Combined immunotherapy with controlled interleukin-12 gene therapy and immune checkpoint blockade in recurrent glioblastoma: An open-label, multi-institutional phase I trial

Abstract

Background: Veledimex (VDX)-regulatable interleukin-12 (IL-12) gene therapy in recurrent glioblastoma (rGBM) was reported to show tumor infiltration of CD8+ T cells, encouraging survival, but also up-regulation of immune checkpoint signaling, providing the rationale for a combination trial with immune checkpoint inhibition.

Methods: An open-label, multi-institutional, dose-escalation phase I trial in rGBM subjects (NCT03636477) accrued 21 subjects in 3 dose-escalating cohorts: (1) neoadjuvant then ongoing nivolumab (1mg/kg) and VDX (10 mg) (n = 3); (2) neoadjuvant then ongoing nivolumab (3 mg/kg) and VDX (10 mg) (n = 3); and (3) neoadjuvant then ongoing nivolumab (3 mg/kg) and VDX (20 mg) (n = 15). Nivolumab was administered 7 (±3) days before resection of the rGBM followed by peritumoral injection of IL-12 gene therapy. VDX was administered 3 hours before and then for 14 days after surgery. Nivolumab was administered every two weeks after surgery.

Results: Toxicities of the combination were comparable to IL-12 gene monotherapy and were predictable, dose-related, and reversible upon withholding doses of VDX and/or nivolumab. VDX plasma pharmacokinetics demonstrate a dose-response relationship with effective brain tumor tissue VDX penetration and production of IL-12. IL-12 levels in serum peaked in all subjects at about Day 3 after surgery. Tumor IFNγ increased in post-treatment biopsies. Median overall survival (mOS) for VDX 10 mg with nivolumab was 16.9 months and for all subjects was 9.8 months.

Conclusion: The safety of this combination immunotherapy was established and has led to an ongoing phase II clinical trial of immune checkpoint blockade with controlled IL-12 gene therapy (NCT04006119).

Keywords: clinical trial; controlled gene expression; gene therapy; glioblastoma; immunotherapy.

Fig. 1

Upon screening and determination of eligibility, each accrued subject underwent an intravenous infusion of nivolumab at either 1 mg/kg (cohort 1) or 3mg/kg (cohorts 2 and 3), seven days (±3 days) before scheduled craniotomy. The activator ligand, veledimex (VDX), was administered 3 hours (±2 hours) before planned craniotomy for tumor resection at either 10 mg (cohort 1 and 2) or 20 mg (cohort 3). Pericavitary injection of the IL-12 immunogene therapy vector (Ad-RTS-hIL-12) was then carried out as previously described. Subjects then orally took VDX daily for 14 days postoperatively at 10 mg (cohorts 1 and 2) or 20 mg (cohort 3). Nivolumab was then administered every 2 weeks at either 1 mg/kg (cohort 1) or 3mg/kg (cohorts 2 and 3).

Fig. 2

A. Veledimex peak plasma PK results. (Left, VDX 10 mg and nivolumab 1 mg/kg; middle, VDX 10 mg and nivolumab 3 mg/kg; and right, VDX 20 mg and nivolumab 3 mg/kg.). There is a dose-response relationship for plasma veledimex concentration. The VDX 10 mg and nivolumab 1 mg/kg cohort has a similar veledimex plasma concentration to VDX 10 mg and nivolumab 3 mg/kg cohort, and for the VDX 20 mg and nivolumab 3 mg/kg cohort, the veledimex plasma concentration is more than double the level of the VDX 10 mg with nivolumab 1 or 3 mg/kg cohorts (mean ± SEM). B. Veledimex tumor pharmacokinetics (PK). Veledimex tumor concentration is similar to the pattern observed on plasma except for VDX 10 mg and nivolumab 3 mg/kg cohort is slightly higher than VDX 10 mg and nivolumab 1 mg/kg cohort. C&D. Serum cytokine levels. A dose-response of serum cytokine IL-12 and IFN-γ levels was seen between VDX 10 mg and 20 mg dosing with the nivolumab dose combinations. Peak levels of serum IL-12 were observed on Day 3 and Day 7 in VDX 20 mg with nivolumab 3 mg/kg cohort and VDX 10 mg with nivolumab 1or 3 mg/kg cohorts, respectively. Peak levels of serum IFNγ were observed on Day 7 and Day 14 in VDX 10 mg with nivolumab 1or 3 mg/kg cohorts and the VDX 20 mg with nivolumab 3 mg/kg cohort, respectively. (Data are presented as mean ± SEM by day of collection.) The tables below the panels show the number of subjects whose blood samples were available for analyses at indicated timepoints. E. Tumor cytokine levels. Tumor IL-12 and IFNγ pre-IL-12 (though post an adjuvant dose of nivolumab) and post-treatment (Ad + VDX and nivolumab) in subject 103 from the VDX 10 mg and nivolumab initially 1 mg/kg dose-escalated to 3 mg/kg and subject 108 from the VDX 20 mg and nivolumab 3 mg/kg cohorts, respectively.

Fig. 3

Heat Map constructed from quantitative image analyses of multiplex immunofluorescence (MultiOmyx™ High-Order Multiplexing) images from 4 matched pairs of tumor samples pre- and post-treatment. Ratios are expressed as positive cells per mm² of tissue (density). The panel consists of markers covering the following immunophenotypes: CD3⁺ (pan T cells), CD3⁺ CD4⁺ (helper T cells), CD3⁺ CD8⁺ (cytotoxic T cells), CD3⁺ CD8⁺ CD56⁺ (NKT cells), CD3⁺ CD4⁺ FOXP3⁺ (regulatory T cells), and PD-1⁺ and PD-L1⁺ total immune cells. CD56⁺ cells may include NK cells but have been confounded by expression of NCAM on tumor cells (not shown). Structural marker Glial Fibrillary Acidic Protein (GFAP) expression is expected to be higher in tumor than normal adjacent brain tissue or nontumor gliosis. The apparent row width varies because the number of bands depends on the number of regions of interest (ROI) analyzed per sample, as compared with conventional heat maps for a homogenized tumor sample or liquid specimen (see Materials and Methods). Refer to Supplementary Table 1 for corresponding subject characteristics and the treatment group assigned.

Fig. 4

Kaplan-Meier survival analysis for overall survival in VDX 10 mg (blue) and 20 mg (red) dose groups. Median overall survival (mOS) for VDX 10 mg in combination with nivolumab either at 1 or 3 mg/kg (n = 6) was 16.9 months (3.8, 24.0 months, CI) whereas for VDX 20 mg in combination with nivolumab mOS (n = 15) was 8.5 months (4.4, 17.4 months, CI). mOS among all subjects (n = 21) was 9.8 months (5.2,17.4 months, CI). Censored (alive) subjects at data cutoff are shown as a black dot. The data cutoff was 16 October 2020.