31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Salmonella Bacterial Monotherapy Reduces Autochthonous Prostate Tumor Burden in the TRAMP Mouse Model

<div><p>Attenuated <i>Salmonella typhimurium</i> injected in the circulatory system of mammals selectively targets tumors. Using weekly intraperitoneal injections of attenuated <i>Salmonella</i> strain CRC2631, we tested for regression and/or inhibition of tumor development in the TRAMP prostate tumor mouse model, which utilizes SV40 early region expression for autochthonous formation of prostate tumors that progress into metastatic, poorly differentiated prostatic carcinomas in an immunocompetent murine model. Thirteen weekly intraperitoneal administrations of 10⁵–10⁷ CFU CRC2631 into 10 week old mice were well tolerated by the TRAMP model. Sacrifice and histological analysis of TRAMP prostates at 22 weeks indicated that <i>Salmonella</i> monotherapy at administrated levels decrease visible tumor size (>29%) but did not significantly inhibit previously described SV40 expression-driven TRAMP tumor progression to undifferentiated carcinomas when histologically examined. In conclusion, this work demonstrates baseline results for CRC2631 <i>Salmonella</i> monotherapy using the immunocompetent TRAMP prostate tumor model in preparation for study of combination therapies that resolve autochthonously generated TRAMP prostate tumors, further reduce tumor size, or inhibit prostate tumor progression.</p></div

Neuroendocrine-type carcinoma in the periurethral region of a TRAMP mouse.

<p>Histologic sections of the periurethral region from a transgenic mouse stained with hematoxylin and eosin (H&E) at 4X (A), 10X (B), 40X (C) and 100X (D). (A): Note discrete tumor (arrow) within the epithelium of the periurethral region. The outline in (A) is the magnified region shown in (C) and (D).</p

Effect of weekly Salmonella dosages on prostate tumor development in the 5 month TRAMP prostate.

<p>Effect of weekly Salmonella dosages on prostate tumor development in the 5 month TRAMP prostate.</p

Bacterial Strains and Animal Models.

<p>Bacterial Strains and Animal Models.</p

Histology of prostate tumor development in TRAMP mice.

<p>Histologic sections of the dorsal lobes of the prostate from transgenic mice stained with hematoxylin and eosin at 40X magnification. <b>Pathologic grades</b>: PIN, prostatic epithelial neoplasia; WD, well-differentiated adenocarcinoma; PHY, phylloides-like; PDC, poorly differentiated neuroendocrine-type carcinoma. <b>Slides</b>: (A) Normal tissue, (B) Hyperplastic tissue, (C) PIN, (D) WD, (E) PHY and (F) PDC (neuroendocrine-type). <b>Observations</b>: (C) Note tufting of epithelial cells, increased mitoses, hyperchromatic nuclei, stratification of nuclei and cribiform structures (arrow). (D) Note neoplastic cells with round nuclei; tumor type is characterized by increased numbers of small glands and thickening of the stroma. (E) Note staghorn luminal patterns of neoplastic cells. (F) Note the high nuclear:cytoplasmic ratio of neoplastic cells, loss of glandular differentiation and marked cell pleomorphism.</p

Tumor volume decreases with increasing <i>Salmonella</i> dosage.

<p>Caliper measurement of prostate-associated tumor volumes extracted from TRAMP mouse groups at end of study with visible excess tumor growth. Mean tumor volumes: PBS control (n = 1/16 with visible excess growth) 9.57 cm³, 10⁵ injection group (n = 5/17 with visible excess growth) 7.45 cm³, 10⁶ injection group (n = 5/17 with visible excess growth) 6.43 cm³, 10⁷ injection group (n = 2/13 with visible excess growth) 5.94 cm³.</p

Weekly <i>Salmonella</i> injections tolerated by TRAMP mouse model.

<p>Survival curve of TRAMP mouse groups (n = 20) aged 10–22 weeks (98 days) during study period with weekly IP injection of 100μl PBS (control) or 100μl PBS with <i>Salmonella</i> at indicated concentrations. Surviving mice were sacrificed at the end of week 22.</p