CD47 Blockade Enhances Therapeutic Activity of TCR Mimic Antibodies to Ultra-Low Density Cancer Epitopes through Cytokine Feed Forward Mechanisms

INTRODUCTION

T Cell Receptor mimic (TCRm) antibodies to low-density peptide epitopes from undruggable intracellular proteins presented in the context of major histocompatibility (MHC) molecules are therapeutically effective in mouse models of human cancers. CD47 blockade by use of a high affinity SIRPα variant protein (CV1) has been shown to improve the effects of monoclonal antibodies to high-density antigens in tumor models by enhancement of antibody dependent cellular phagocytosis (ADCP). We asked if combination therapy with a TCRm antibody to Preferentially Expressed Antigen in Melanoma (PRAME) could enhance activity of both drugs in vitro and in vivo. Additionally, we explored the role of macrophage-secreted cytokines in the enhanced in vivo activity.

METHODS

We performed in vitro ADCP assays with human acute myeloid and acute lymphoid leukemia cell lines containing antigens of interest using the two agents alone and in combination. We performed therapy experiments in NSG mice using the same leukemia cell lines transformed with a luciferase vector and measured tumor burden through bioluminescent imaging. Survival was measured. We examined cell-surface expression of epitopes of interest and HLA on cell lines in vitro after incubation with IFNγ and TNFα using flow cytometry and performed in vitro ADCP assays with the leukemia cell lines after pretreatment with IFNγ.

RESULTS

CV1 and TCRm antibody showed additive effects in vitro with a statistically significant increase in phagocytosis in both antigen positive cell lines with combination therapy versus single agent therapy. CV1 and TCRm antibody showed greater than additive therapeutic effects in vivo with a 3-log reduction in leukemia burden relative to control untreated mice and a 5-10 fold reduction relative to single agent groups. After therapy was stopped, mice treated with the combination had statistically significant increases in survival (p<0.0001). IFNγ and TNFα led to up-regulation of cell surface HLA-A*02:01. Additionally, the cytokines led to up-regulation of the PRAME derived epitope of interest. Pretreatment of human leukemia cell lines with IFNγ led to statistically significant increases in ADCP in vitro.

CONCLUSIONS

The elimination of anti-phagocytic signal produced by CD47 blockade with the high affinity SIRPα variant CV1, combined with the pro-phagocytic signal of Fc receptor engaging TCRm was effective even with an ultra-low density epitope (700-3000 sites per cell) in vitro and in vivo. A greater than additive effect was seen in two tumor models. These results support the potency of this drug combination. The large enhancement in activity in vivo vs. in vitro may be explained by macrophage-released cytokines leading to increased presentation of epitopes of interest and increased tumor kill.