Abstract 1459: Construction and assessment of a novel Listeria monocytogenes-based vaccine targeting squamous cell carcinoma
Authors: Hessin Alyaa F. , Freitag Nancy E. 
Published: August 2019

Introduction: Immunotherapy represents a potential therapeutic option for many tumors including squamous cell carcinoma (SCC). L. monocytogenes is a potent delivery vehicle that can elicit cellular immunity specific for the encoded antigens, and overcome the immunosuppressive cancer environment. Here we test the hypothesis that L. monocytogenes strain lacking 2 secreted chaperones (ΔprsA2 ΔhtrA) would be able to induce a specific immune response against SCC and would represent a safe vaccine due to its hyper-attenuation.

Methods: Recombinant L. monocytogenes ΔprsA2 ΔhtrA strains were engineered to express Cyp2e1 squamous carcinoma antigen using standard molecular techniques. All genes were verified by DNA sequencing, and secreted proteins were assessed by western blot. Bacterial growth assays were tested in broth and intracellularly in J774 mouse macrophage cells. The safety of the vaccine strains were tested in vivo by counting the CFUs in target organs and measuring the liver function tests in serum. The efficacy was then assessed in a subcutaneous mouse model of tumor growth together with L. monocytogenes ΔactA prfA* strain and ΔprsA2 ΔhtrA strain with no tumor antigen. Mice were first challenged with KLN205 tumor cells, and then vaccinated with 3 immunizations weekly. The tumors volumes were recorded with a caliper every 3 days and organs weights were measured at the end of the experiment.

Results: The Cyp2e1 antigen was detectable in L. monocytogenes supernatant fractions, and the secretion of tumor antigens did not impact the growth of mutant strains in broth culture. Bacterial replication within infected macrophage cells showed defective intracellular growth for L. monocytogenes ΔprsA ΔhtrA expressing tumor antigen while L. monocytogenes ΔactA prfA strains exhibited modest levels of inhibition in comparison to wild type. Mice infected with ΔprsA2 ΔhtrA strains had no CFUs recovered from liver or spleen while half of the mice infected with ΔactA prfA strains exhibited bacterial burdens within the liver and no bacteria were recovered from the spleen. Consistently, all the infected mice exhibited normal liver function as measured by AST and ALT levels. The results confirm the safety of ΔprsA2 ΔhtrA strains in vivo. Additionally, ΔprsA2 ΔhtrA vaccine strains significantly halted the SCC growth when compared to the control mice. Preliminary data suggests that tumor antigen-specific differences in tumor size and spleens weights could be visualized between the groups of mice immunized with L.monocytogenes recombinant strains expressing Cyp2e1 antigen versus no antigen. These data suggest that the highly attenuated L. monocytogenes ΔprsA2 ΔhtrA strains may indeed be efficient in stimulating SCC regression.

Conclusion: L. monocytogenes ΔprsA2 ΔhtrA strains represent a novel and safe vaccine that altered the immunosuppressive microenvironment of SCC and inhibited tumor growth.

Citation Format: Alyaa F. Hessin, Nancy E. Freitag. Construction and assessment of a novel Listeria monocytogenes-based vaccine targeting squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1459.