Efficiently targeting neuroblastoma with the combination of anti-ROR1 CAR NK cells and N-803 in vitro and in vivo in NB xenografts

Abstract

The prognosis for children with recurrent and/or refractory neuroblastoma (NB) is dismal. The receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is highly expressed on the surface of NB cells, provides a potential target for novel immunotherapeutics. Anti-ROR1 chimeric antigen receptor engineered ex vivo expanded peripheral blood natural killer (anti-ROR1 CAR exPBNK) cells represent this approach. N-803 is an IL-15 superagonist with enhanced biological activity. In this study, we investigated the in vitro and in vivo anti-tumor effects of anti-ROR1 CAR exPBNK cells with or without N-803 against ROR1⁺ NB models. Compared to mock exPBNK cells, anti-ROR1 CAR exPBNK cells had significantly enhanced cytotoxicity against ROR1⁺ NB cells, and N-803 further increased cytotoxicity. High-dimensional analysis revealed that N-803 enhanced Stat5 phosphorylation and Ki67 levels in both exPBNK and anti-ROR1 CAR exPBNK cells with or without NB cells. In vivo, anti-ROR1 CAR exPBNK plus N-803 significantly (p < 0.05) enhanced survival in human ROR1⁺ NB xenografted NSG mice compared to anti-ROR1 CAR exPBNK alone. Our results provide the rationale for further development of anti-ROR1 CAR exPBNK cells plus N-803 as a novel combination immunotherapeutic for patients with recurrent and/or refractory ROR1⁺ NB.

Keywords: IL-15 superagonist; MT: Regular Issue; ROR1; chimerical antigen receptor; cytotoxicity; expanded natural killer cells; neuroblastoma; targeted immunotherapy.

Graphical abstract

Figure 1

Construction and anti-ROR1 CAR expression on expanded NK cells (A) Schema of anti-ROR1 CAR construct in pcDNA3.1 consisting of anti-ROR1 single chain fragment variable (scFv), 41BB, CD3zeta, and truncated CD19. (B) The anti-ROR1 CAR exPBNK cells were generated by electroporating exPBNK with anti-ROR1 CAR mRNA using Maxcyte electroporator. Mock exPBNK cells were generated by electroporating exPBNK cells with RNase-free H₂O. Anti-ROR1 CAR expression was evaluated by flow cytometry analysis using an FITC-conjugated goat anti-mouse IgG, F(ab′)₂ fragment-specific antibody at days 1, 2, 4, 6, and 10. The top panel shows the representative flow cytometry dot plots of forward scatter (FSC) vs. CAR. The bottom panel summarizes the percentage of anti-ROR1 CAR expression on exPBNK cells. Data are presented as mean ± SEM, n = 3. ∗∗∗p < 0.001.

Figure 2

Anti-ROR1 CAR enhances exPBNK cell in vitro cytolytic activity against ROR1⁺ NB cells The exPBNK cells electroporated with anti-ROR1 CAR mRNA (anti-ROR1-CAR) or H₂O (mock) were incubated for 4 h with ROR1⁺ NB cell lines: SH-SY5Y (A), SKNFI (B), SKNBE(2)N (C), ROR1⁺ osteosarcoma cell line HOS (D), and ROR1⁻ MCF-7 (E) cell line. In vitro cytotoxicity was measured by 7-AAD/CFSE Cell-Mediated Cytotoxicity Assay. The representative flow cytometry contour plots are shown in the left panel of (A). At indicated by E:T ratios, the cytotoxicity of anti-ROR1 CAR exPBNK cells was significantly higher than mock NK cells against ROR1⁺ NB and osteosarcoma cells but not ROR1⁻ MCF-7 cells. Data are presented as mean ± SEM, n = 3. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 3

Anti-ROR1 CAR NK cells had significantly enhanced intracellular expression of CD107a, IFN-γ, granzyme B targeting ROR1⁺ NB cells The exPBNK cells electroporated with anti-ROR1 CAR mRNA (anti-ROR1-CAR) or H₂O (mock) were incubated with SH-SY5Y, SKNFI, or SKNBE(2)N at an E:T = 10:1 ratio for 4 h. (A) CD107a expression in exPBNK or anti-ROR1 CAR NK cells was detected with anti-human CD107a–APC antibody gated on CD56 cells (labeled with an anti-CD56–FITC antibody) by flow cytometry and significantly increased (p < 0.001, p = 0.004, p = 0.003, respectively). (B) IFN-γ expression in exPBNK or anti-ROR1 CAR NK cells was detected with anti-human IFN-γ-Alexa Fluor 647 antibody gated on CD56 cells (labeled with an anti-CD56-FITC antibody) by flow cytometry and significantly increased (p = 0.002, p = 0.005, p = 0.004, respectively). The same cells stained with isotype-matched controls were used for gating. (C) Granzyme B expression in exPBNK or anti-ROR1 CAR NK cells was detected with anti-human granzyme B-APC antibody gated on CD56 cells (labeled with an anti-CD56-FITC antibody) by flow cytometry and significantly increased (p = 0.007, p = 0.002, p = 0.003, respectively). (D) Perforin expression in exPBNK or anti-ROR1 CAR NK cells was detected with anti-human perforin-APC antibody gated on CD56 cells (labeled with an anti-CD56-FITC antibody) by flow cytometry and significantly increased (p = 0.001, p < 0.001, p < 0.001, respectively). The same cells stained with isotype-matched controls were used for gating. Data are presented as mean ± SEM, n = 3.

Figure 4

N-803 significantly enhanced the in vitro cytotoxicity with the enhanced release of granzyme B, IFN-γ, and perforin of anti-ROR1 CAR NK against NB cells (A) Mock NK or anti-ROR1 CAR NK cells were incubated with or without 3.5 ng/mL N-803 for 48 h and then used for in vitro cytotoxicity assays against SKNFI at an E:T = 3:1. N-803 significantly enhanced the in vitro cytotoxicity of anti-ROR1 CAR NK cells against SKNFI-Luc (p = 0.0001) compared to anti-ROR1 CAR NK cells. (B) After 24 h co-culture under the condition as described in (A), the supernatants were collected for ELISA assays to determine the released granzyme B level. Granzyme B level was significantly enhanced (p = 0.0009). (C) After 24 h co-culture under the condition as described in (A), the supernatants were collected for ELISA assays to determine the released IFN-γ level. IFN-γ level was significantly enhanced (p = 0.0147). (D) After 24 h co-culture under the condition as described in (A), the supernatants were collected for ELISA assays to determine the released perforin level. Perforin level was significantly enhanced (p = 0.0166). (E) Mock NK or anti-ROR1 CAR NK cells were incubated with or without 3.5 ng/mL N-803 for 48 h and then used for in vitro cytotoxicity assays against CHLA-255 at an E:T = 1:1. N-803 significantly enhanced the in vitro cytotoxicity of anti-ROR1 CAR NK cells against CHLA-255 (p = 0.0004) compared to anti-ROR1 CAR NK cells. (F) After 24 h co-culture under the condition as described in (E), the supernatants were collected for ELISA assays to determine the released granzyme B level. Granzyme B level was significantly enhanced (p = 0.0028). (G) After 24 h co-culture under the condition as described in (E), the supernatants were collected for ELISA assays to determine the released IFN-γ level. IFN-γ level was significantly enhanced (p = 0.0004). (H) After 24 h co-culture under the condition as described in (E), the supernatants were collected for ELISA assays to determine the released perforin level. Perforin level was significantly enhanced (p = 0.0140). Data are presented as mean ± SEM, n = 3–4.

Figure 5

High-dimensional analysis of N-803 activated anti-ROR1 CAR NK cells with/without NB by mass cytometry Anti-ROR1 CAR NK cells or NK cells were co-cultured with or without SKNFI and N-803 for 2 days. After the cells were stained and fixed, the samples were run on a CyTOF2. The experiment was repeated 3 times using NK cells from 3 different donors. (A) The representative viSNE plots show pStat5 in anti-ROR1 CAR NK cells or NK cells under the indicated conditions by mass cytometry. (B) The representative viSNE plots show Ki67 levels in anti-ROR1 CAR NK cells or NK cells under the indicated conditions by mass cytometry.

Figure 6

The combination of anti-ROR1 CAR NK + N-803 significantly extended the survival of NB xenografted NSG mice (A) 1 × 10⁶ of SKNFI-Luc cells were intraperitoneally injected in NSG mice on day 0. After confirming the tumor engraftment at day 7, 5 × 10⁶ exPBNK cells, 5 × 10⁶ anti-ROR1 CAR exPBNK cells, or PBS was intraperitoneally injected to each mouse once a week for 6 weeks. The mice treated with anti-ROR1 CAR NK cells (n = 8) significantly extended the survival of SKNFI xenografted mice compared to the control groups that were treated with PBS (n = 8, p = 0.0019) or mock NK cells (n = 8, p = 0.0073). (B) 1 × 10⁶ of SKNFI-Luc cells was intraperitoneally injected in NSG mice on day 0. After confirming the tumor engraftment at day 7, 5 × 10⁶ exPBNK cells, 5 × 10⁶ anti-ROR1 CAR exPBNK cells, 5 × 10⁶ exPBNK cells + 0.2 mg/kg N-803, 5 × 10⁶ anti-ROR1 CAR exPBNK cells + 0.2 mg/kg N-803, or PBS was intraperitoneally injected into each mouse once a week for 6 weeks. The mice treated with anti-ROR1 CAR NK cells + N-803 (n = 8) significantly extended the survival of SKNFI xenografted mice compared to the control groups that were treated with NK + N-803 (n = 9, p = 0.018) or anti-ROR1 CAR NK cells alone (n = 7, p = 0.055). (C) Representative bioluminescence images of mice of each group of (B) are shown at day 7, day 81, and day 137. (D) 1 × 10⁶ of CHLA255-Luc cells was intraperitoneally injected in NSG mice on day 0. After confirming the tumor engraftment at day 7, 5 × 10⁶ exPBNK cells, 5 × 10⁶ anti-ROR1 CAR exPBNK cells, 5 × 10⁶ exPBNK cells + 0.2 mg/kg N-803, 5 × 10⁶ anti-ROR1 CAR exPBNK cells + 0.2 mg/kg N-803, or PBS was intraperitoneally injected into each mouse once a week for 6 weeks. The mice treated with anti-ROR1 CAR NK cells + N-803 (n = 9) significantly extended the survival of CHLA255 xenografted mice compared to the control groups that were treated with mock NK + N-803 (n = 9, p = 0.032) or anti-ROR1 CAR NK cells alone (n = 10, p < 0.021).