Site-Specific Photoaffinity Bioconjugation for the Creation of 89Zr-Labeled Radioimmunoconjugates

Abstract

Purpose: Site-specific approaches to bioconjugation produce well-defined and homogeneous immunoconjugates with potential for superior in vivo behavior compared to analogs synthesized using traditional, stochastic methods. The possibility of incorporating photoaffinity chemistry into a site-specific bioconjugation strategy is particularly enticing, as it could simplify and accelerate the preparation of homogeneous immunoconjugates for the clinic. In this investigation, we report the synthesis, in vitro characterization, and in vivo evaluation of a site-specifically modified, ⁸⁹Zr-labeled radioimmunoconjugate created via the reaction between an mAb and an Fc-binding protein bearing a photoactivatable 4-benzoylphenylalanine residue.

Procedures: A variant of the Fc-binding Z domain of protein A containing a photoactivatable, 4-benzoylphenylalanine residue - Z(35BPA) - was modified with desferrioxamine (DFO), combined with the A33 antigen-targeting mAb huA33, and irradiated with UV light. The resulting immunoconjugate - DFO^Z(35BPA)-huA33 - was purified and characterized via SDS-PAGE, MALDI-ToF mass spectrometry, surface plasmon resonance, and flow cytometry. The radiolabeling of DFO^Z(35BPA)-huA33 was optimized to produce [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33, and the immunoreactivity of the radioimmunoconjugate was determined with SW1222 human colorectal cancer cells. Finally, the in vivo performance of [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 in mice bearing subcutaneous SW1222 xenografts was interrogated via PET imaging and biodistribution experiments and compared to that of a stochastically labeled control radioimmunoconjugate, [⁸⁹Zr]Zr-DFO-huA33.

Results: HuA33 was site-specifically modified with Z(35BPA)-DFO, producing an immunoconjugate with on average 1 DFO/mAb, high in vitro stability, and high affinity for its target. [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 was synthesized in 95% radiochemical yield and exhibited a specific activity of 2 mCi/mg and an immunoreactive fraction of ~ 0.85. PET imaging and biodistribution experiments revealed that high concentrations of the radioimmunoconjugate accumulated in tumor tissue (i.e., ~ 40%ID/g at 120 h p.i.) but also that the Z(35BPA)-bearing immunoPET probe produced higher uptake in the liver, spleen, and kidneys than its stochastically modified cousin, [⁸⁹Zr]Zr-DFO-huA33.

Conclusions: Photoaffinity chemistry and an Fc-binding variant of the Z domain were successfully leveraged to create a novel site-specific strategy for the synthesis of radioimmunoconjugates. The probe synthesized using this method - DFO^Z(35BPA)-huA33 - was well-defined and homogeneous, and the resulting radioimmunoconjugate ([⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33) boasted high specific activity, stability, and immunoreactivity. While the site-specifically modified radioimmunoconjugate produced high activity concentrations in tumor tissue, it also yielded higher uptake in healthy organs than a stochastically modified analog, suggesting that optimization of this system is necessary prior to clinical translation.

Keywords: Immunopet; Photoaffinity labeling; Positron emission tomography; Site-selective bioconjugation; Site-specific bioconjugation; Zirconium-89.

Figure 1.

(A) Ribbon illustration of a Z domain (gray) binding the Fc region of human IgG₁ (figure prepared based on PDB 5U4Y [13]). (B) The chemical structure of the unnatural amino acid benzoylphenylalanine (BPA). (C) The reaction scheme of the benzophenone group of BPA entering a diradical triplet intermediate state upon irradiation with UV light and cross-linking with a ligand to form a covalent linkage.

Figure 2.

(A) The site-specific bioconjugation and radiosynthesis of [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 (top) and (B) the random lysine bioconjugation and radiosynthesis of [⁸⁹Zr]Zr-DFO-huA33.

Figure 3.

Structural, biological, and radiochemical evaluation of the mAbs. (A) SDS-PAGE analyzing the composition of huA33, DFO-huA33, and DFO^Z(35BPA)-huA33. (B) Fluorescence-associated cell sorting of huA33, huA33, DFO-huA33, and DFO^Z(35BPA)-huA33 with A33-expressing SW1222 cells and an AlexaFlour^™ 488 secondary antibody. (C) Longitudinal stability study of [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 in PBS over 5 days. (D) Cell-based immunoreactivity assay comparing [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 and [⁸⁹Zr]Zr-DFO-huA33 with A33-expressing SW1222 cells.

Figure 4.

In vivo evaluation of the radioimmunoconjugates. Representative coronal PET images acquired 24, 48, 72, 96, and 120 h after the administration of (A) [⁸⁹Zr]Zr-DFO-Z(35BPA), (B) [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33, or (C) [⁸⁹Zr]Zr-DFO-huA33 to athymic nude mice bearing subcutaneous SW1222 colorectal cancer xenografts. (D) Ex vivo distribution data collected 120 h after the administration of [⁸⁹Zr]Zr-DFO^Z(35BPA)-huA33 (red) and [⁸⁹Zr]Zr-DFO-huA33 (blue) to athymic nude mice bearing subcutaneous SW1222 xenografts in the right shoulder. Statistical significance was determined via a two-tailed t test with a Welch’s correction using GraphPad Prism: * = p <0.05; *** = p <0.001.