Purification and characterization of checkpoint blockade enhancing molecules from Coprobacillus cateniformis.

Abstract

Checkpoint blockade treatment enhancement is one of the frontline areas of study in cancer treatment, however it comes with two large drawbacks, the limited number of cancer lines in which efficacy is shown and the difficulty in tolerating the treatment. An increase in the efficacy of checkpoint blockade treatment has been shown to correlate to the presence of individual members of the human gut microbiota and the microbial contributions to immune training and regulation within the host. Coprobacillus cateniformis has been shown to enhance anti-PD-L1 and anti-PD-L2 treatments in a mouse tumor model, even when cotreated as a dead bacteria indicating the presence of an immunomodulatory molecule on the surface of the bacteria. I compared three chemical extraction techniques to determine the most efficient and effective method for molecular isolation. After isolation, I used low pressure size exclusion chromatography paired with a second ion exchange chromatography method to remove superfluous bacterial extract products. Further characterization of the bacterial product was performed through chemical analysis via gas chromatography mass spectroscopy, enzymatic degradation, and NMR spectroscopy. The sum total of the chemical analysis led to the characterization of a small surface associated bacterial lipid that may be attached to a xylose polysaccharide that we have shown increases the efficacy of anti-PD-L1 and anti PD-L2 treatments in our mouse tumor models.