Effective combination treatment of GD2-expressing neuroblastoma and Ewing's sarcoma using anti-GD2 ch14.18/CHO antibody with Vγ9Vδ2+ γδT cells

Abstract

Gamma delta T lymphocytes (γδT cells) have pleiotropic properties including innate cytotoxicity, which make them attractive effectors for cancer immunotherapy. Combination treatment with zoledronic acid and IL-2 can activate and expand the most common subset of blood γδT, which express the Vγ9Vδ2 T cell receptor (TCR) (Vδ2 T cells). Vγ9Vδ2 T cells are equipped for antibody-dependent cell-mediated cytotoxicity (ADCC) through expression of the low-affinity FcγR CD16. GD2 is a highly ranked tumor associated antigen for immunotherapy due to bright expression on the cell surface, absent expression on normal tissues and availability of therapeutic antibodies with known efficacy in neuroblastoma. To explore the hypothesis that zoledronic acid, IL-2 and anti-GD2 antibodies will synergize in a therapeutic combination, we evaluated in vitro cytotoxicity and tumor growth inhibition in the GD2 expressing cancers neuroblastoma and Ewing's sarcoma. Vδ2 T cells exert ADCC against GD2-expressing Ewing's sarcoma and neuroblastoma cell lines, an effect which correlates with the brightness of GD2 expression. In an immunodeficient mouse model of small established GD2-expressing Ewing's sarcoma or neuroblastoma tumors, the combination of adoptively transferred Vδ2+ T cells, expanded in vitro with zoledronic acid and IL-2, with anti-GD2 antibody ch14.18/CHO, and with systemic zoledronic acid, significantly suppressed tumor growth compared to antibody or γδT cell-free controls. Combination treatment using ch14.18/CHO, zoledronic acid and IL-2 is more effective than their use in isolation. The already-established safety profiles of these agents make testing of the combination in GD2 positive cancers such as neuroblastoma or Ewing's sarcoma both rational and feasible.

Keywords: Ewing's Sarcoma; Gamma-delta T cells; antibody-dependent cytotoxicity; neuroblastoma; translational.

Figure 1.

Killing of Ewing's sarcoma cell lines in 4 h chromium release assays by (A) Vδ1+ γδT cells and (B) Vδ1–/Vγ2– γδT cells. Representative data showing one of five donors.

Figure 2.

(A) 24 h treatment with 5 μM zoledronic acid sensitizes the Ewing's sarcoma cell line TC71 to antibody independent lysis by Vδ2+ γδT cells (n = 7, ***p = 0.001). (B) Comparative GD2 staining of Ewing's sarcoma and Neuroblastoma cell lines determined by flow cytometry and expressed as specific fluorescence intensity (SFI). SFI is calculated by dividing the median fluorescence intensity of a sample stained with PE-anti-GD2 with the median fluorescence intensity of a sample of the same cells stained with PE-isotype control.

Figure 3.

(A) Killing of wild-type Ewing's sarcoma cell lines by expanded Vδ2+ γδT cells opsonized with ch14.18/CHO anti-GD2 antibody or in the presence of a control antibody (Rituximab) (n = 3–6, **p = 0.0099). (B) Killing of isogenic GD2^bright and GD2^dim DC-ES6 Ewing's cell lines by Vδ2+ γδT cells in the presence of ch14.18/CHO anti-GD2 opsonizing antibody or Rituximab control antibody (n = 3, ***p = 0.0033). (C) IFNγ expression of Vδ2+ γδT cells in the presence of GD2^bright DC-ES6. Elimination of the DC-ES6 population and production of IFNγ is only seen when DC-ES6 is opsonized with ch14.18/CHO. (D) Correlation between the GD2 stain (PE-SFI) of Ewing's sarcoma and neuroblastoma cell lines and the degree to which Vδ2+ γδT cells exert ADCC or AIC against them at effector:target ratio 10:1. R value calculated by Spearman correlation.

Figure 4.

(A) To determine whether the correlation between ADCC and antigen expression was due to cellular stress imparted by the binding of antibody to GD2, we examined the killing of neuroblastoma and Ewing's sarcoma lines opsonized with ch14.18/CHO (20 μg/mL). High levels of spontaneous cell death were only seen in the GD2⁺ neuroblastoma lines, despite GD2 expression being higher in some of the Ewing's sarcoma lines (n = 9–42, depending on cell line, ****p = <0.0001). (B) To confirm that killing of neuroblastoma cell lines was not simply due to the binding and activation of residual complement in the serum, we compared the effects in the presence or absence of serum, and saw no significant difference (n = 5). The data is expressed as the “% of maximum killing” and represents the ⁵¹Cr release from opsonized cells incubated in medium for 4 h as a percentage of the ⁵¹Cr release of the same cells treated with Triton X, minus the paired result from cells treated with control antibody.

Figure 5.

(A) Treatment schedule of mice used in in vivo experiments (B) Engraftment of Vγ9Vδ2+ γδT cells in NSG mice bearing TC71 Ewing's sarcoma (representative of six mice sampled) or Kelly neuroblastoma (representative of eight mice sampled) xenografts. Samples taken after culling, following three serial intravenous injections of 1 × 10⁶ γδT cells. (C) Fold change in tumor size of TC71 xenografts in NSG mice receiving Vγ9Vδ2 + zoledronate, Vγ9Vδ2 + zoledronate + ch14.18/CHO or zoledronate + ch14.18/CHO (D) – fold change in tumor size of Kelly xenografts in NSG mice receiving Vγ9Vδ2 + zoledronate, Vγ9Vδ2 + zoledronate + ch14.18/CHO or zoledronate + ch14.18/CHO.