AACR 2025_Poster_250407 v3

INTRODUCTION

• Hepatoblastoma (HB) and pediatric hepatocellular carcinoma (HCC) are aggressive childhood liver cancers with limited treatment options and poor survival outcomes.

• Oncolytic virus therapies (OVTs) have shown promise in cancer treatment, though efficacy and selectivity challenges remain.

• Humane Genomics has developed a synthetic virology platform enabling the advanced engineering of vesicular stomatitis virus (VSV)-based therapies. The resulting therapy shows robust therapeutic efficacy and selectivity using innovative two-factor authentication (2FA) strategy combining engineered glycoproteins for cancer-specific infection and protein-responsive aptazymes for selective replication.

• HGI627 is our lead candidate for liver cancer featuring a synthetic glycoprotein retargeted to glypican-3 (GPC3) and a beta-catenin (CTNNB1)-dependent aptazyme as a genetic ON switch.

GOALS

• Validate the target specificity of HGI627 in vitro using cancer versus normal cell lines. • Assess therapeutic efficacy of HGI627 in vivo in a Hep3B hepatocellular carcinoma model. • Evaluate safety profile and potential maximum tolerated dose (MTD) of HGI627 in vivo.

METHODS

VIRUSES

• HGI627 is a vesicular stomatitis Indiana virus (VSIV) engineered using our platform for targeted treatment of liver cancer. Key modifications include deletion of the native VS/V-G glycoprotein, replacement with detargeted, engineered glycoprotein, incorporation of a synthetic anti-GPC3 nanobody for tumor-specific retargeting, and integration of a cancer protein–responsive aptazyme that acts as a genetic ON switch to restrict replication to tumor cells.

IN VITRO

• HGI627 was constructed and rescued using a synthetic virology platform, and viral titers were measured by TCD50 assay on Hep3B cells and calculated using Spearman-Karber. • Endpoint dilution assays were performed and immunocytochemistry (ICC) staining was carried out using anti-VSV to evaluate selectivity. • Cytotoxicity and selectivity were evaluated using real-time imaging on liver cancer (e.g. Hep3B, HepG2) and off-target/normal cells.

IN VIVO

• Efficacy of HGI627 was assessed in NSG mice bearing subcutaneous Hep3B hepatocellular carcinoma xenograft. A single intraperitoneal (IP) dose of 10^10 TCID50 was administered when average tumor volume reached 80-100mm³. • Histology was performed by HistoWiz using a Standard Operation Procedure and fully automated workflow. Staining with anti-mitochondria was performed to differentiate human Hep3B xenograft from mouse cells. • Acute toxicity and maximum tolerated dose (MTD) were evaluated in NSG mice following single and multiple IP doses of 10^10 TCID50 to assess safety and tolerability.

RESULTS

IN VITRO

• HGI627 demonstrated robust lytic activity in vitro in GPC3+/CTNNB1+ cells with minimal to no cytotoxicity in off-target and normal cells.

IN VIVO

• In vivo, a single intraperitoneal injection of 1×10^10 TCID50 HGI627 achieved a 100% complete response and significantly extended survival to day 49 compared to median survival of 15 days in vehicle group. • In the safety study, even multiple high-dose administrations of HGI627 were well tolerated, and no signs of clinical toxicity were observed up to 28 days; importantly, this highest dosing group received an equivalent of 300-fold more than the proposed final first-in-human dose.

FIGURE 1. SELECTIVE LYSIS OF GPC3+/CTNNB1+ LIVER CANCER CELLS

IN VIVO

FIGURE 2. EFFICACY STUDY OF HGI627 IN SUBCUTANEOUS HEP3B CDX MODEL

FIGURE 3. SURVIVAL AND TUMOR BURDEN ANALYSIS OF HGI627

• HGI627 treatment achieved 100% survival through Day 49, compared to a median survival of 15 days in the vehicle group. • Vehicle-treated tumors progressed rapidly to endpoint, while HGI627-treated tumors peaked in volume shortly post-treatment and then exhibited a durable, complete response through Day 49.

REFERENCES

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[2] Czauderna P, Mackinlay G, Perilongo G, Brown J, Shafford E, Aronson D, Pritchard J, Chapchap P, Keeling J, Plaschkes J, Otte JB; Liver Tumors Study Group of the International Society of Pediatric Oncology. Hepatocellular carcinoma in children: results of the first prospective study of the International Society of Pediatric Oncology group. J Clin Oncol. 2002 Jun 15;20(12):2798-804. PMID: 12065556.

[3] Basu R, Moles CM. Rational selection of an ideal oncolytic virus to address current limitations in clinical translations. Int Rev Cell Mol Biol. 2023;379:241-261. Pub 2023 Apr 1. PMID: 27541726.

[4] Moles CM, Basu R, Weijmarshausen P, Ho B, Farhat M, Flaaat T, Smith BF. Leveraging Synthetic Virology for the Rapid Engineering of Vesicular Stomatitis Virus. Viruses. 2024 Oct 21;16(10):1641. PMID: 39459973.