Background Cytokines, key orchestrators of anti-tumor immunity, are yet to fully realise their potential as cancer therapies largely due to difficulties with tumor penetration, clearance and resultant toxicity due to the high systemic doses required. Tight regulation by decoy receptors presents a further barrier for some cytokines. Live bacterial immunotherapies offer a solution with their intrinsic tumor homing ability, genetic programmability to yield sustained local delivery and potential synergy with intrinsic bacterial immune stimulation. Protein delivery by bacterial hosts however often leads to formation of inactive inclusion bodies. Prokarium’s Directed Evolution pipeline addresses this challenge by optimizing cytokine expression in live-attenuated Salmonella bacteria whilst preserving functionality.

Methods Directed evolution-generated candidate IL-15 and IL-18 variants were cloned into relevant plasmids and screened in a 3-stage process: 1) Selection of optimal signal peptides for secretion by measuring HiBit-tagged protein in growth media by luminescence; 2) Solubility assessment by tripartite β-lactamase antibiotic resistance assay; 3) Activity evaluation in reporter and primary immune cells with purified proteins. In vivo activity of candidate strains was determined by splenocyte flow cytometry analysis following intraperitoneal administration in mice.

Results We built a directed evolution pipeline for optimization of payload secretion, solubility and activity, and validated it with IL-15 and IL-18 cytokines. In silico evolved candidates were screened against a 56-member library to define the specific signal peptide for optimal secretion. High throughput solubility assessment identified unique amino acid mutations in IL-15 and IL-18 candidates that significantly enhanced solubility in bacteria. Multiple soluble candidates retained equivalent activity to wild-type human protein when tested on primary human and mouse NK cells and/or reporter cells. For IL-18, further protein engineering identified the minimal amino acid mutations required for decoy resistance, abolishing interaction with IL-18BP. Finally, we show that Salmonella strains delivering IL-15 variants enhance activation of NK cell and T cell responses in vivo.

Conclusions We developed a synthetic biology pipeline for engineering immunomodulatory payloads to be efficiently secreted from Salmonella whilst retaining high levels of activity. By integrating this pipeline into Prokarium’s Living Cures platform, we are designing a novel class of programmable bacterial immunotherapies poised to address the challenges associated with safely and effectively delivering immunotherapies to the tumor microenvironment.

Ethics Approval Animal studies were conducted under UK Home Office Animals (Scientific Procedures) Act 1986 project licence PP8366809.