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

Abstract

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 [ 68 Ga]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 68 Ga/ 89 Zr/ 177 Lu 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. [ 68 Ga]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 [ 68 Ga]Ga-NOTA-C2, both [ 68 Ga]Ga-NOTA-ABDC2 and [ 89 Zr]Zr-DFO-ABDC2 showed prolonged circulation and increased tumor uptake, with the highest uptake of [ 89 Zr]Zr-DFO-ABDC2 occurring at 72 h post-injection. Moreover, [ 177 Lu]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 [ 68 Ga]Ga-NOTA-C2 and [ 89 Zr]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.

Site-Specific Modification of Single Domain Antibodies by Enzyme-Immobilized Magnetic Beads

“…We demonstrate that magnetic beads with zwitterionic surfaces (MB3) exhibit superior performance for nanobody modification. By using the magnetic beads in our previous work, 56 additional amine functionalization and poly(acrylic acid) (PAA) coatings were performed to obtain an MB3 sample with zwitterionic surfaces. As shown by Figure S7, the protein recovery of MB3 was apparently higher than that of the commercial MB2 sample after the magnetic beads were incubated with the same amount of BSA protein.…”

“…To demonstrate the generality of the immobilization method, we purchased two types of magnetic beads (MB1 with NHS groups and MB2 with carboxyl groups) from different vendors. We further prepared another type of magnetic beads with zwitterionic surfaces (MB3), because many studies have demonstrated that zwitterionic surfaces can effectively reduce nonspecific adsorption of proteins. , On basis of the magnetic beads in our previous work, additional amine functionalization and poly­(acrylic acid) (PAA) coating were performed to generate a zwitterionic surface. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) were used to characterize the different samples.…”

ImmunoPET Imaging of CD47 with VHH-Derived Tracers in Pancreatic Cancers

“…Female athymic nude mice (5 week-old; GemPharmatech) and female NCG-hCD47 humanized mice (5 week-old; GemPhar- S1). 25 We used surface plasmon resonance to determine the affinities of C2 and ABDC2 to the target protein. C2 binds to human CD47, but not murine CD47, with a high K D value of 23.50 pM.…”

“…Female athymic nude mice (5 week-old; GemPharmatech) and female NCG-hCD47 humanized mice (5 week-old; GemPhar- We have developed a series of VHHs with high and specific binding affinities to human CD47, including a clone termed C2 used in the study (Figure S1). 25 We used surface plasmon resonance to determine the affinities of C2 and ABDC2 to the target protein. C2 binds to human CD47, but not murine CD47, with a high K D value of 23.50 pM.…”

“…23,24 We have developed a series of VHHs with high binding affinities to human CD47. 25 This study aimed to validate the specific diagnostic value and pharmacokinetics of [ 68 Ga]Ga-NOTA-C2 and [ 89 Zr]Zr-DFO-ABDC2, two VHH-derived tracers, in preclinical PDAC models.…”