Targeting GD2-positive Refractory/Resistant Neuroblastoma and Osteosarcoma with Anti- CD3 x Anti-GD2 Bispecific Antibody Armed T cells

Abstract

Background: Since treatment of neuroblastoma (NB) with anti-GD2 monoclonal antibodies provides a survival benefit in children with minimal residual disease and our preclinical study shows that anti-CD3 x anti-GD2 bispecific antibody (GD2Bi) armed T cells (GD2BATs) were highly cytotoxic to GD2+ cell lines, we conducted a phase I/II study in recurrent/refractory patients to establish safety and explore the clinical benefit of GD2BATs.

Methods: The 3+3 dose escalation study (NCT02173093) phase I involved 9 evaluable patients with NB (n=5), osteosarcoma (OST) (n=3), and desmoplastic small round cell tumors (DSRCT) (n=1) with twice weekly infusions of GD2BATs at 40, 80, or 160 x 10⁶ GD2BATs/kg/infusion with daily interleukin 2 (300,000 IU/m²) and twice weekly granulocyte-macrophage colony stimulating factor (250 μg/m²). Phase II portion of the trial was conducted in patients with NB at the dose 3 level of 160 x 10⁶ GD2BATs/kg/infusion but failed to enroll the planned number of patients.

Results: Nine of 12 patients in the phase I completed therapy. There were no dose limiting toxicities (DLTs). All patients developed mild and manageable cytokine release syndrome (CRS) with grade 2-3 fevers/chills, headaches, and occasional hypotension up to 72 hours after GD2BAT infusions. GD2-antibody associated pain was not significant in this study. The median OS for patients in the Phase I and limited Phase II was 18.0 and 31.2 months, respectively, whereas the combined OS was 21.1 months. There was a complete bone marrow response with overall stable disease in one of the phase I patients with NB. Ten of 12 phase II patients were evaluable for response: 1 had partial response. Three additional patients were deemed to have clinical benefit with prolonged stable disease. More than 50% of evaluable patients showed augmented immune responses to GD2+ targets after GD2BATs as measured by interferon-gamma (IFN-γ) EliSpots, Th1 cytokines, and/or chemokines.

Conclusions: Our study demonstrated safety of up to 160 x 10⁶ cells/kg/infusion of GD2BATs. Combined with evidence for the development of post treatment endogenous immune responses, this data supports further investigation of GD2 BATs in larger Phase II clinical trials.

Keywords: Targeted T cells; anti-CD3 x anti-GD2 bispecific antibody; bispecific antibodies; hu3F8; neuroblastoma; osteosarcoma.

Figure 1

A) Protocol Schema. Shows the treatment schema for the Phase I/II study. B) Survival Curves for Phase I and Phase II. Shows the K-M Curves for Phase I (Blue, OS =18.0 months), Phase II (Red, OS=31.2 months), and All patients (Green, OS=21.0 months).

Figure 2

A) Shows a bone marrow response with clearance of neuron-specific enolase (NSE) positive tumor cells in the bone marrow of a NB patient after 8 infusions of GD2 BATs. B) Shows the metabolic PET changes after 5 infusions of GD2 BAT in an OST patient. C) Flow cytometry staining for CD4+ and CD8+ to evaluate presence of tumor infiltrating lymphocytes in the tumor for Pt # 20115. Most intranasal tumor infiltrating lymphocytes were CD8⁺. Cells were gated on CD3⁺ cells and analyzed for FITC positive CD4 and PE positive CD8 T cells. D) Summarizes the proportion of CD3+A1G4+ cells in the peripheral blood of patient # 8 pre-infusion and at various times during and after GD2 BATs infusions., % of CD3⁺A1G4⁺ cells shows the persistence of GD2 BATs in peripheral blood. CD3⁺A1G4⁺ cells were measured by using 2 color FACS gated on lymphocytes using anti-CD3 and A1 G4 anti-idiotypic antibody for hu3F8.

Figure 3

Anti-GD2 Cytotoxicity (IFN-γ EliSpots). Each panel shows the pre-IT and post-IT IFN-γ EliSpots responses from PBMC after overnight stimulation. Phase II portion: Panel a, b, and c show responses of PBMC from the phase II NB patients to KCNR (p<0.02), MG63 (p<0.02), and K562 (NS). Phase I/II combined: Panels d, e, and f show IFN-γ EliSpots responses of PBMC from all 18 of the phase I/II patients pre-IT and post-IT after overnight exposure to KCNR (p<0.03), MG83 (p<0.04), and K562 (Not significant, NS), respectively.

Figure 4

Cytokines/chemokines: Panel a (IL-12) shows significant changes between pre-IT and post-IT levels of IL-12 in all 18 Phase I/II patients (p<0.002). Panel b (IL-12) shows a significant change between pre-IT and post-IT IL-12 levels for 10 NB in the phase II patients analyzed separately (p<0.02). Panel c (MIP-1β) shows post-IT levels of MIP-1β increased significantly over pre-IT levels in 28 phase I/II patients(p<0.02). Panel d (IL-10) shows the levels of IL-10 significantly increased from pre-IT to post-IT in all 18 phase I/II patients (p<0.02). Panels e, f, and g show the pre-IT and post-IT serum levels of IL-6, TNF-α, and IP10 (all NS).