Development of a new fusion-enhanced oncolytic immunotherapy platform based on herpes simplex virus type 1

Abstract

Background: Oncolytic viruses preferentially replicate in tumors as compared to normal tissue and promote immunogenic cell death and induction of host systemic anti-tumor immunity. HSV-1 was chosen for further development as an oncolytic immunotherapy in this study as it is highly lytic, infects human tumor cells broadly, kills mainly by necrosis and is a potent activator of both innate and adaptive immunity. HSV-1 also has a large capacity for the insertion of additional, potentially therapeutic, exogenous genes. Finally, HSV-1 has a proven safety and efficacy profile in patients with cancer, talimogene laherparepvec (T-VEC), an oncolytic HSV-1 which expresses GM-CSF, being the only oncolytic immunotherapy approach that has received FDA approval. As the clinical efficacy of oncolytic immunotherapy has been shown to be further enhanced by combination with immune checkpoint inhibitors, developing improved oncolytic platforms which can synergize with other existing immunotherapies is a high priority. In this study we sought to further optimize HSV-1 based oncolytic immunotherapy through multiple approaches to maximize: (i) the extent of tumor cell killing, augmenting the release of tumor antigens and danger-associated molecular pattern (DAMP) factors; (ii) the immunogenicity of tumor cell death; and (iii) the resulting systemic anti-tumor immune response.

Methods: To sample the wide diversity amongst clinical strains of HSV-1, twenty nine new clinical strains isolated from cold sores from otherwise healthy volunteers were screened across a panel of human tumor cell lines to identify the strain with the most potent tumor cell killing ability, which was then used for further development. Following deletion of the genes encoding ICP34.5 and ICP47 to provide tumor selectivity, the extent of cell killing and the immunogenicity of cell death was enhanced through insertion of a gene encoding a truncated, constitutively highly fusogenic form of the envelope glycoprotein of gibbon ape leukemia virus (GALV-GP-R^-). A number of further armed derivatives of this virus were then constructed intended to further enhance the anti-tumor immune response which was generated following fusion-enhanced, oncolytic virus replication-mediated cell death. These viruses expressed GMCSF, an anti-CTLA-4 antibody-like molecule, CD40L, OX40L and/or 4-1BB, each of which is expected to act predominantly at the site and time of immune response initiation. Expression of these proteins was confirmed by ELISA and/or western blotting. Immunogenic cell death was assessed by measuring the levels of HMGB1 and ATP from cell free supernatants from treated cells, and by measuring the surface expression of calreticulin. GALV-GP-R^- mediated cell to cell fusion and killing was tested in a range of tumor cell lines in vitro. Finally, the in vivo therapeutic potential of these viruses was tested using human A549 (lung cancer) and MDA-MB-231(breast cancer) tumor nude mouse xenograft models and systemic anti-tumor effects tested using dual flank syngeneic 4434 (melanoma), A20 (lymphoma) mouse tumor models alone and in combination with a murine anti-PD1 antibody, and 9 L (gliosarcoma) tumors in rats.

Results: The twenty nine clinical strains of HSV-1 isolated and tested demonstrated a broad range of tumor cell killing abilities allowing the most potent strain to be identified which was then used for further development. Oncolytic ability was demonstrated to be further augmented by the expression of GALV-GP-R^- in a range of tumor cell lines in vitro and in mouse xenograft models in nude mice. The expression of GALV-GP-R^- was also demonstrated to lead to enhanced immunogenic cell death in vitro as confirmed by the increased release of HMGB1 and ATP and increased levels of calreticulin on the cell surface. Experiments using the rat 9 L syngeneic tumor model demonstrated that GALV-GP-R^- expression increased abscopal uninjected (anenestic) tumor responses and data using mouse 4434 tumors demonstrated that virus treatment increased CD8+ T cell levels both in the injected and uninjected tumor, and also led to increased expression of PD-L1. A combination study using varying doses of a virus expressing GALV-GP-R^- and mGM-CSF and an anti-murine PD1 antibody showed enhanced anti-tumor effects with the combination which was most evident at low virus doses, and also lead to immunological memory. Finally, treatment of mice with derivatives of this virus which additionally expressed anti-mCTLA-4, mCD40L, m4-1BBL, or mOX40L demonstrated enhanced activity, particularly in uninjected tumors.

Conclusion: The new HSV-1 based platform described provides a potent and versatile approach to developing new oncolytic immunotherapies for clinical use. Each of the modifications employed was demonstrated to aid in optimizing the potential of the virus to both directly kill tumors and to lead to systemic therapeutic benefit. For clinical use, these viruses are expected to be most effective in combination with other anti-cancer agents, in particular PD1/L1-targeted immune checkpoint blockade. The first virus from this program (expressing GALV-GP-R^- and hGM-CSF) has entered clinical development alone and in combination with anti-PD1 therapy in a number of tumor types (NCT03767348).

Keywords: Anenestic effect; Herpes simplex-1; Immunotherapy; Oncolytic viruses.

Fig. 1

Schematic representation of the viruses constructed in this study. The genome structures of the viruses constructed and tested. The construction of each virus is described in detail in the Additional file 1

Fig. 2

The effects of GALV-GP-R⁻ expression on human tumor cell lines in vitro and human tumor xenograft models in vivo. a Images of cell lines infected with Virus 12 (expresses GFP) top panel and (a) Images of cell lines infected with Virus 10 (expresses GFP and GALV-GP-R⁻). b Images representing the cell killing effects of Virus 12 and (b) Virus 10 in a panel of tumor cells. c Individual tumor growth curves from mice treated with either vehicle, Virus 19 (expresses mGM-CSF) or Virus 16 (expresses mGM-CSF and GALV-GP-R⁻) in the A549 lung cancer model and (d) in the MDA-MB-231 breast cancer model. The dose level of virus was in each case 5 × 10³ pfu in 50 μl given 3x every other day. Statistical differences between groups were measured by one-way ANOVA at day 41 for the A549 model and at day 38 for the MDA-MB-231 model. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

Markers of immunogenic cell death in cells treated with either Virus 23 (expresses hGM-CSF) or Virus 17 (expresses hGM-CSF and + GALV-GP R-) in vitro. a Levels of ATP release measured by luminescence in a panel of cell lines treated at the indicated MOI at 24 h post infection and (a) at 48 h post infection observed in cell-free supernatants treated with Virus 23 (indicated by the clear bars) and Virus 17 (indicated by the solid bars). b ELISA measuring HMGB1 (pg/ml) levels in cell-free supernatants from cells treated for 48 h with MOI 0.0001–1. c Histogram showing the expression levels of surface calreticulin (CRT) in cells treated at indicated MOI 0.01 for 48 h. Data show un-permeabilized, viable cells stained with CRT and measured by FACS. Statistical differences between groups were determined by using two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

Effects of GALV-GP-R⁻ expression in an immune competent tumor model. a Tumor growth curves of rat 9 L tumors treated with either vehicle (PBS), Virus 19 (expresses mGM-CSF) or Virus 16 (expresses mGM-CSF and GALV-GP R⁻). Virus or vehicle were injected into the right tumor only. b A repeat of the experiment in (A), treating with either vehicle or Virus 16 but with longer follow up until day 60. 5 × 10⁶ pfu of virus in 50 μl was given 5x every other day in each case. Statistical differences between groups were measured by one-way ANOVA at day 31 for a and at day 35 for b. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

Tumors from Virus 16 treated animals demonstrate increased levels of CD8+ T cells and PD-L1. a Immunohistochemistry staining for CD8 (red), CD4 (green) and foxp3 (pink) of injected and un-injected 4434 tumors from mice either treated with mock or with Virus 16 (expresses mGM-CSF and GALV-GP R⁻) 10 days after treatment. b Relative frequency of PD-L1+ cells in mice bearing 4434 bi-flank tumours treated in the right flank with Virus 16 or vehicle on days 1, 3 and 5 and collected on days 3, 7, 10 and 16 after the first day of treatment. c The relative frequency of tumor infiltrating CD8+ cells, gated from the viable cell population, from tumours collected on days 3, 7, 10 and 16. d The relative frequency of CD8+ cells from lymph nodes at days 3, 7, 10 and 16. Statistical differences between groups were determined by using two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 6

Effects of combination treatment with Virus 16 and anti-PD1. a-d Individual tumor growth curves of injected (right) and contralateral/un-injected (left) tumors from BALB/C mice bearing A20 lymphoma tumors treated with either (a) vehicle or anti-PD1, b Virus 16 (5 × 10⁶ pfu/dose 3x), or Virus 16 (5 × 10⁶ pfu/dose 3x) and anti-PD1, c Virus 16 (5 × 10⁵ pfu/dose 3x) or Virus 16 (5 × 10⁵ pfu/dose 3x) and anti-PD1 and (d) Virus 16 (5 × 10⁴ pfu/dose 3x) or Virus 16 (5 × 10⁴ pfu/dose 3x) and anti-PD1. Statistical differences between groups were measured by one-way ANOVA with multiple comparisions at day 28. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 7

Expression of anti-CTLA-4 or immune co-stimulatory pathway activating ligands further increases the efficacy of Virus 16 in vivo. a Western blot showing the expression of of anti-mouse CTLA-4 as detected in cell lysates from cells infected with Virus 27. b Individual tumor growth curves of injected and contralateral tumors from BALB/C mice bearing A20 lymphoma tumors treated with either vehicle, Virus 16 (expresses GM-CSF and GALV-GP R-), Virus 27 (additionally expresses anti-mCTLA-4, Virus 32 (additionally expresses mCD40L), Virus 33 (additionally expresses m4-1BBL) or Virus 35 (additionaly expresses mOX40L). The dose level of virus was in each case 5 × 10⁴ pfu in 50 μl given 3x every other day. Statistical differences between groups were measured by one-way ANOVA at Day 40. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001