Targeted RPT of CEACAM5-Expressing Human Xenografts Using an Albumin-Binding Domain-Fused C9 Affibody: Proof-of-Principle Study.

The carcinoembryonic antigen cell adhesion molecule 5 (CEACAM5) is highly overexpressed in carcinomas of different origins. Affibody molecules are non-immunoglobulin-based affinity proteins capable of in vivo recognition of cancer-associated targets. Affibody C9 was previously generated to bind selectively to CEACAM5. In an earlier in vivo study, C9 was engineered as a dimer, and the effects on the biodistribution from the addition of an N- or C-terminal-fused albumin-binding domain (ABD035) were investigated using 99mTc labeling. The variant ABD035-C9-C9 with N-terminal ABD035 placement (20,296 Da; affinities of 2.5 and 1.9 nM to CEACAM5 and albumin, respectively) was found to provide the most favorable biodistribution for the delivery of cytotoxic payloads. In this study, we tested the hypothesis that ABD035-C9-C9 labeled with a cytotoxic radionuclide would be suitable for targeted radiopharmaceutical therapy (RPT). Methods: ABD035-C9-C9, labeled with the β-emitting radionuclide 177Lu via DOTA or DOTAGA chelators, was evaluated. CEACAM5-positive (BxPC3, LS174T) and CEACAM5-negative (HT-29) cell lines were used to assess binding specificity, kinetics, and internalization in vitro. The biodistribution of [177Lu]Lu-ABD035-C9-C9-DOTA and [177Lu]Lu-ABD035-C9-C9-DOTAGA was compared in BALB/c nu/nu mice with BxPC3 xenografts. The biodistribution of [177Lu]Lu-ABD035-C9-C9-DOTA was measured for dosimetry calculation. Modeling was performed to maximize tumor control probability. The effect of a single injection of 12 MBq of [177Lu]Lu-ABD035-C9-C9-DOTA on the growth of BxPC3 xenografts was evaluated. Results: In vitro, both [177Lu]Lu-ABD035-C9-C9-DOTA and [177Lu]Lu-ABD035-C9-C9-DOTAGA constructs demonstrated saturable, CEACAM5-dependent binding, with a minor difference in internalization rate (∼40% internalization after 24 h). In vivo, [177Lu]Lu-ABD035-C9-C9-DOTA showed significantly lower kidney uptake than the DOTAGA-conjugated variant. [177Lu]Lu-ABD035-C9-C9-DOTA demonstrated a significantly reduced renal uptake (14-fold, 4 h postinjection) and an increased uptake in BxPC3 xenografts (3.6-fold) compared with a [177Lu]Lu-C9-C9-DOTA reference construct without ABD035 This experimental RPT study demonstrated a significant increase in the survival of tumor-bearing mice and mild, transient toxicity. Conclusion: ABD035-C9-C9-DOTA is a promising candidate for the development of targeted RPT of CEACAM5-expressing tumors.