Abstract 4993: Novel fully human anti-GD2 monoclonal antibodies with potent therapeutic activity against neuroblastoma, sarcoma and melanoma
Authors: Ragupathi Govind , Wu Xiaohong , Sawada Ritsuko , Livingston Philip O. , Scholz Wolfgang W. 
Published: July 2016

Background: Gangliosides such as GD2, GD3 and GM2 are promising targets for antibody-mediated cancer therapy since they are expressed at high levels on the surface of several cancers, including neuroblastomas, sarcomas and melanomas. Anti-GD2 monoclonal antibodies have shown promising clinical outcomes in neuroblastoma and a chimeric anti-GD2 antibody (Unituxin™) was recently approved by FDA. However, murine-derived antibodies discovered so far show adverse effects that limit their clinical utility. Fully human antibodies derived from immunized patients might be able to overcome these limitations, since epitope specificity has evolved in the human tissue environment. Here we describe the characterization and functional evaluation of two potential development candidates.

Methods: PBMCs from patients immunized with GD2 lactone-keyhole limpet hemocyanin (GD2L-KLH) conjugate vaccine were utilized to isolate fully human monoclonal antibodies (humAbs). HumAbs were selected for 1) specificity against GD2 initially by ELISA and cell surface binding by flow cytometry 2) by glycan array and affinity assays and 3) by effector functions in complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC) assays. The therapeutic potential of lead antibodies was further evaluated in xenograft models in SCID mice using GD2 positive CHLA255 neuroblastoma as well as with TC71 and SaoS2 sarcoma cell lines.

Results: Antibodies that bind specific to purified GD2 by ELISA were expressed as recombinant antibodies in CHO cells. Many recovered antibodies showed cross-reactivity with several gangliosides in ELISA assays. Two antibodies with high specificity for GD2, 1B7 and 31F9 were selected for further studies. Binding to native antigen expressed on the cell surface of different cell lines was confirmed by FACS analysis. 31F9 was monospecific for GD2 (affinity by Surface Plasmon Resonance was ∼4 nM) while 1B7 showed dual specificity for GD2 and GM2 with affinities of ∼1 nM for GD2 and ∼ 370 nM for GM2, respectively. These two antibodies induced CDC ranged between 35% and 80%, and ADCC with human PBMCs between 40% and 65% on CHLA255, TC71 and SaoS2 cell lines. Survival following IV or SC challenge with SaoS2 and TC71 cells (respectively) was at least doubled following six IP treatments with 200 mcg of either 1B7 or 31F9 twice weekly. Inhibition of CHLA255 tumor growth was less dramatic.

Conclusions: Vaccinated patients produce antigen-specific IgG antibodies that were further analyzed on the molecular level. Antibodies with single and dual specificity (GD2 and GM2) were recovered and shown to be very active in functional assays. Based on these favorable in vitro and in vivo studies, 1B7 and 31F9 humAbs merit further development efforts to evaluate potential utility for treatment of GD2 positive cancers.

Citation Format: Govind Ragupathi, Xiaohong Wu, Ritsuko Sawada, Philip O. Livingston, Wolfgang W. Scholz. Novel fully human anti-GD2 monoclonal antibodies with potent therapeutic activity against neuroblastoma, sarcoma and melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4993.