Purpose. Tumor Endothelial Marker-1 (TEM-1) is a promising target for immunotherapy, being upregulated by tumor cells, tumor vasculature and tumor-associated microenvironment. Our purpose was to test in-vitro and in-vivo a single-chain variable fragment directed against TEM1 (scFv78-Fc) labeled with either 111In or 152Tb, a positron-emitting CERN-produced radionuclide. Methods. 152Tb was produced by proton-induced spallation of tantalum targets followed by mass separation, and purified using cation exchange chromatography. A concentrated solution of scFv78-Fc in pH 9.0 buffer and 10 eq. of CHX-A’’-DTPA chelator were conjugated by heating at 42°C for 1 h, and then purified with a 50 kDa filter. 111In or 152Tb were complexed by additional 1 h heating at 42°C. The maximum binding sites (Bmax) of purified TEM1 antigen and equilibrium dissociation constant (Kd) were determined with Scatchard plot. Immunoreactivity (IR) was determined by Lindmo plot. Two human cell lines, RD-ES (Ewing Sarcoma) and SK-N-AS (Neuroblastoma) were xenografted in NOD-SCID common gamma KO female mice. TEM1 expression was confirmed by fluorescence-activated cell sorting; antigen positivity was additionally proven by immunohistochemistry in excised tumors. Biodistribution was assessed 5, 24 and 48 h after i.v. injection of 0.1 MBq 111In-CHX-A’’-DTPA-scFv78-Fc (0.2 µg). The non-tumor antigenic sink was evaluated by co-injecting different amounts of unlabeled scFv78-Fc (range 0.2 – 200 µg). Additionally, after validation of 152Tb activity quantification with a NEMA NU4 phantom, tumor-bearing mice were injected with 7 MBq 152Tb-CHX-A’’-DTPA-scFv78-Fc for µPET imaging and dosimetry (µSPECT/PET/CT Albira, Bruker). Results. Radiolabeling yield was >80% for both 111In- and 152Tb-labeled products (iTLC). 99% pure products were obtained after ultrafiltration. Bmax and Kd were between 2-6 nmol/mg antigen and between 2-8 nM, respectively. IR was retained after radiolabeling (>70%). TEM1 expression was higher in SK-N-AS than in RD-ES cells. The best tumor-to-non-target ratios were obtained by co-injecting 25 µg of unlabeled CHX-A’’-DTPA-scFv78-Fc. 24-h percent of injected 111In-CHX-A’’-DTPA-scFv78-Fc activity/g varied in the range 8-16, 16-22 and 10-14 for tumor, liver and kidneys, respectively. Tumor-to-background ratios increased slightly between 24 and 48 hours (average 24-h and 48-h tumor-to-liver ratios were 0.55 and 0.64, respectively). µPET imaging of 152Tb-CHX-A’’-DTPA-scFv78-Fc gave results comparable to those of biodistribution, showing enhanced tumor uptake and similar tumor-to-liver ratios. Conclusion. The scFv78-Fc anti-TEM1 was successfully labeled with 111In and 152Tb. Final products showed specific binding and preserved immunoreactivity. Biodistribution and µPET imaging showed favorable tumor uptake in two human tumor cell lines. TEM1 has promise as a target for radiolabeled antibodies.
Preclinical evaluation of a single-chain variable anti TEM-1 fragment labeled with In-111 and Tb-152
F Cicone
;
2017-01-01
Abstract
Purpose. Tumor Endothelial Marker-1 (TEM-1) is a promising target for immunotherapy, being upregulated by tumor cells, tumor vasculature and tumor-associated microenvironment. Our purpose was to test in-vitro and in-vivo a single-chain variable fragment directed against TEM1 (scFv78-Fc) labeled with either 111In or 152Tb, a positron-emitting CERN-produced radionuclide. Methods. 152Tb was produced by proton-induced spallation of tantalum targets followed by mass separation, and purified using cation exchange chromatography. A concentrated solution of scFv78-Fc in pH 9.0 buffer and 10 eq. of CHX-A’’-DTPA chelator were conjugated by heating at 42°C for 1 h, and then purified with a 50 kDa filter. 111In or 152Tb were complexed by additional 1 h heating at 42°C. The maximum binding sites (Bmax) of purified TEM1 antigen and equilibrium dissociation constant (Kd) were determined with Scatchard plot. Immunoreactivity (IR) was determined by Lindmo plot. Two human cell lines, RD-ES (Ewing Sarcoma) and SK-N-AS (Neuroblastoma) were xenografted in NOD-SCID common gamma KO female mice. TEM1 expression was confirmed by fluorescence-activated cell sorting; antigen positivity was additionally proven by immunohistochemistry in excised tumors. Biodistribution was assessed 5, 24 and 48 h after i.v. injection of 0.1 MBq 111In-CHX-A’’-DTPA-scFv78-Fc (0.2 µg). The non-tumor antigenic sink was evaluated by co-injecting different amounts of unlabeled scFv78-Fc (range 0.2 – 200 µg). Additionally, after validation of 152Tb activity quantification with a NEMA NU4 phantom, tumor-bearing mice were injected with 7 MBq 152Tb-CHX-A’’-DTPA-scFv78-Fc for µPET imaging and dosimetry (µSPECT/PET/CT Albira, Bruker). Results. Radiolabeling yield was >80% for both 111In- and 152Tb-labeled products (iTLC). 99% pure products were obtained after ultrafiltration. Bmax and Kd were between 2-6 nmol/mg antigen and between 2-8 nM, respectively. IR was retained after radiolabeling (>70%). TEM1 expression was higher in SK-N-AS than in RD-ES cells. The best tumor-to-non-target ratios were obtained by co-injecting 25 µg of unlabeled CHX-A’’-DTPA-scFv78-Fc. 24-h percent of injected 111In-CHX-A’’-DTPA-scFv78-Fc activity/g varied in the range 8-16, 16-22 and 10-14 for tumor, liver and kidneys, respectively. Tumor-to-background ratios increased slightly between 24 and 48 hours (average 24-h and 48-h tumor-to-liver ratios were 0.55 and 0.64, respectively). µPET imaging of 152Tb-CHX-A’’-DTPA-scFv78-Fc gave results comparable to those of biodistribution, showing enhanced tumor uptake and similar tumor-to-liver ratios. Conclusion. The scFv78-Fc anti-TEM1 was successfully labeled with 111In and 152Tb. Final products showed specific binding and preserved immunoreactivity. Biodistribution and µPET imaging showed favorable tumor uptake in two human tumor cell lines. TEM1 has promise as a target for radiolabeled antibodies.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
