Val-boroPro accelerates T cell priming via modulation of dendritic cell trafficking resulting in complete regression of established murine tumors.

Although tumors naturally prime adaptive immune responses, tolerance may limit the capacity to control progression and can compromise effectiveness of immune-based therapies for cancer. Post-proline cleaving enzymes (PPCE) modulate protein function through N-terminal dipeptide cleavage and inhibition of these enzymes has been shown to have anti-tumor activity. We investigated the mechanism by which Val-boroPro, a boronic dipeptide that inhibits post-proline cleaving enzymes, mediates tumor regression and tested whether this agent could serve as a novel immune adjuvant to dendritic cell vaccines in two different murine syngeneic murine tumors. In mice challenged with MB49, which expresses the HY antigen complex, T cell responses primed by the tumor with and without Val-boroPro were measured using interferon gamma ELISPOT. Antibody depletion and gene-deficient mice were used to establish the immune cell subsets required for tumor regression. We demonstrate that Val-boroPro mediates tumor eradication by accelerating the expansion of tumor-specific T cells. Interestingly, T cells primed by tumor during Val-boroPro treatment demonstrate increased capacity to reject tumors following adoptive transfer without further treatment of the recipient. Val-boroPro -mediated tumor regression requires dendritic cells and is associated with enhanced trafficking of dendritic cells to tumor draining lymph nodes. Finally, dendritic cell vaccination combined with Val-boroPro treatment results in complete regression of established tumors. Our findings demonstrate that Val-boroPro has antitumor activity and a novel mechanism of action that involves more robust DC trafficking with earlier priming of T cells. Finally, we show that Val-boroPro has potent adjuvant properties resulting in an effective therapeutic vaccine

Val-boroPro induces complete regression in multiple tumor models and requires inhibition of intracellular PPCE.

<p>(A) C57BL/6 female mice were inoculated with 1×10⁶ MB49 on day 0. Mice were treated with saline (open squares) or 20 µg Val-boroPro 5×/week during weeks 1 through 4 (days 3–28, closed squares), week 1 only (days 3–7, open triangles), or weeks 2 through 4 (days 10–28, inverted solid triangles) (n = 6/group). All treatment groups are statistically different than saline (Anova, p<0.05 for late treament vs saline and p<0.01 for early treatment groups vs saline). The days 10–28 group is also statistically different than both early treatment groups (p<0.05). (B) C57BL/6 mice were inoculated with 1×10⁶ M3-9-M on day 0 and treated with 20 ug Val-boroPro (dashed line) or saline (solid line) for four weeks (n = 5/group). Tumor volumes are statistically different between the Val-boroPro and saline groups at p<0.001 for all times beyond day 10. (C) Mice were inoculated with MB49 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g001" target="_blank">Figure 1A</a> and treated with 20 ug Val-boroPro (open squares), 20 ug PT-630 (open triangles), or saline (closed squares) (n = 5/group). (D) Mice were inoculated with M3-9-M as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g001" target="_blank">Figure 1B</a> and treated with PT-630 (open triangles) or saline closed squares) (n = 5/group). Mean tumor volumes show that PT-630 treatment was no different from saline. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g001" target="_blank">Figures 1A–D</a> are representative of 3 or more experiments.</p

Val-boroPro is an effective adjuvant to tumor primed dendritic cell vaccination.

<p>(A) C57BL/6 female mice were challenged with MB49 (1×10⁶) on day 0 and treated with 20 µg Val-boroPro or saline from days 3–7 (closed squares) or 10–14 (n = 5/group). On day 12, late-treatment groups received either a male-derived HY-expressing DC vaccine or a female-derived DC vaccine IP (1×10⁵). Tumors were significantly smaller in mice receiving male DC vaccine plus Val-boroPro during week 2 (open circles) compared to no treatment (open squares), male DC alone (closed triangles) or female DC plus Vale-boroPro during week 2 (closed circles) (p<0.01, Anova with Tukey post-test) (B) C57BL/6 mice were injected intramuscularly with M3-9-M and treated with Val-boroPro starting on day 10. Bone marrow-derived DCs pulsed with irradiated M3-9-M were given IP on day 12 (n = 8/group). Tumors were significantly smaller in mice receiving combined treatment (open triangles) compared to no treatment (closed squares) or either treatment alone (open squares or closed triangles) (p<0.01, Anova). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g007" target="_blank">Figures 7A and 7B</a> are both representative of 2 experiments.</p

Val-boroPro treatment is associated with an acceleration of tumor-induced priming.

<p>(A) Female mice were inoculated with MB49 and treated with Val-boroPro (closed bars) or saline (open bars) (n = 12/group). Spleens and lymph nodes were harvested from mice on days 10, 17, and 24 for IFN-gamma ELISPOT analysis (*p<0.05, **p<0.01, ***p<0.001, Mann-Whitney). (B) Mice were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g003" target="_blank">Figure 3A</a>. On day 17, greater numbers of HY-reactive CD8⁺ T cells were observed in the lymph nodes of saline-treated (open bars) mice compared to Val-boroPro treated (closed bars) mice (*p<0.05, **p<0.01, Mann-Whitney, n = 5/group). (C) Composite data showing combined (UTY+SMCY+DBY) relative to tumor volume from mice treated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g001" target="_blank">figure 1A</a> with saline (open squares top, open bars bottom) or Val-boroPro (closed squares top, closed bars bottom) (n = 12/group). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g003" target="_blank">Figures 3A–C</a> are representative of 3 experiments.</p

Antitumor activity with Val-boroPro is associated with increased trafficking of DCs.

<p>(A–B) B6CD45.1 congenic mice were treated with 20 µg Val-boroPro for three days, and CD11c⁺ cells from spleens and LNs were magnetic bead purified and injected at a dose of 5×10⁶ into lateral tarsals of C57BL/6 (CD45.2⁺) recipients. Immediately following CD11c⁺ cell injection, recipients were treated with Val-boroPro or saline (n = 3/group). Popliteal lymph nodes were harvested 15 hours following Val-boroPro treatment for flow cytometric analysis. (A) Representative dot plots. (B) Bar graph showing statistically more CD45.1⁺ adoptive transferred DCs in the popliteal LN in recipients of DCs from Val-boroPro treated donors also receiving a single injection of Val-boroPro compared to all other group (*p<0.05, **p<0.01, Mann-Whitney). (C) Purified CD11c⁺ cells from Val-boroPro treated GFP⁺ mice were injected intratumorally into established MB49 tumors. Mice were treated immediately thereafter with one dose of Val-boroPro (n = 3/group). Tumor-draining inguinal lymph nodes were harvested 15 hours later and imaged by fluorescent microscopy. Representative lymph nodes from mice receiving CD11c⁺ cells from Val-boroPro treated donors and Val-boroPro post injection. Experiments displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g008" target="_blank">Figures 8A–C</a> were conducted 2 times.</p

Val-boroPro mediated tumor regression is T cell dependent.

<p>(A) Female C57BL/6 mice were challenged on day 0 with 1×10⁶ MB49 and treated with 20 µg Val-boroPro during weeks 1 through 4 (5× per week). Mice that were treated with 20 µg Val-boroPro and subsequently rejected MB49 were then rechallenged with MB49 (1×10⁶) or 76-9 rhabdomyosarcoma (5×10⁵) on day 56 post-primary challenge (n = 5/group). Saline control, initial treatment (closed squares), naïve mice (that did not previously reject tumors) injected with MB49 (closed triangles) or 76-9 (closed diamonds) at time of rechallenge, mice that previously rejected MB49 during Val-boroPro and rechallenged with MB49 (open circles) or 76-9 (inverted open triangles). (B) Mice were inoculated with MB49 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g001" target="_blank">Figure 1A</a> and treated with Val-boroPro or saline (closed squares) (n = 5/group). CD4⁺ and/or CD8⁺ T cells were depleted with anti-CD4 and/or anti-CD8 monoclonal antibodies as outlined in methods. Tumor volumes in Val-boroPro treated mice without T cell depletion (closed squares) were significantly smaller than either CD4 (closed triangles) or CD8 (inverted open triangles) depleted groups (p<0.05) and CD4/CD8 depleted group (closed circles) (p<0.001) Open circles represent anti-CD4/anti-CD8 without Val-boroPro treatment. (C) Male (closed circles) and female (closed squares) mice were challenged subcutaneously with 10⁶ HY-expressing tumor MB49 on day 0 and were treated with 20 µg Val-boroPro 5×/week for two weeks (n = 5/group). P<0.01 for all timepoints beyond day 15. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g002" target="_blank">Figures 2A–C</a> are representative of 3 experiments.</p

Myeloid dendritic cells (CD11b⁺CD11c⁺) are increased in TDLN and are required for tumor regression.

<p>(A) C57BL/6 mice were challenged with 1×10⁶ MB49 on day 0 and treated with Val-boroPro (closed bars) or saline (open bars) during week one (n = 4/group). Tumor-draining lymph nodes were harvested and analyzed by flow cytometry on day 7 (p<0.05, Mann-Whitney) (B) C57BL/6 mice were inoculated with 10⁶ MB49 and injected with clodronate IP (0.1 mL/10 g body weight) every other day from day –1 to 9 following tumor inoculation. Control groups were injected with empty lysosomes or saline (sham). Val-boroPro was administered orally for 1 week (days 3–7). Survival was significantly different in Val-boroPro treated mice given clodronate (thick solid line) compared to those treated with Val-boro-Pro and given empty lysosomes (gray solid line) (*p<0.05, Logrank test, n = 5/group). (C) CD11c-diphtheria toxin (DT) chimeric mice were generated by transplanting bone marrow from CD11c-DT transgenic mice into lethally irradiated C57BL/6 recipients (n = 5/group). Female chimeras were inoculated with 10⁶ MB49 on day 0 and treated with Val-boroPro or saline during week one (days 3–7) with or without IP injections of DT (8 ng/1 g body weight) every other day from day –1 to 9. Tumor volumes were significantly larger in Val-boroPro treated chimeric mice receiving DT (closed circles) compared to Val-boro-Pro treated chimeric mice receiving saline (closed squares) (p<0.001 for all timepoints beyond day 10). Tumor volumes were not statistically different between saline treated (open squares) and DT treated (open circles) chimerics not treated with Val-boroPro. Experiments displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g007" target="_blank">Figures 7A–C</a> were each conducted twice.</p

Treatment with Val-boroPro alters lymphocyte and myeloid cell population in secondary lymphoid tissues.

<p>C57BL/6 mice were treated with 20 µg Val-boroPro (solid bars) or saline (open bars) 5×/week for four weeks, and spleens (n = 15/group) and lymph nodes (n = 8/group) were harvested and analyzed by flow cytometry. <b>(</b>*p<0.05, **p<0.01, ***p<0.001 and reflect comparisons between Val-boroPro treated and Saline treated groups by Mann-Whitney). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058860#pone-0058860-g006" target="_blank">Figure 6</a> was conducted twice.</p