Screening and Preclinical Evaluation of Novel Radiolabeled Anti-Fibroblast Activation Protein-α Recombinant Antibodies.

Background: Fibroblast activation protein-α (FAPα) is selectively overexpressed in tumor-associated fibroblasts in more than 90% of epithelial tumors, and may be a good target for anticancer treatment, for example, using an anti-FAPα recombinant antibody (rAb) labeled with radionuclides. In the present report, the radiolabeling and preclinical evaluation of novel anti-FAPα rAbs were investigated. Materials and Methods: Two novel anti-FAPα VHHs (AMS002-1 and AMS002-2) with high binding affinity to FAPα were selected from an antibody phage library. The anti-FAPα VHHs were then fused with the Fc fragment of human IgG4 to create two VHH-Fc rAbs. The VHH-Fc rAbs were radiolabeled with ⁸⁹Zr and ¹⁷⁷Lu. The radiolabeled products were evaluated by radioligand-binding assays using FAPα-expressing cells. The biodistribution and tumor-targeting properties were investigated by small-animal PET/CT. AMS002-1-Fc, which showed promising tumor-targeting properties in ⁸⁹Zr-microPET imaging, was radiolabeled with ¹⁷⁷Lu for efficacy study on HT1080 tumor-bearing mice and monitored with SPECT/CT imaging. Results: The two VHH-Fc rAbs with good affinity with K_D values in low nanomolar range were identified. Both PET/CT imaging with ⁸⁹Zr-AMS002-1-Fc rAb and SPECT/CT imaging with ¹⁷⁷Lu-AMS002-1-Fc rAb demonstrated highest tumor uptakes at 72 h p.i. and long tumor retention in the preclinical models. Furthermore, ex vivo biodistribution analysis revealed high tumor uptake of ⁸⁹Zr-AMS002-1-Fc at 48 h p.i. with the value of 6.91% ± 2.08% ID/g. Finally, radioimmunotherapy with ¹⁷⁷Lu-AMS002-1-Fc rAb delayed the tumor growth without significant weight loss in mice with HT1080 xenografts. The tumor size of untreated control group was 2.59 times larger compared with the treatment group with ¹⁷⁷Lu-AMS002-1-Fc at day 29. Conclusion: ⁸⁹Zr/¹⁷⁷Lu-AMS002-1-Fc represent a pair of promising radiopharmaceuticals for theranostics on FAPα-expressing tumors.