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

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.