A practical approach to pancreatic cancer immunotherapy using resected tumor lysate vaccines processed to express α-gal epitopes

<div><p>Objectives</p><p>Single-agent immunotherapy is ineffective against poorly immunogenic cancers, including pancreatic ductal adenocarcinoma (PDAC). The aims of this study were to demonstrate the feasibility of production of novel autologous tumor lysate vaccines from resected PDAC tumors, and verify vaccine safety and efficacy.</p><p>Methods</p><p>Fresh surgically resected tumors obtained from human patients were processed to enzymatically synthesize α-gal epitopes on the carbohydrate chains of membrane glycoproteins. Processed membranes were analyzed for the expression of α-gal epitopes and the binding of anti-Gal, and vaccine efficacy was assessed <i>in vitro</i> and <i>in vivo</i>.</p><p>Results</p><p>Effective synthesis of α-gal epitopes was demonstrated after processing of PDAC tumor lysates from 10 different patients, and tumor lysates readily bound an anti-Gal monoclonal antibody. α-gal(+) PDAC tumor lysate vaccines elicited strong antibody production against multiple tumor-associated antigens and activated multiple tumor-specific T cells. The lysate vaccines stimulated a robust immune response in animal models, resulting in tumor suppression and a significant improvement in survival without any adverse events.</p><p>Conclusions</p><p>Our data suggest that α-gal(+) PDAC tumor lysate vaccination may be a practical and effective new immunotherapeutic approach for treating pancreatic cancer.</p></div

B cell and T cell expansion in response to tumor lysate vaccination.

<p>ELISPOT assays for (a) anti-MUC1 Ab secreting B cells and (b) MUC1-specific activated T cells and <i>in vitro</i> and <i>in vivo</i> depletion of CD8⁺ T cells, detected as IFN-γ secreting lymphocytes. ELISPOT assays for (c) anti-mesothelin Ab secreting B cells and (d) mesothelin-specific activated T cells and <i>in vitro</i> and <i>in vivo</i> depletion of CD8⁺ T cells, detected as IFN-γ secreting lymphocytes. Data represent the mean ± SD of five independent splenocyte preparations; bars, SD. Statistical analyses were performed using Student’s <i>t</i>-test. N.S.: not significant.</p

Immunohistological findings of original PDAC tumors obtained from patients treated with or without neoadjuvant chemoradiotherapy.

<p>H&E stained sections of PDAC tumors clearly demonstrate viable PDAC cells in the tumor treated without NACRT. However, grade IIa destruction of PDAC cells (Evans classification) was detected in the tumor treated with NACRT. Expression of MUC1 and mesothelin was observed in PDAC tumors treated with or without NACRT. The expression levels of these TAAs were similar between PDAC tumors treated with or without NACRT. Representative images of four individual patients are shown. Scale bars = 100 μm.</p

Anti-PDAC cell IgG subclasses production induced by tumor lysate vaccination.

<p>(a) Induced anti-PANC-1 IgG subclasses. (b) Induced anti-MIAPaCa-2 IgG subclasses. (c) Induced anti-BxPC-3 IgG subclasses. Representative data from five experiments with similar results are shown. ELISA results represent one data set from a group of 10 vaccinated mice.</p

Anti-MUC1 IgG and anti-mesothelin IgG production induced by tumor lysate vaccination.

<p>(a) ELISA for anti-MUC1 IgG production. (b) ELISA for anti-mesothelin IgG production. Representative data from ten experiments with similar results are shown. ELISA results represent one or three data sets from n = 10 mice/group (one data set: naïve KO mice, α-gal(-) or α-gal(+) N-ly; three data sets: α-gal(-) or α-gal(+) PDAC-ly).</p

ELISA analysis of anti-PDAC cell IgG production induced by tumor lysate vaccination.

<p>(a) Anti-PANC-1 IgG production, (b) anti-MIAPaCa-2 IgG production, (c) anti-BxPC-3 IgG production. Vaccination with α-gal(+) PDAC-ly resulted in marked increase in the production of anti-PDAC cell IgG compared with α-gal(-) PDAC-ly vaccination. Vaccinations with either α-gal(-) or α-gal(+) N-ly did not elicit a significant anti-PDAC cell IgG response. Representative data from ten experiments with similar results are shown. ELISA results represent one or three data sets from n = 10 mice/group (one data set: naïve KO mice, α-gal(-) or α-gal(+) N-ly; three data sets: α-gal(-) or α-gal(+) PDAC-ly).</p

<i>In vivo</i> tumor growth and survival of adoptive transferred NOD/SCID mice challenged with live PANC-1 cells.

<p>(a) Size of subcutaneous tumors of NOD/SCID mice to which splenocytes were adoptively transferred from normal pancreatic tissue lysate vaccinated KO mice. □: α-gal(+) N-ly vaccinated KO mice, △: α-gal(-) N-ly vaccinated KO mice. The tumor sizes of five individual recipients in each group after adoptive transfer are shown. (b) Size of subcutaneous tumors of NOD/SCID mice to which splenocytes were adoptively transferred from PDAC tumor lysate vaccinated KO mice. ●: α-gal(+) PDAC-ly vaccinated KO mice, ○: α-gal(-) PDAC-ly vaccinated KO mice. The tumor sizes of five individual recipients in each group after adoptive transfer are shown. S: sacrifice, †: cancer death. (c) Survival curves of adoptive transferred NOD/SCID mice after tumor cell challenge with live PANC-1 cells. The curves were generated by the Kaplan-Meier method and assessed by the log-rank test.</p