An AutologousIn SituTumor Vaccination Approach for Hepatocellular Carcinoma. 1. Flt3 Ligand Gene Transfer Increases Antitumor Effects of a Radio-Inducible Suicide Gene Therapy in an Ectopic Tumor Model

Abstract

Hepatocellular carcinoma (HCC) often presents as a diffuse or multifocal tumor making it difficult to control by surgery or radiation. Radio-inducible herpes simplex virus thymidine kinase (HSV-TK) gene therapy has been shown to enhance local tumor control after radiation therapy (RT), while limiting the expression of the transgene in the irradiated tumor tissues. To prevent liver tumor recurrence and control systemic disease while limiting the potential bystander toxicity of HSV-TK therapy, we proposed to stimulate endogenous dendritic cell (DC) proliferation with systemic adenovirus Flt3 ligand (Adeno-Flt3L) gene therapy, followed by primary tumor radiation therapy combined with a radio-inducible HSV-TK gene therapy. We hypothesized that adenovirus-expressing Flt3L gene therapy will stimulate DC proliferation, allowing the upregulated DCs to locally harness tumor antigens released from HSV-TK/RT-treated HCC cells, thereby converting irradiated tumors to an autologous in situ tumor vaccine in mice with primary liver tumors. To test this hypothesis, an expression vector of HSV-TK was constructed under the control of a radio-inducible promoter early-growth response (Egr-TK) and a recombinant adenovirus-expressing human Flt3L was constructed. The Adeno-Flt3L [10(9) plaque forming units (pfu)] was administered intravenously on days 1 and 8 after radiation therapy. The murine hepatoma cell line (BNL1ME) was stably transfected by Egr-TK or Egr-Null (encoding no therapeutic gene). Palpable tumors in BALB/c mice were treated with a localized dose of 25 Gy of radiation followed by ganciclovir (GCV, 100 mg/kg, 14 days). Four treatment cohorts were compared: Egr-Null/GCV + RT + Adeno-LacZ; Egr-Null/GCV + RT + Adeno-Flt3L; Egr-TK/GCV + RT + Adeno-LacZ; and Egr-TK/GCV + RT + Adeno-Flt3L. There was no primary tumor regression in the Egr-Null tumors after radiation therapy alone. In contrast, Egr-TK tumors had nearly complete tumor regression for 3 weeks after radiation therapy (P < 0.01), however, long-term follow-up demonstrated primary tumor recurrence and death secondary to pulmonary metastasis. Flt3L expression was confirmed by serum bioassay (mean = 88 ng/mL) in these animals and Western blotting of tissue culture medium in Adeno-Flt3L-infected BaF/huFlt3L cells. Radiation therapy with Adeno-Flt3L gene therapy effectively retarded primary tumor growth when compared to radiation therapy alone. The trimodality therapy (Egr-TK/GCV + RT + Adeno-Flt3L) was the most efficacious with 40% complete tumor regression (>100 days) and <20% pulmonary metastases, indicating the development of sustained antitumor immune response. These studies provide a rationale for triple modality therapies with radiation-inducible HSV-TK gene therapy and Adeno-Flt3L when used in combination with primary tumor radiation therapy for improved local and systemic control of HCC.

Radiation improves antitumor effect of immune checkpoint inhibitor in murine hepatocellular carcinoma model

“…Although RT led to antitumor effect by inflammatory response, immune-escape often occurs and tumors may acquire resistance which promotes relapse [38, 39]. HCC also acquires frequent resistance to RT, and has resulted in recurrence [40]. One of the mechanisms of radio-resistance was upregulated PD-L1 in tumor microenvironment [24, 41].…”

Immunobiology of Radiotherapy: New Paradigms

“…Flt3L is a potent cytokine for stimulating tumor-specific responses and inducing proliferation of DCs. Kawashita et al ( 7 ) demonstrated the efficacy of hepatocellular tumor control treated with radiation-inducible suicide gene therapy combined with Ad-Flt3L transfer. CD40L binds to CD40 on DCs to induce activation and maturation of DCs for tumor-specific response ( 7 ).…”

“…Kawashita et al ( 7 ) demonstrated the efficacy of hepatocellular tumor control treated with radiation-inducible suicide gene therapy combined with Ad-Flt3L transfer. CD40L binds to CD40 on DCs to induce activation and maturation of DCs for tumor-specific response ( 7 ). In a followup study, Kawashita et al ( 8 ) demonstrated an augmented antitumor immune response when CD40L was combined with radiation-inducible gene therapy plus Adeno-Flt3L in an orthotopic tumor model of hepatocellular carcinoma ( 8 ).…”

An Autologous In Situ Tumor Vaccination Approach for Hepatocellular Carcinoma. 2. Tumor-Specific Immunity and Cure after Radio-Inducible Suicide Gene Therapy and Systemic CD40-Ligand and Flt3-Ligand Gene Therapy in an Orthotopic Tumor Model

“…Mice, cell lines, plasmids and adenoviral vectors were used as described in the companion article (16). In brief, Balb/c (H-2 d ) six-to eight-week-old male mice were purchased from Jackson Laboratories (Bar Harbor, ME).…”

“…Res. 2014; 182) (16), we demonstrated that intravenous administration of a recombinant adenovirus-expressing human Flt3L (Adeno-Flt3L) induced a Th1-biased, immune response and enhanced tumor control after radio-inducible suicide gene therapy in an ectopic model of HCC. A cautionary note for Flt3L immunotherapy is that it induces immature DCs that are good at antigen uptake but can be anergic to T cells.…”