Developing a theranostic nanobody targeting FAP for cancer imaging and therapy

Fibroblast activation protein (FAP) is a pan-cancer target. Its selective expression on the majority of solid tumors with minimal to absent expression in healthy tissues positions FAP as a promising target for radiotheranostic applications. Nanobodies (Nbs) have unique characteristics, including small size, high affinity, stability, and ease of modification, making them ideal candidates for cancer diagnostics and targeted radiotherapeutics. Llama-derived Nbs were generated and screened against full-length FAP, with three unique candidates selected from the library for further characterization. The lead candidate Nb159 was engineered for site-specific radiolabeling with ⁸⁹Zr for PET imaging and with ¹⁷⁷Lu coupled with PEG for therapeutic evaluation in mice bearing FAP-positive U87 tumor xenografts.

Nb159 exhibited exceptional picomolar binding affinity to FAP with stable interaction and slow dissociation. PET imaging with [⁸⁹Zr]Zr-Nb159 demonstrated specific tumor uptake, peaking at 1 h post-injection, with rapid renal clearance and minimal uptake in non-target organs. A competitive binding study confirmed its specificity to FAP on U87 tumors, as pre-injection with a tenfold molar excess of unlabeled Nb159 reduced tumor uptake by ~ 55% (3.78 ± 0.50 to 1.67 ± 0.26%ID/g). PEGylation of Nb159 improved its pharmacokinetic profile, yielding prolonged tumor accumulation and significantly reduced renal retention when co-injected with lysine. PET imaging further demonstrated target-specific uptake in FAP-positive U87 xenografts, which exhibited higher signal than FAP-negative HCT116 tumors, with SUV_mean at 48 h of 0.45 ± 0.04 versus 0.09 ± 0.01 (P < 0.0001). In the therapeutic study, [¹⁷⁷Lu]Lu-PEG-Nb159 demonstrated significant tumor growth inhibition with no observable toxicity. Mice treated with a single dose of [¹⁷⁷Lu]Lu-PEG-Nb159 survived significantly longer compared to either [¹⁷⁷Lu]Lu-DOTA (23 days, P < 0.001, HR: 0.06107) or vehicle (21 days, P < 0.0001, HR: 0.04017).

The lead candidate Nb159 holds promise as a versatile platform for FAP-targeted radiotheranostics, with [⁸⁹Zr]Zr-Nb159 serving as an effective companion diagnostic and [¹⁷⁷Lu]Lu-PEG-Nb159 demonstrating promising therapeutic potential. These findings support further development of Nb159-based radiopharmaceuticals for treatment of FAP positive tumors.