per group) of CD44^hiCD122⁺ CD8 T cells (top), CD44^hiCD122⁺CD62L⁺ CD8 T cells (middle), and CD11b⁺KLRG1⁺CD27⁻ NK cells (bottom) in the peripheral blood of mice surviving long-term (day 110) after primary tumor implantation and tumor rechallenge. *p<0.05, **p<0.01, ****p<0.0001.