In vivo imaging of T-cell coregulator B7-H4 reveals protumor macrophage status in prostate cancer

Abstract

BackgroundB7-H4 is a cell surface ligand overexpressed by tumors to inhibit T cell functions and evade the immune system. B7-H4 is minimally expressed in normal tissues but is highly expressed by various cancer cells and tumor-associated macrophages (TAM). Despite its importance as an immune checkpoint inhibitor, no imaging techniques specifically targeting B7-H4 have been established. To close this gap, we sought to assess the ability of a monoclonal antibody (mAb) based immunoPET radiotracer to visualize B7-H4 in human and murine prostate cancer models. MethodsAnti-B7-H4 mAb clone 2H9 was functionally characterized for binding to the human and mouse B7-H4 protein. The antibody was conjugated with chelator p-SCN-Bn-Deferoxamine (DFO) and labeled with radioisotope Zirconium-89 (89Zr) to obtain immunoPET tracer 89Zr-2H9-mAb. The biolayer interferometry method was used to test the binding kinetics of DFO-2H9-mAb compared to that of parental 2H9 mAb. A group of six athymic nude mice with human DU145 prostate tumor xenograft underwent MicroPET imaging after tail vein injection of [~]150{micro}Ci 89Zr-2H9-mAb or non-binding 89Zr-Isotype-mAb. Next, immunocompetent C57BL/6J mice with TRAMP-C2 tumors each were injected with either PBS (n=8), cold 2H9 mAb (10mg/kg) to block B7-H4 (n=6), or chlodronate liposome (15mg/kg) to cause total macrophage depletion (n=6), followed by 89Zr-2H9-mAb MicroPET imaging. An ex vivo biodistribution assay was performed after 144 hr post radiotracer injection. Tumor radiotracer binding, quantified as a percentage injected dose per gram (%ID/g), was compared between different experimental groups using two-way ANOVA with Bonferroni or Tukey corrections. ResultsImmunoconjugation yielded a 2.59 {+/-} 0.08 chelator-to-antibody ratio, and the binding of DFO conjugated 2H9-mAb was similar to that of parental 2H9 mAb, with unaffected affinity in targeting B7-H4 protein moiety. The radiochemical purity of 89Zr-2H9-mAb tracer was yielded >95% with an average specific activity of 5{micro}Ci/{micro}g antibody. DU145 tumor xenografts demonstrated significantly stronger radiotracer binding at 24, 48, 72, 96, and 120 hr than the non-binding isotype control group. In TRAMP-C2 tumor xenografts, the radiotracer binding in B7-H4 blocked tumors was significantly lower than in the non-blocked PBS-injected group. Macrophage depletion resulted in a significant decrease in tumor binding compared to the control group. 89Zr-2H9-mAb could efficiently distinguish tumors with high sensitivity, showing a high correlation between PET imaging and bio-distribution. More importantly, the immunohistochemistry of the harvested tumor revealed no significant difference between the three groups, as discernible through in vivo PET imaging. ConclusionThis study highlights the potential of B7-H4 immunoPET imaging for monitoring immunotherapy response. With the emerging potential of B7-H4 blocking as an immunotherapeutic, immunoPET imaging could be readily expanded to patient stratification and therapy monitoring. B7-H4 imaging could augment our understanding of B7-H4 dynamics in response to various therapeutic interventions in clinical trials. The new B7-H4 immunoPET probe is, in principle, clinically translatable.