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Bee Hyaluronidase and PS2-armed VCN-12 is superior to VCN-01 in tumor cytotoxicity and immunogenicity Laura Moya Mar Tarinas Stan Peters Rafael Moreno Marcel Costa Silvia Torres Pau Fiol Josep M. Piulats Lu s Rojas Ana Mato Victoria Maliandi Paz Moreno Carlos L pez Sheila Connelly Manel Cascall Collaborators: Cristina Fillat Javier Garc a Castro Manuel Ram rez Orellana Judith Peris Barrios Juan Fueyo PID2020-119692RB-C21
Tumors HP-1 (pancreatic) Day -11 Day 1 Day 4 Virus administration (IT) Right tumor 2.5E10vp/animal Inoculation Right flank Inoculation Left flank Monitor tumor growth Same 2 animals eradicated initial treated tumor and would not establish new tumor VCN-12 improves survival and induces complete responses in hamsters with HP1 tumors VCN-12 induces long-lasting antitumor immune responses in responders * * Historical control Tumor rechallenge 2 cured hamsters Day 1 Rechallenge with HP-1 tumor cells Monitor tumor growth Day -43 Tumor disappearance Summary Parasporin 2 pore-forming toxin activated by furin linkers and secreted from plasmid-transfected or oncolytic adenovirus infected cells has cytotoxic and immunogenic cell death properties.
Tumors HP-1 (pancreatic) Day -11 Day 1 Day 4 Virus administration (IT) Right tumor 2.5E10vp/animal Inoculation Right flank Inoculation Left flank Treated tumors Non-treated tumors Monitor tumor volume VCN-12 shows more antitumor activity than VCN-01 in treated and contralateral tumors in an immunocompentent animal model # * *p-value<0.05 (VCN-12 vs PBS) #p-value<0.05 (VCN-12 vs VCN-01) Survival Rejections Syrian hamster Hamster sc.
PBS ICOVIR15K-PS2 vs ICOVIR15K body weight toxicity ****vs. PBS *vs. PBS #vs. ICO15K ICOVIR15K-PS2 vs ICOVIR15 antitumor efficacy VCN-12: Next Generation OV Based on VCN-01 LITR E2F boxes E1A- 24 MLP RITR L1 L2 L3 L4 L5 Fiber RGDK VCN-01: PH20 IIIaSA LITR RITR E2F boxes E1A- 24 MLP L1 L2 L3 L4 L5 Fiber RGDK E1B/19K E3/19K VCN-12: Bee Hyal Parasporin2 IIIaSA T2A VCN-12 modifications vs VCN-01: - Replace PH20 with Bee hyaluronidase greater specific activity - Insert the furin-modified pore-formin toxin Parasporin2 as a second transgene - Deletion of E1B-19K and E3-19K create space in viral genome PS2 activity in SN (CT26 cells): CT26 HepG2 Hyaluronidase and PS2 secreted from VCN-12-infected cells Hyaluronidase activity in SN (turbidometry) 2x Increased cytotoxicity of VCN-12 vs VCN01 in a panel of tumor cell lines SK-mel28 8x 3.4x 10x HT29 SW1116 3.5x SK-CO1 13.7x A549 HP1 6x CMT64-RG6-hCAR 6x HKT-1097 Human Hamster Mouse VCN-12 shows a similar toxicity profile to VCN-01 No differences between VCN-12 and VCN-01 in: - Biochemical, hematological and coagulation parameters - Liver pathology analysis Liver transaminases VCN-12 VCN-12 Body weight loss *p-value<0.05 (IIIa-BH-PS2 vs PBS) *p-value<0.05 (VCN-12 vs PBS) p-value<0.05 (VCN-01 vs PBS) #p-value<0.05 (VCN-12 vs VCN-01) # Syrian hamster Hamster sc.
(2017) Parasporin 2 pore formation requires several unknown receptors Created with BioRender.com PS2 may kill non-infected bystander cells and reduce viral immunodominance Created with BioRender.com Parasporin2 (PS2) with furin-mediated activation Furin-sensitive linkers C-term Inhibitory domain N-term Inhibitory domain CT26 Fritolysis Furin cut Furin cut Furin-cleavable linkers enhance cytotoxicity CT26 cells , MTS assay % Metabolic activity A549 CT26 PS2 induces membrane calreticulin (marker of ICD) CT26 (mouse colorectal adenocarcinoma) PS2 induces ATP release (marker of ICD) HepG2 (human hepatocellular carcinoma) A549 (human lung adenocarcinoma) RealTime-Glo extracellular ATP assay with SNs diluted : PS2 induces HMGB1 release (marker of ICD) CT26 (mouse colorectal adenocarcinoma) HepG2 (human hepatocellular carcinoma) A549 (human lung adenocarcinoma) Lumit HMGB1 immunoassay with SNs : PS2 induces fritolysis in multiple mouse tumor cell lines CMT64-RG6 (mouse lung adenocarcinoma) CT26 (mouse colorectal adenocarcinoma) MC38 (mouse colorectal adenocarcinoma) TRAMPC2 (mouse prostate adenocarcinoma) B16-CAR (mouse melanoma) Sensitive Resistant KPC (mouse pancreatic ductal adenocarcinoma) CT26 (mouse colorectal adenocarcinoma) MC38 (mouse colorectal adenocarcinoma) CMT64-RG6 (mouse lung adenocarcinoma) TRAMPC2 (mouse prostate adenocarcinoma) B16-CAR (mouse melanoma) Sensitive Resistant KPC (mouse pancreatic ductal adenocarcinoma) PS2 induces cytotoxicity in multiple mouse tumor cell lines CT26 mixed with 293GL CT26 mixed with 293GL-PS2 A549 mixed with 293GL-PS2 PS2 induces bystander fritolysis d0 1e+06 CMT64.6 d12 (~50-70 mm3) C57BL/6J 3 IT injections PS2FuT/Control SN d16 d19 Tumor volume (mm 3 ) SNs containig PS2 have antitumor activity PS2 confers ICD-mediated antitumor immunity Antitumor immune responses were detected in a PS2FuT-treated mouse Created with BioRender.com ICOVIR-15K-SA-PS2 SA pA Fiber RGDK E1a 24 MLP L1 L2 L3 L4 L5 E2F boxes PS2FuT N-term Inhibitory domain C-term Inhibitory domain Furin cut Furin cut Created with BioRender.com PS2-armed OAd intratumoral replication and gene expression upon iv admin *vs.
Cell Mol Immunol 18, 1106 1121 (2021) - Strong cytocidal activity against a broad range of human cancer cell lines - Minimal cytotoxicity toward normal hepatocytes and A549 cells - Inactive 37-kDa protoxin is processed to a 30-kDa active form by proteinase K - Exposure to the toxin causes striking morphological changes in susceptible cells, including swelling, blebbing, and lysis Rationale for pore-forming toxin parasporin-2 (PS2) From Akiba and Okumura, J Invertebr Pathol.