Humanized T Cell Receptor-like Antibody Hu8F4 Reduces Leukemia By Fc-Mediated Mechanism

Abstract

8F4, a T cell receptor (TCR) - like antibody which targets the leukemia-associated antigen PR1 (VLQELNVTV) when presented in the context of MHC class I (HLA-A2:01), is a mouse IgG2a monoclonal antibody (mAb) which mediates both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in vitro and eliminates AML in PDX (patient derived xenograft) mouse models. Recognizing the potential therapeutic value of 8F4, we produced a humanized IgG1 (Hu8F4) as a first-in-class mAb for preclinical development and clinical use. Characterization of Hu8F4 by ELISA and surface plasmon resonance showed high affinity (KD=6.5 nM) and specificity of Hu8F4 binding to PR1/HLA-A2 monomer. Staining of peptide-pulsed T2 cells confirmed Hu8F4 specificity to PR1, but not to control peptide MART1. To study anti-leukemia activity of Hu8F4 we used HLA-A2-transfected leukemia cell lines U937 (U937-A2) and MV411 (MV411-A2), as well as THP1 (endogenous HLA-A2+) as targets. In contrast to mouse 8F4, Hu8F4 did not induce CDC in vitro. Rather, Hu8F4 mediated both potent antibody-dependent cellular phagocytosis (ADCP) and ADCC of U937-A2 and THP1 cells in NFAT reporter assays, using FcgRI- (EC50 = 0.002 µg/ml), FcgRII- (EC50 = 1.49 µg/ml), and FcgRIII- (EC50 = 0.04 µg/ml) transfected Jurkat cells, as well as pooled human PBMC (EC50 = 0.95 µg/ml) and donor-derived macrophages (EC50 = 1.1 µg/ml), as effectors. Based on the ADCP activity of Hu8F4, we sought to examine the potential synergy of anti-CD47 antibody, a macrophage checkpoint inhibitor, with Hu8F4. Treatment of U937-A2 and THP1 target cells with anti-CD47 F(ab')2 neutralizing antibody made cells susceptible to Hu8F4-mediated phagocytosis by NSG mouse bone marrow derived macrophages (BMDM) in vitro. To confirm the critical role of CD47 in Hu8F4-mediated ADCP we created CD47 KO U937-A2 and THP1 AML lines. Within 96 hours of treatment with Hu8F4 both CD47 KO cell lines were completely phagocytosed by NSG BMDM, thus confirming the role of phagocytosis as a mechanism. To test Hu8F4 activity in vivo, we established xenografts using luciferase-transfected U937-A2 and MV411-A2 cell lines in NSG mice. Hu8F4 was then administered starting on either day 3 (U937-A2) or day 7 (MV411-A2). Treatment with Hu8F4 resulted in elimination of leukemia as measured by FACS and BLI and increased median survival (from 33 days to >85 days for U937-A2, p=0.0075; and from 125 days to >225 days for MV411-A2, p=0.0033). To further test Hu8F4 efficacy, we established PDX in NSG mice using cells from HLA-A2+ refractory/relapsed AML patients. Hu8F4 treatment reduced leukemia by 90.3% to 99.9% when assessed by FACS in four out of five PDX and resulted in increased median survival of the mice (from 41 days to 49 days, p=0.0002) in one PDX tested. To study the mechanism of action of Hu8F4, we produced a 3 aa mutant (Hu8F4 PG LALA), which completely abrogated binding to Fcg receptors. Hu8F4 PG LALA, as well as Hu8F4 F(ab')2, showed no in vivo activity against U937-A2 or MV411-A2 xenografts, confirming that the anti-leukemia activity of Hu8F4 is dependent on its ability to bind Fc receptors. Because FcgR may be expressed on AML we tested fratricide as a possible in vivo mechanism of Hu8F4. We utilized the CRISPR /Cas9 system to edit out both FcgRI and II in the U937-A2 cell line that is naturally deficient in FcgRIII (DKO U937-A2). We then used a retroviral system to stably express FcgRI or FcgRII in U937-A2 to create FcgRI-U937-A2 and FcgRII-U937-A2, respectively, and tested their susceptibility to Hu8F4 in the xenograft model. Compared to FcR-expressing wild-type U937-A2, Hu8F4 showed similar anti-leukemia activity in all three xenografts. There was no significant difference noted in the anti-leukemia potency of Hu8F4 between DKO U937-A2 or single FcR-expressing xenografts compared with FcgRI/FcgRII expressing wild-type U937-A2 xenografts, suggesting that fratricide is an unlikely mechanism in this model. Here we demonstrate high specificity, affinity, and remarkable anti-AML activity of Hu8F4, a first-in-class humanized TCR mimic monoclonal antibody that is currently being tested in a phase I clinical trial for HLA-A2+ refractory/relapsed AML and MDS. This study provides evidence for the importance of FcgR-mediated mechanisms (ADCC and ADCP) in the activity of Hu8F4. In addition, our results support future combination trials with treatments that promote phagocytosis.