Abstract A140: A T-cell utilizing bispecific anti-CD3/GD2 construct mediates superior in vitro efficacy compared to CH14.18 mAb in neuroblastoma patients after allogeneic SCT

Abstract

Abstract Introduction: Whereas immunotherapy in high-risk/relapsed neuroblastoma with standard antibody formats targeting the tumor associated antigen GD2 is currently used after autologous/allogeneic stem cell transplantation (SCT), T-cells have rarely been addressed as effectors. Bispecific antibodies can bring T-cells into close proximity to tumor cells and activate them through the CD3 region, which ultimately leads to targeT-cell elimination. They lack the Fc-domain and cannot trigger ADCC or CDC as NK-cells. An example for bispecific antibodies is NG-CU (developed at the Department of Immunology, University of Tuebingen), which recognizes CD3 and GD2 as target antigens. The tumor antigen specific antibody domain, derived from a fab fragment of GD2 14.18 antibody and the T-cell recruiting antibody domain, derived from single chain variable fragment (scFv fragment) of UCHT-1 (CD3) are linked by modified CH2 domain. We investigated whether the bispecific antibody NG-CU might be an alternative to the therapeutic monoclonal antibody CH14.18, which mediates CDC and ADCC through NK-cells. Method: Different antibody concentrations and effector to target ratios (E:T) were evaluated using the xCELLigence RTCA system, PBMCs (healthy donors and patients after allogeneic SCT) and the neuroblastoma cell lines LS and LAN-1. CDC was evaluated using autologous serum.Results: NG-CU showed enhanced cytotoxicity compared to CH14.18. Median specific lysis (n=3) after 12/24/48 hrs (effectors: healthy PBMCs, E:T=5:1) with LS cells was: 55/73/77% (CH14.18, 1 µg/ml) vs. 70/100/100% (NG CU, 100ng/ml). P values with Mann-Whitney test: p=0,0244; p<0,0001; p<0,0001. With LAN-1 cells: 56/69/88% (CH14.18, 1 µg/ml) vs. 9/46/99% (NG-CU, 100 ng/ml). P values with Mann-Whitney test: p<0,0001; p=0,1139; p=0,0464.Taking CDC into account by adding autologous serum, cytotoxicity of CH14.18 was drastically increased. ADCC and CDC eradicated targets with 64% after 4 hrs and 93% after 48 hrs (E:T=5:1). However, using a lower E:T ratio (1:1) lysis of CH14.18 remained static, but not for NG-CU. Median lysis (n=3) after 12/24/48 hrs: 71/74/77% (CH14.18) vs. 25/42/67% (NG-CU). After >48 hrs targets were completely eradicated by NG-CU, while targets treated with CH14.18 still proliferated.Using patient PBMCs, significant lysis with both constructs was detected dependent on percentages and total numbers of T- and NK-cells. In patients early after SCT, NK-cells represented the majority of effectors. Here, CH14.18 produced higher lysis, whereas later on, associated with increasing T-cell counts, NG-CU was more effective. With both antibody constructs complete eradication of neuroblastoma cells (LS+LAN-1) was detectable. Conclusion: NG-CU showed enhanced cytotoxicity compared to CH14.18 even in lower concentrations and E:T ratios. The bispecific design represents an alternative to classical CH14.18 based therapies. Dependent on the differential recovery of effector cells post-transplant, either NK-cell based or T-cell based antibody constructs might result in optimal antitumor activity. Citation Format: Anne-Marie Lang, Gundram Jung, Ursula Seidel, Florian Heubach, Armin Rabsteyn, Patrick Schlegel, Christian Seitz, Emmanuelle Moraes Ribeiro, Rupert Handgretinger, Peter Lang. A T-cell utilizing bispecific anti-CD3/GD2 construct mediates superior in vitro efficacy compared to CH14.18 mAb in neuroblastoma patients after allogeneic SCT [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 A140.