Wyatt Paulishak, PhD: No financial relationships to disclose
Introduction/Rationale: Lyophilization (freeze-drying) is a common method of preparing bacteria for storage at higher temperatures with lyoprotectant and buffer selection greatly impacting viability. Listeria monocytogenes (LM) is a Gram-positive intracellular bacterium being investigated preclinically and clinically as an anticancer vaccine platform and is stored at ultra-low temperatures (ULT). ULT storage greatly limits LM therapy accessibility globally due to a lack of necessary storage equipment in many areas of the world.
Methods: Here, we investigated preparations of lyophilized (freeze-dried) LM for stability at cold-chain viable temperatures while maintaining the required infectivity for anticancer effect. Using a murine subcutaneous renal cell carcinoma model we further compared the antitumor efficacy of non-lyophilized versus lyophilized LM LLO-CD105A vaccine and the effects of lyophilization on immune recruitment to the tumor microenvironment.
Results: Through development of lyophilization media, we found that the choice of lyoprotectant and buffer dramatically impacts LM viability following lyophilization and during storage. Of those tested, the HEPES 2.5% sucrose medium (3mM HEPES 2.5% w/v sucrose) demonstrated the best viability while maintaining the infectivity and antitumor efficacy of LM LLO-CD105A vaccine in a subcutaneous renal cell carcinoma model. Lyophilized LM demonstrated near identical efficacy to non-lyophilized LM but resulted in higher myeloid-derived suppressor cell infiltration into the tumor microenvironment.
Conclusion: The methods described herein are easily adoptable in both laboratory and industry settings for the preparation of LM and stable storage at non-ultra-low temperatures. These methods address a critical need for translation of LM cancer therapies for global distribution and enable further development of novel LM therapeutic approaches.
