Combinatorial immunotherapy with anti-ROR1 CAR NK cells and an IL-21 secreting oncolytic virus against neuroblastoma

Abstract

Children with recurrent/metastatic neuroblastoma (NB) have a dismal survival (<25%). Novel therapies are desperately needed. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is highly expressed on NB. C021 is a selective oncolytic herpes simplex virus modified to overexpress human interleukin-21 (hIL-21), a cytokine that enhances natural killer (NK) cell cytotoxicity. In the current study, we successfully engineered ex-vivo-expanded NK cells to express a chimeric antigen receptor (CAR) against ROR1 using mRNA electroporation and investigated the efficacy of anti-ROR1-CAR-NK cells combined with C021 in targeting ROR1⁺ NB. We found that C021-infected NB cells secreted hIL-21 in vitro and in vivo. Compared to the non-cytokine-secreting parental virus C134, C021 significantly enhanced the in vitro cytotoxicity (p < 0.05) of anti-ROR1-CAR-NK cells with increased interferon (IFN)-γ (p < 0.05), granzyme B (p < 0.05), and perforin (p < 0.05) secretion against NB cells. Furthermore, the combination of C021 and anti-ROR1-CAR-NK cells significantly extended the survival of human NB xenografted NSG mice compared to controls (mock NK, ROR1-CAR-NK, C134, C021, C134+ROR1-CAR-NK, and C021+mock NK). Our results suggest that cytokine-secreting oncolytic virus in combination with CAR-NK cells is a novel, effective immunotherapeutic approach for high-risk NB.

Keywords: CAR; IL-21; ROR1; chimeric antigen receptor; interleukin-21; natural killer cells; neuroblastoma; oncolytic herpes simplex virus.

Graphical abstract

Figure 1

The effects of C134 and C021 on NB cells and ex-vivo-expanded NK cells in vitro (A) Schematic representation of C134 and C021 constructs. C021 contains the hIL-21 gene and dsRed (linked by a 2A element) within both copies of the γ₁34.5 deletion locus of C134. (B) C021-infected CHLA-255 cells secreted significantly higher levels of hIL-21 on day 2 at MOI = 0.0001 (p < 0.001) compared to day 1, 3, or 6. n = 3. In this and the subsequent panels, markers represent the mean values, error bars indicate the standard deviation (SD) of triplicate samples in a representative experiment. The same trend was seen in three independent biological replicates. Results were compared using the two-tailed Student t-test with p < 0.05 considered as significant. (C) C021-infected CHLA-255 cells secreted significantly higher levels of hIL-21 on day 2 at MOI = 0.001 (p < 0.001) compared to day 1, 3, or 6. n = 3. (D) C021 significantly reduced the viability of CHLA-255 cells at day 2, 3, or 6 (p < 0.001) compared to day 1 at MOI = 0.0001. n = 3. (E) C021 significantly reduced the viability of CHLA-255 cells at day 2, 3, or 6 (p < 0.001) compared to day 1 at MOI = 0.001. n = 3. (F) At MOI = 0.025, C021-infected SKNFI cells secreted significantly higher levels of hIL-21 on day 3 (p < 0.001) compared to day 1, 2, 5, 7, 9, or 11. n = 3. (G) Both C134 and C021 infections significantly reduced the viability of SKNFI cells at 24 h at MOI = 0.01, 0.1, or 1 compared to MOI = 0.001 (p < 0.001). n = 3. (H) Ex-vivo-expanded NK cells were incubated with C134 or C021 at the indicated MOI. NK viability was measured at 24 and 48 h by CellTiter 96 AQueous one solution cell proliferation assay. n = 3. ∗p < 0.05 and ∗∗∗p < 0.001.

Figure 2

C021 significantly enhanced in vitro cytotoxicity with the enhanced release of granzyme B, IFN-γ, and perforin of anti-ROR1-CAR-NK cells against NB cells (A) Mock NK or anti-ROR1-CAR-NK cells (CAR) were incubated with SKNFI cells at E:T = 3:1 with or without C134 or C021 (MOI = 0.025) for 24 h. The percentage of killing of SKNFI cells was measured by Britelite plus reporter gene assay. C021 significantly enhanced the in vitro cytotoxicity of anti-ROR1-CAR-NK cells against SKNFI cells compared to controls. In this and the subsequent panels, columns represent the mean values, error bars indicate the standard deviation (SD) of triplicate samples in a representative experiment. The same trend was seen in three independent biological replicates. Results were compared using the two-tailed Student t-test with p < 0.05 considered as significant. (B–D) After 24 h co-culture under the condition as described in (A), the supernatants were collected for ELISAs to determine the released granzyme B (B), IFN-γ (C), and perforin (D) levels. (E) Mock NK or anti-ROR1-CAR-NK cells (CAR) were incubated with CHLA-255 cells at E:T = 1:1 with or without C134 or C021 (MOI = 0.001) for 24 h. The percentage of killing of CHLA-255 cells was measured by Britelite plus reporter gene assay. C021 significantly enhanced the in vitro cytotoxicity of anti-ROR1-CAR-NK cells against CHLA-255 cells compared to controls (p = 0.0286 vs. CAR and CAR+C134, p < 0.0001 vs. C021). (F–H) After 24 h co-culture under the condition as described in (E), the supernatants were collected for ELISAs to determine the released granzyme B (F), IFN-γ (G), and perforin (H) levels. n = 4.

Figure 3

The combination of anti-ROR1 CAR NK+C021 significantly extended the survival of NB xenografted NSG mice (A) hIL-21 expression in C021-injected NB xenograft tumors. 2 × 10⁷ of CHLA-255-Luc cells were subcutaneously injected into the right flank of NSG mice. After the tumor diameter reached 1 ± 0.3 cm, one dose of 1 × 10³, 10⁴, or 10⁵ PFU C021 was intratumorally injected into CHLA-255-Luc xenografted NSG mice. Two days later, tumors were collected and homogenized in 1 mL RPMI1640 medium. The tumor samples were centrifuged, and the supernatant was collected and used for IL-21 ELISAs. Columns represent the mean values, error bars indicate the standard deviation (SD) of all the samples. Results were compared using the two-tailed Student t-test with p < 0.05 considered as significant. (B) Experimental schema. 2 × 10⁷ of CHLA-255-Luc cells were subcutaneously injected into the right flank of NSG mice. After the tumor diameter reached 1 ± 0.3 cm, one dose of PBS or 1 × 10⁴ PFU C134 or C021 was intratumorally injected into CHLA-255-Luc xenografted mice. PBS, 5 × 10⁶ expanded peripheral blood natural killer (exPBNK) cells, or 5 × 10⁶ anti-ROR1-CAR-NK cells were intraperitoneally injected to each mouse 2 or 9 days after viral injection. (C) The Kaplan-Meier survival curves of mice receiving treatment are shown using animal sacrifice as the terminal event. Survival curves were analyzed using the log rank (Mantel-Cox) test. The mice treated with anti-ROR1-CAR-NK+C021 (n = 5) significantly extended the survival of CHLA255 xenografted mice as compared to the control groups, which were treated with mock NK+C021 (n = 5, p < 0.05), anti-ROR1-CAR-NK cells alone (n = 5, p < 0.05), or C021 alone (n = 5, p < 0.01). NK, mock NK; CAR, anti-ROR1-CAR-NK. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.