Intravesical ALT-803 and BCG Treatment Reduces Tumor Burden in a Carcinogen Induced Bladder Cancer Rat Model; a Role for Cytokine Production and NK Cell Expansion

<div><p>Intravesical Bacillus Calmette-Guérin (BCG) has been shown to induce a specific immunologic response (<i>i.e.</i>, activation of IL-2 and effector T-cells), while preclinical studies using ALT-803 (mutated IL-15 analogue combined with IL-15Rα-Fc fusion) have shown promising results by prolonging the agent's half-life and stimulating CD8+ T-cells. Based on these results, we hypothesized that the intravesical administration of ALT-803 along with BCG will generate an immunologic response leading to significant bladder tumor burden reduction. Using a well-established carcinogen induced rat non-muscle invasive bladder cancer (NMIBC) model, we studied the effects of intravesical ALT-803 with and without BCG. Rat tissues were evaluated to document treatment response. Intravesical ALT-803 was safe and well tolerated alone and in combination with BCG. As a single treatment agent, ALT-803 reduced tumor burden by 35% compared to control whereas BCG alone only reduced tumor burden by 15%. However, the combination of ALT-803 plus BCG reduced tumor burden by 46% compared to control. Immune monitoring suggested that the antitumor response was linked to the production and secretion of IL-1α, IL-1β and RANTES, which in turn, induced the proliferation and activation of NK cells. Lastly, tumoral responses of the combinational treatment were associated with 76% reduction in angiogenesis, which is significantly higher than when assessed with either agent alone. The enhanced therapeutic index seen with this duplet provides justification for the development of this regimen for future clinical trials.</p></div

Study treatment schema.

<p>Treatment with PBS, BCG, ALT-803, or ALT-803 plus BCG commenced one week after completion of the eight weeks exposure to 0.05% N-butyl-N-(4- hydroxybutyl) nitrosamine in drinking water. Rats were randomly assigned into four groups and treated weekly for six consecutive weeks according to the schedule. Rats were sacrificed and necropsied two weeks later. CTRL, control.</p

Treatment-related changes in key lymphocytic cell tumoral infiltration.

<p>Tumoral infiltration of cells positive for CD3⁺, CD8a⁺, CD161 (NK cell), or CD163 (macrophage) (200×) were noted. No changes were noted in CD4⁺ T-cells (<i>data not shown</i>). ALT-803 alone increased tumoral CD8a⁺ expressing cells, whereas ALT-803 plus BCG increased tumoral expression of NK cells (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096705#pone.0096705.s001" target="_blank">Table S1</a>). <i>Insert</i>, left panel positive control from rat spleen and right panel negative control from rat spleen without primary antibody.</p

Treatment-related activation of peripheral immune cells in female rats with carcinogen induced NMIBC.

<p><b>A</b>) Peripheral blood mononuclear cells (PBMC) were isolated and analyzed by flow cytometry for expression of CD3⁺, CD4⁺, CD8a⁺ T-cells, NKG2D- positive (NK cells) and F480 expression cells (macrophages). ALT-803 alone, as well as, ALT- 803 plus BCG resulted in an increase in NKG2D-positive cells and a decrease in macrophages compared to PBS. <b>B</b>) Splenocytes were isolated and analyzed by flow cytometry for expression of CD3⁺, CD4⁺, CD8⁺, NKG2D-positive and F480 expression cells. Similar to the PBMC results, ALT-803 alone, as well as, ALT-803 plus BCG resulted in an increase in NKG2D- positive cells and a decrease in macrophages compared to PBS. *, <i>p</i><0.05, **, <i>p</i><0.01, ***, <i>p</i><0.001.</p

Changes in serum and urinary cytokine profiles following intravesical treatment.

<p><b>A</b>) Serum cytokines - Serum samples from female rats with carcinogen induced NMIBC was compared among the four treatment groups. Serum levels of a panel of cytokines were measured by Rat Inflammatory Cytokines Multi-Analyte ELISArray Kit (SABiosciences, Valencia CA). Mean ± SEM of each cytokine is reported for each treatment group. <b>B</b>) Urinary cytokines - Voided urine samples from female rats with carcinogen induced NMIBC was compared among the four treatment groups. Rat Inflammatory Cytokines Multi-Analyte ELISArray Kit measured urine levels of a panel of cytokines. Mean ± SEM of each cytokine is reported for each treatment group. *, <i>p</i><0.05, **, <i>p</i><0.01, ***, <i>p</i><0.001.</p

Treatment-related changes in key biomarkers in bladders of female rats with carcinogen induced NMIBC.

<p>Analysis of bladder angiogenesis (MVD), proliferation index (PI) and apoptosis index (AI) by IHC (200×) as described in the Material and Methods. ^*, <i>p</i>#, <i>p</i><0.05 compared to BCG, <b>^‡</b>, <i>p</i><0.05 compared to ALT-803.</p

Antitumor activity of BCG, ALT-803 and ALT-803 plus BCG.

<p>NMIBC were induced in female Sprague Dawley rats by the exposure to 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine in drinking water. At week 9, rats were randomized to one of four treatment groups (PBS, BCG, ALT-803, or ALT-803 plus BCG). Each group received weekly intravesical therapy for six consecutive weeks. On week 16, rats were sacrificed. <b>A</b>) Prior to bladder resection, 200 µl of sterile PBS was instilled into bladder and imaging of the bladder was performed with the Vevo 2100 Ultrasound System from VISUALSONICS (22–55 MHz probe). Thickness of the bladder wall was recorded in millimeters. Non-parametric Kraskal-Wallis test followed by post-hoc Dunnet's test was performed. Representative images from the four groups (<i>top left</i>) along with mean bladder wall thickness (<i>top right</i>) are illustrated. <b>B</b>) Resected bladders were fixed in 10% buffered formalin and embedded in paraffin and subsequently stained with hematoxylin and eosin. Detailed histopathologic examination of each bladder was performed to assess tumor burden. Representative images from the four groups are illustrated. The greatest reduction in bladder tumor burden was seen in ALT-803 and ALT-803 plus BCG. <b>C</b>) Bladder wall thickness was reduced in groups treated with ALT-803, BCG and ALT-803 plus BCG compared to control. Greatest reduction in bladder wall thickness was seen in the group treated with ALT-803 and ALT-803 plus BCG (<i>p</i> = 0.0001). <b>D</b>) Bladder weight was reduced in groups treated with BCG alone, ALT-803 alone and ALT-803 plus BCG compared to control. Greatest reduction in bladder weight was seen in the group treated with ALT-803 plus BCG. *, <i>p</i><0.05, **, <i>p</i><0.01, ***, <i>p</i><0.001.</p

Representative immunostaining showing examples of prostate tumors with (A) negative staining intensity, (B) weak staining intensity, and (C) moderate staining intensity.

<p>Representative immunostaining showing examples of prostate tumors with (A) negative staining intensity, (B) weak staining intensity, and (C) moderate staining intensity.</p

Association between FOXA1 staining level and biochemical recurrence following salvage radiation therapy.

<p>Association between FOXA1 staining level and biochemical recurrence following salvage radiation therapy.</p

Representative immunostaining showing examples of prostate tumors with (A) 0–25% staining percentage, (B) 26–50% staining percentage, (C) 51–75% staining percentage, and (D) 76–100% staining percentage.

<p>Representative immunostaining showing examples of prostate tumors with (A) 0–25% staining percentage, (B) 26–50% staining percentage, (C) 51–75% staining percentage, and (D) 76–100% staining percentage.</p