04:12 PM Abstract No. 226 Glypican-3 optical molecular imaging enables in vivo detection and monitoring of response to adoptive immunotherapy

Abstract

We have previously described synthesis of a bispecific fusion protein for catheter-directed targeting of glypican-3 (GPC3) expressed on hepatocellular carcinoma (HCC) tumors and CD3 T-cell receptors (GPC3-CD3-bsAb) (1). We hypothesized that in vivo optical fluorescent imaging of GPC3 in mouse models of human HCC would enable effective monitoring of adoptive immunotherapy mediated by GPC3-CD3-bsAb. A modified GPC3 specific small peptide was labeled with Hylite488 or Cylyte5 fluorescent dyes (GPC3-FL) and used for in-vitro and in vivo imaging, respectively. In-vitro competitive binding assay was performed to assess specific probe binding to GPC3 on human HCC tumor cells (HepG2 (GPC3+) and SNU449 (GPC3-)). For in vivo imaging, HCC mouse xenograft models were created through implantation of HepG2 and SNU449 cells in the flanks of nude mice (n=5 each group). Optical imaging was performed after tail-vein injection of 6.25μg of GPC3-FL on IVIS Spectrum in vivo imaging system. Mice were imaged at baseline, followed by GPC3 blockage using higher concentrations of non-labeled GPC3 specific peptide and after adoptive immunotherapy with concurrent injection of GPC3-CD3-bsAb and activated human T-cells. In-vitro assays confirmed dose dependent blockage of GPC3-FL binding to GPC3+ HepG2 cells by GPC3 specific antibodies. Optical imaging of HepG2 and SNU449 tumor bearing mice demonstrated a significantly higher target to background ratio (TBR) for GPC3+ HepG2 tumors compared to GPC3- SNU449 tumors (2.99±0.36 vs 1.16±1.15, p=0.01). Concurrent injection of non-labeled GPC3 specific peptide and GPC-FL showed a marked decrease in tumor TBRs in GPC3+ HepG2 tumors (1.45±0.18 vs. 2.99±0.36, p=0.004). Following an adoptive immunotherapy treatment course, fluorescent signal in treated tumors markedly decreased in the treatment group compared to controls (4.36±0.9 before treatment vs 1.8±0.4, p=0.049). Optical imaging using GPC3-FL is feasible for in vivo detection of GPC3 positive HCC cells and allows non-invasive monitoring of tumor response to adoptive immunobtherapy, which cannot be achieved using available morphological methods of imaging.