Development and Characterization of Nanobody-Derived CD47 Theranostic Pairs in Solid Tumors

Overexpression of CD47 is frequently observed in various types of human malignancies, inhibiting myeloid-mediated elimination of tumor cells and affecting the prognosis of cancer patients. By mapping biomarker expression, immuno-positron emission tomography has been increasingly used for patient screening and response monitoring. By immunization alpacas with recombinant human CD47, we prepared a CD47-targeting nanobody C2 and developed [⁶⁸Ga]Ga-NOTA-C2, followed by an exploration of the diagnostic value in CD47-expressing tumor models including gastric-cancer patient-derived xenograft models. By fusing C2 to an albumin binding domain (ABD), we synthesized ABDC2, which had increased in vivo half-life and improved targeting properties. We further labeled ABDC2 with ⁶⁸Ga/⁸⁹Zr/¹⁷⁷Lu to develop radionuclide theranostic pairs and evaluated the pharmacokinetics and theranostic efficacies of the agents in cell- and patient-derived models. Both C2 and ABDC2 specifically reacted with human CD47 with a high K _D value of 23.50 and 84.57 pM, respectively. [⁶⁸Ga]Ga-NOTA-C2 was developed with high radiochemical purity (99 >%, n = 4) and visualized CD47 expression in the tumors. In comparison to the rapid renal clearance and short half-life of [⁶⁸Ga]Ga-NOTA-C2, both [⁶⁸Ga]Ga-NOTA-ABDC2 and [⁸⁹Zr]Zr-DFO-ABDC2 showed prolonged circulation and increased tumor uptake, with the highest uptake of [⁸⁹Zr]Zr-DFO-ABDC2 occurring at 72 h post-injection. Moreover, [¹⁷⁷Lu]Lu-DOTA-ABDC2 radioimmunotherapy suppressed the tumor growth but was associated with toxicity, warranting further optimization of the treatment schedules. Taken together, we reported a series of nanobody-derived CD47-targeted agents, of which [⁶⁸Ga]Ga-NOTA-C2 and [⁸⁹Zr]Zr-DFO-ABDC2 are readily translatable. Optimization and translation of CD47-targeted theranostic pair may provide new prospects for CD47-targeted management of solid tumors.