IL-12 encoding oNDV synergizes with CAR-T cells in orthotopic models of non-small cell lung cancer

Abstract

Systemic administration of oncolytic viruses (OVs) is a promising approach for targeting metastatic solid tumors, but their anti-tumor activity is limited by pre-existing neutralizing antibodies against common human viruses. Therefore, investigators have developed OVs derived from non-human host viruses. Successful implementation of this strategy requires that the viral vector selectively infects and replicates within human cancer cells. Newcastle disease virus (NDV) is an avian paramyxovirus that, as NDV-based OVs (oNDVs), has demonstrated safety and activity against multiple human tumors in clinical trials. Their use as a single agent, however, is insufficient to cure tumors. Similarly, chimeric antigen receptor-modified T cells (CAR-T cells) enable systemic targeting of cancer cells but have limited anti-tumor effects against bulky solid tumors, in part due to the immunosuppressive tumor environment. In this study, we evaluated the anti-tumor effects of combining systemic oNDV and CAR-T cell treatments. In models of non-small cell lung carcinoma (NSCLC), we found that oNDV itself and interleukin (IL)-12 derived from oNDVs enhance HER2-directed CAR-T cell anti-tumor activity and persistence in vitro and in vivo, leading to superior control of NSCLC tumors compared with either agent alone in vivo. Our data indicate that oNDV enhances the anti-tumor effects of HER2.CAR-T cells, thus controlling the growth of orthotopic NSCLC tumors.

Keywords: CAR-T cell; MT: Regular Issue; combination therapy; immunotherapy; non-small cell lung carcinoma; oncolytic Newcastle disease virus.

Graphical abstract

Figure 1

oNDV transgene expression and lytic effect in NSCLC (A) A panel of NSCLC cell lines (A549, H1650, H1703, and H441) were infected with 0.1 PFU/cell oNDVEGFP or oNDVIL12. GFP expression in live cells was monitored via fluorescent microscopy, and medium was collected for IL-12p70 ELISA 24, 48, and 72 h post-infection. Data are presented as means ± SD (n = 4, representative images shown). (B) NSCLC cells were infected with increasing doses of oNDVEGFP or oNDVIL12. Viable cells were analyzed at 72 h by MTS assay. Data are presented as means ± SD (n = 4). (C) A549 and H1650 cells expressing firefly luciferase (ffluc) were orthotopically transplanted via tail vein injection to NSG mice. A total of 1 × 10⁷ or 1 × 10⁸ PFUs of oNDVIL12 or vehicle control were systemically administered 14 days after tumor inoculation. Serum samples were collected 4, 11, and 25 days after injection of oNDV, and IL-12p70 levels in serum were measured by ELISA. Data are presented as means ± SD (n = 5). (D) Bioluminescence of A549 or H1650 tumor cells was monitored at different time points. Data are presented as means ± SD (n = 5). (E) Kaplan-Meier survival curve after administration of oNDV. The humane endpoint was established as body weight <80% from baseline (n = 5). p values were determined using the log rank Mantel-Cox test. Statistical significance set at p < 0.05, ns > 0.05. The experiment was repeated for purposes of tissue collection, with similar trends for all replicates.

Figure 2

HER2.CAR-T cells control NSCLC growth both in vitro and in vivo (A) HER2 expression was analyzed by flow cytometry on NSCLC cell lines. Isotype antibody staining served as a negative control. NSCLC cell lines were seeded into 48-well plates. After 24 h, HER2.CAR-T cells derived from three healthy donors were co-cultured with tumors cells (effector-to-target ratio [E:T] = 1:20). The co-culture was then stained with Annexin, and the total red objective area was measured to quantify cell death via Incucyte over 72 h. Data are presented as means ± SD (n = 3). (B) A549 and H1650 cells expressing firefly luciferase (ffluc) were orthotopically transplanted via tail vein injection to NSG mice. This experiment was performed concomitantly with the study shown in Figure 1, and the control animals are the same. A total of 1 × 10⁶ HER2.CAR-T cells or vehicle control was systemically administered 14 days after tumor inoculation. Bioluminescence of A549 or H1650 tumor cells was monitored at different time points. Data are presented as means ± SD (n = 5). (C) Kaplan-Meier survival curve after administration of HER2.CAR-T cells. The humane endpoint was established as body weight <80% from baseline (n = 5). p values were determined using the log rank Mantel-Cox test. Statistical significance set at p < 0.05, ns > 0.05. The experiment was repeated for purposes of tissue collection, with similar trends for all replicates. (D) T cells were isolated from the lungs of 2–3 mice 12 weeks post-infusion to ensure sufficient T cell numbers for analysis, and HER2.CAR expression and markers of T cell activation/exhaustion from bulk HER2.CAR-positive T cells were analyzed by flow cytometry.

Figure 3

Combination of oNDVIL12 and HER2.CAR-T cell enhances anti-tumor activity of HER2.CAR-T cells (A) NSCLC cell lines expressing firefly luciferase (ffluc) were infected with 0.1 PFU/cell of oNDVEGFP or oNDVIL12. HER2.CAR-T cells were added to all conditions 24 h post-infection (E:T = 1:20; uninfected cells [−] served as a negative control). Cells were harvested 72 h post-co-culture, and viable cancer cells were analyzed by luciferase assay. Data are presented as means ± SD (n = 4), relative to untreated tumor cells. (B) A549 cells expressing ffluc were orthotopically transplanted via tail vein injection to NSG mice. On day 10 after tumor inoculation, 1 × 10⁸ PFU oNDVIL12 was administered via tail vein injection, with subsequent doses given at weeks 2 and 3 (total of 3 × 10⁸ PFUs) (green arrows). HER2.CAR-T cells were systemically administered 7 days after the first dose of oNDV (blue arrow), and mice without oNDV treatment served as controls. Bioluminescence of A549 tumor cells was monitored at different time points. Data are presented as means ± SD (n = 5). (C) A549 cells were orthotopically transplanted via tail vein injection to NSG mice. On day 10 after tumor inoculation 1 × 10⁸ PFU oNDVIL12 was administered via tail vein injection, with subsequent doses given at weeks 2 and 3 (total of 3 × 10⁸ PFUs). HER2.CAR-T cells expressing ffluc were systemically administered 7 days after the first dose of oNDV, and mice without oNDV treatment served as controls. Bioluminescence of HER2.CAR-T cells was monitored at different time points. Data are presented as means ± SD (n = 5). (D) Serum samples were collected 6, 13, 20, 27, and 49 days after initial injection of oNDV, and IL-12p70 and IFNγ levels in serum were measured by ELISA. Data are presented as means ± SD (n = 15, data were combined from three independent experiments). (E) Kaplan-Meier survival curve after treatment. The humane endpoint was established as body weight <80% from baseline (n = 15, data were combined from three independent experiments). p values were determined using the log rank Mantel-Cox test. Statistical significance set at p < 0.05, ns > 0.05.

Figure 4

IL-12 derived from oNDV improves CAR-positive T cell persistence in vivo (A) A549 cells were orthotopically transplanted via tail vein injection to NSG mice. On day 10 after tumor inoculation, 1 × 10⁸ PFU oNDVIL12 was administered via tail vein injection, with subsequent doses given at weeks 2 and 3 (total of 3 × 10⁸ PFUs). HER2.CAR-T cells expressing firefly luciferase (ffluc) were systemically administered 7 days after the first dose of oNDV, and mice without oNDV treatment served as controls. Lungs from treated mice were collected 2, 6, and 9 weeks post-administration of CAR-T cells and subjected to RNA sequencing. Lungs from untreated mice and pre-infusion CAR-T cells served as controls (n = 3 for each condition). (B and C) A549 cells were orthotopically transplanted via tail vein injection to NSG mice. On day 10 after tumor inoculation, 1 × 10⁸ PFU oNDVIL12 was administered via tail vein injection, with subsequent doses given at weeks 2 and 3 (total of 3 × 10⁸ PFUs). HER2.CAR-T cells were systemically administered 7 days after the first dose of oNDV, and mice without oNDV treatment served as controls. T cells were harvested from lungs of 2–3 mice 2, 6, and 9 weeks post-administration of CAR-T cells to ensure sufficient T cell numbers and stained for flow cytometry and phenotyping.