Abstract 1335: Dissecting the mechanisms of anti-tumor protection mediated by an antibody targeting the transferrin receptor 1
Authors: Leoh Lai Sum , Daniels-Wells Tracy R. , Martinez-Maza Otoniel , Penichet Manuel L. 
Published: August 2015

Abstract
The transferrin receptor 1 (TfR1/CD71) is a type II transmembrane homodimeric protein involved in iron uptake and regulation of cell growth. The elevated levels of TfR1 expression on malignant cells, together with its extracellular accessibility, make TfR1 an attractive target for antibody therapy. We developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which exhibits direct cytotoxic activity against certain human malignant B cells in vitro. This anti-tumor activity is due to the induction of TfR1 degradation and lethal iron deprivation. Importantly, ch128.1 also shows extraordinary anti-tumor activity in two xenograft models of the incurable B-cell malignancy multiple myeloma in immunosuppressed mice (SCID-Beige) resulting in up to 100% survival using a single dose. Interesting, this anti-tumor activity occurs even against malignant cell lines that show limited or no sensitivity to ch128.1 in vitro. The mechanism of in vivo protection of ch128.1 is currently unknown. In order to explore the possible role of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) in the anti-tumor mechanism of ch128.1 and to delineate the residues involved in these effector functions, we developed three constructs with mutations in the ch128.1 heavy chain: 1) L234A/L235A, 2) P331S, and 3) L234A/L235A/P331S. We now show that ch128.1 is capable of inducing ADCC and CDC against malignant B cells in vitro suggesting the potential contribution of these antibody effector functions to its effects in vivo. Additionally, we show that the triple mutant consistently displayed significant ADCC reduction in vitro, while the other mutants showed a weaker impairment, suggesting that all three residues are relevant contributors to ADCC. Lack of CDC in the P331S mutant confirmed the critical role of this residue, while the other residues show a lesser role in CDC in vitro. These results provide, for the first time, insights into the human IgG3 effector functions in the context of TfR1 targeting of malignant cells. Intriguingly, we have recently observed that the strong in vivo protection of ch128.1 against disseminated malignant B cells is abolished when the triple ch128.1 mutant (L234A/L235A/P331S) is used as a therapeutic at the same dose as that of ch128.1. These results point to a functional role for the antibody Fc fragment in anti-tumor protection mediated by ch128.1. Further studies aimed to provide a better understanding of the in vivo activity exhibited by ch128.1 are in progress. This antibody may be potentially used in the therapy of incurable human B-cell malignancies, such as multiple myeloma and certain non-Hodgkin lymphomas.
Citation Format: Lai Sum Leoh, Tracy R. Daniels-Wells, Otoniel Martinez-Maza, Manuel L. Penichet. Dissecting the mechanisms of anti-tumor protection mediated by an antibody targeting the transferrin receptor 1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1335. doi:10.1158/1538-7445.AM2015-1335