Background Tigilanol Tiglate (TT) is a novel small molecule under development for local treatment of solid tumours via intratumoral (I.T.) injection. TT is a protein kinase C (PKC)/C1 domain activator that disrupts tumour vasculature, leading to haemorrhagic necrosis of the lesion.¹ Strikingly, in both preclinical syngeneic mouse models and cutaneous/subcutaneous tumours presenting in the veterinary clinic, I.T. injection of TT results in complete and enduring ablation of target tumours in >70% of patients.^1,2,3 TT has completed a Phase I/IIa dose-escalation trial in humans (ACTRN12614000685617), with strong evidence of local anti-cancer efficacy and signs of abscopal effects in some patients.⁴ However, the underlying mechanism of action (MOA) of TT, together with its immunotherapeutic potential in oncology, is not fully understood.

Methods A combination of microscopy, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, LDH release assays and mixed lymphocyte reactions were used to probe the MOA of TT in vitro. TT-mediated damage associated molecular pattern (DAMP) release/externalization was assessed using luciferase (ATP), ELISA (HMGB1), flow cytometry and immunohistochemical (calreticulin) approaches. In vivo experimentation with TT utilized CT-26 and B16-F10-OVA tumor bearing mice, with or without anti-PD1/anti-CTLA4 treatment.

Results Our data demonstrates that therapeutic concentrations of TT induce death of cancer and endothelial cell lines, both in vitro and in vivo, via oncosis. Whilst largely PKC-independent, PKC/C1 domain signaling appears necessary for timely oncolysis in vitro and efficacious tumor ablation in vivo. Our results also show that TT binds to ER membranes, causing ER stress with subsequent activation of the integrated stress response. This is followed by mitochondrial membrane potential loss, ATP depletion, organelle swelling, oncosis and terminal necrosis. We also found that TT treatment promoted the release/externalization of DAMPs (HMGB1, ATP, calreticulin) from cancer cells in vitro and in vivo, characteristics indicative of immunogenic cell death (ICD). Confirmation of ICD in vivo was obtained through rechallenge experiments using CT-26 tumour bearing mice, which also demonstrated that TT promoted the development of tumour-specific T cells. In addition to stimulating immune cell infiltration into tumours, TT significantly improved treatment response in the B16-F10-OVA mouse melanoma model when combined with immune checkpoint blockade.

Conclusions These data indicate that TT is an oncolytic small molecule with the potential to enhance responses to immunotherapy. TT is currently undergoing Phase I/II trials in head and neck cancers (ACTRN12619001407189), soft tissue sarcomas, Stage III melanoma in-transit (NCT05234437) and non-resectable Stage IIIB to IV M1c melanoma (TT/pembrolizumab combination: NCT04834973).⁵

References

1. Boyle GM, D’Souza MMA, Pierce CJ, Adams RA, Cantor AS, Johns JP, Maslovskaya L, Gordon VA, Reddell PW, Parsons PG. Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models. PLOS ONE 2014;9:e108887.

2. Cullen JK, Boyle GM, Yap PY, Elmlinger S, Simmons JL, Broit N, Johns J, Ferguson B, Maslovskaya LA, Savchenko AI, Mirzayans PM, Porzelle A, Bernhardt PV, Gordon VA, Reddell PW, Pagani A, Appendino G, Parsons PG, Williams CM. Activation of PKC supports the anticancer activity of tigilanol tiglate and related epoxytiglianes. Sci Rep 2021;11:207.

3. De Ridder TR, Campbell JE, Burke-Schwarz C, Clegg D, Elliot EL, Geller S, Kozak W, Pittenger ST, Pruitt JB, Riehl J, White J, Wiest ML, Johannes CM, Morton J, Jones PD, Schmidt PF, Gordon VA, Reddell PW. Randomized controlled clinical study evaluating the efficacy and safety of intratumoral treatment of canine mast cell tumors with tigilanol tiglate (EBC-46). J Vet Intern Med 2021;35:415–429.

4. Panizza BJ, de Souza P, Cooper A, Roohullah A, Karapetis CS, Lickliter JD. Phase I dose-escalation study to determine the safety, tolerability, preliminary efficacy and pharmacokinetics of an intratumoral injection of tigilanol tiglate (EBC-46). EbioMedicine 2019;50:433–441.

5. QBiotics Group Ltd. website. https://qbiotics.com/

Ethics Approval All animal procedures were approved in accordance with NHMRC guidelines (Australian Code for the Care and Use of Animals for Scientific Purposes 8th Edition, 2013; National Health and Medical Research Council of Australia) by the QIMR Berghofer Animal Ethics Committee: A0106-042M, A0404-606M and A01047M.