Introduction: Targeting disialoganglioside GD2 with monoclonal antibodies (mAbs) significantly improves survival in high-risk neuroblastoma (NB) patients after autologous or allogeneic SCT. However, GD2 expression is heterogeneous and the recently approved anti-GD2 mAb CH14.18 causes severe adverse effects, e.g., neuropathy and neuropathic pain due to neurotoxicity. Additional or alternative target antigens might improve therapy. B7-H3 belongs to the B7-CD28 family and is thought to function as an immune checkpoint by regulating T and NK cell response. B7-H3 is highly overexpressed on many solid tumors, correlating with poor prognosis and outcome. However, on healthy tissue its protein expression is very limited, thus making B7-H3 an interesting target for cancer immunotherapy. Therefore, we evaluated the use of B7-H3 as an alternative to GD2 and investigated different anti-B7-H3 mAb constructs and mAb-cytokine fusions (immunocytokines) for their ability to elicit antibody-dependenT-cellular cytotoxicity (ADCC). Methods: Derived from a parent anti-B7-H3 clone (HEK5-1B3), five additional mAb constructs were engineered: (1) chimeric with human IL-2 fusion (cHEK5-IL2), (2) chimeric and Fc-optimized (SDIE) w/o fusion (cHEK5opt), (3) cHEK5opt fused with human IL-2 (cHEK5opt-IL2), (4) cHEK5opt fused with human IL-15 (cHEK5opt-IL15), and (5) cHEK5-IL2 produced in rat myeloma YB2/0 to create an optimized low-fucose version (cHEX5LF-IL2). All IL-2 fusions were to the C-terminus of the light chain. The abilities of all six anti-B7-H3 mAb constructs and the GD2-specific mAb CH14.18 to mediate ADCC were compared in vitro in cytotoxicity assays using calcein release assays and the RTCA xCELLigence system. TargeT-cells: NB cell lines expressing high levels of B7-H3 but variable levels of GD2 (LAN-1, Kelly, SH-SY5Y). Effector cells: Human expanded NK cells (eNKs), expanded γ/δ T-cells and patient PBMCs after allogeneic SCT. Results: Except the parent clone, all anti-B7-H3 mAb constructs were able to elicit ADCC. TargeT-cell lysis of LAN-1 (high expression of both GD2 and B7-H3) mediated by the optimized anti-B7-H3 immunocytokines was comparable or even better than that mediated by CH14.18 (effectors: eNKs; calculated after 36 hrs.; in ascending order): Targets + effectors w/o mAb (24 %), parent pHEK5 (29 %), cHEK5opt (44 %), cHEK5-IL2 (76 %), cHEK5opt-IL15 (85 %), CH14.18 (90 %) and cHEK5opt-IL2 (97 %). Recently, we produced the low-fucose immunocytokine cHEX5LF-IL2. In a direct comparison with all other mAb constructs the cHEX5LF-IL2 immunocytokine mediated best targeT-cell lysis against SH-SY5Y (effectors: eNKs; calculated after 48 hrs.; in ascending order): Targets + effectors w/o mAb (31 %), CH14.18 (42 %), parent pHEK5 (46 %), cHEK5-IL2 (80 %), cHEK5opt (85 %), cHEK5opt-IL2 (93 %), cHEK5opt-IL15 (96 %) and low-fucose cHEX5LF-IL2 (100 %). Using expanded γ/δ T-cells of healthy donors we were able to confirm cHEX5LF-IL2 to be the most effective anti-B7-H3 mAb. Interestingly, cHEK5-IL2 showed comparable targeT-cell lysis; however, lysis was only transient while cHEX5LF-IL2 mediated permanent target cell lysis. Using patient PBMCs after receiving allogeneic SCT target cell lysis mediated by cHEX5LF-IL2 and CH14.18 was comparable. Calculated specific lysis of LAN-1 (after 96 hrs.; in ascending order): Targets + effectors w/o mAb (22 %), parent pHEK5 (26 %), cHEK5opt-IL2 (40 %), cHEK5opt (49 %), cHEK5-IL2 (57 %), cHEK5opt-IL15 (70 %), low-fucose cHEX5LF-IL2 (78 %) and CH14.18 (81 %). Conclusion: B7-H3 has been demonstrated to be a suitable alternative target antigen in case GD2 expression is low or absent. Fc-optimized mAbs and mAb-cytokine fusions targeting B7-H3 might increase the efficacy of immunotherapy in GD2-negative tumors and in combinatory approaches. So far, the low-fucose immunocytokine cHEX5LF-IL2 seems to be the most promising anti-B7-H3 construct.

Citation Format: Florian Heubach, Patrick Schlegel, Latifa Zekri, Timo Manz, Sabine Schleicher, Armin Rabsteyn, Gundram Jung, Hans-Jörg Bühring, Stephen D. Gillies, Rupert Handgretinger, Peter Lang. Targeting B7-H3 (CD276) in neuroblastoma: In vitro evaluation of Fc-optimized antibodies and immunocytokines [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A131.