Lysine-Directed Site-Selective Bioconjugation for the Creation of Radioimmunoconjugates

Abstract

The synthesis of radioimmunoconjugates via the stochastic attachment of bifunctional chelators to lysines can yield heterogeneous products with suboptimal in vitro and in vivo behavior. In response to this, several site-selective approaches to bioconjugation have been developed, yet each has intrinsic drawbacks, such as the need for expensive reagents or the complexity of incorporating unnatural amino acids into IgGs. Herein, we describe the use of a simple and facile approach to lysine-directed site-selective bioconjugation for the generation of radioimmunoconjugates. This strategy relies upon on the selective modification of single lysine residues within each light chain of the monoclonal antibody (mAb) with a branched azide-bearing perfluorophenyl ester (PFP-bisN₃) followed by the ligation of dibenzocyclooctyne (DBCO)-bearing payloads to these bioorthogonal handles via the strain-promoted azide-alkyne cycloaddition. This methodology was used to create [⁸⁹Zr]Zr-^SSKDFO-pertuzumab, a radioimmunoconjugate of the HER2-targeting mAb pertuzumab labeled with desferrioxamine (DFO) and the positron-emitting radiometal zirconium-89 (⁸⁹Zr). [⁸⁹Zr]Zr-^SSKDFO-pertuzumab was compared to a pair of analogous probes: one synthesized via random lysine modification ([⁸⁹Zr]Zr-DFO-pertuzumab) and another via thiol-maleimide chemistry ([⁸⁹Zr]Zr-^malDFO-pertuzumab). The bioconjugation strategy was assessed using ESI mass spectrometry, SDS-PAGE, and autoradiography. All three immunoconjugates demonstrated comparable binding to HER2 via flow cytometry and surface plasmon resonance (SPR), and ⁸⁹Zr-labeled variants of each were synthesized in >99% radiochemical yield and molar activities of up to ∼55.5 GBq/μmol (10 mCi/mg). Subsequently, the in vivo behavior of this trio of ⁸⁹Zr-immunoPET probes was interrogated in athymic nude mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. [⁸⁹Zr]Zr-^SSKDFO-pertuzumab, [⁸⁹Zr]Zr-^malDFO-pertuzumab, and [⁸⁹Zr]Zr-DFO-pertuzumab produced positron emission tomography (PET) images with high tumoral uptake and high tumor-to-healthy organ activity concentration ratios. A terminal biodistribution study complemented the PET results, revealing tumoral activity concentrations of 126.9 ± 50.3%ID/g, 86.9 ± 53.2%ID/g, and 92.5 ± 27.2%ID/g at 144 h post-injection for [⁸⁹Zr]Zr-^SSKDFO-pertuzumab, [⁸⁹Zr]Zr-^malDFO-pertuzumab, and [⁸⁹Zr]Zr-DFO-pertuzumab, respectively. Taken together, the data clearly illustrate that this highly modular and facile approach to site-selective bioconjugation produces radioimmunoconjugates that are better-defined and more homogeneous than stochastically modified constructs and also exhibit excellent in vitro and in vivo performance. Furthermore, we contend that this lysine-directed strategy holds several key advantages over extant approaches to site-selective bioconjugation, especially in the context of production for the clinic.

Figure 1.

(A) The lysine-directed site-selective bioconjugation strategy for ^SSKDFO-pertuzumab using PFP-bisN₃, a branched bifunctional synthon bearing two azides and an amine-reactive perfluorophenyl ester; (B) the structure of PFP-bisN₃; (C) schematics of the bioconjugation routes for DFO-pertuzumab and ^malDFO-pertuzumab.

Figure 2.. The structural characterization of the immunoconjugates.

(A) ESI-MS of pertuzumab, ^SSKN₃-pertuzumab, and N₃-pertuzumab; (B) reducing SDS-PAGE of the immunoconjugates; HC = heavy chain and LC = light chain.

Figure 3.. The biological characterization of the immunoconjugates.

(A) Fluorescence-associated cell sorting (FACS) of pertuzumab, DFO-pertuzumab, ^SSKDFO-pertuzumab, ^malDFO-pertuzumab, and non-specific hIgG₁ using HER2-expressing BT-474 cells and an AlexaFluor488-labeled secondary antibody (n = 3); (B) bead-based immunoreactivity of [⁸⁹Zr]Zr-DFO-pertuzumab, [⁸⁹Zr]Zr-^SSKDFO-pertuzumab, and [⁸⁹Zr]Zr-^malDFO-pertuzumab using HER2-coated magnetic beads (n = 3). Statistical significance was determined via a two-tailed t test with a Welch’s correction using GraphPad Prism software. * = p-value < 0.05; ** = p-value < 0.01.

Figure 4.. In vivo evaluation of the radioimmunoconjugates.

(A) Representative PET scans collected 24, 48, 72, 96, 120, and 144 h after the intravenous administration of [⁸⁹Z]Zr-DFO-pertuzumab, [⁸⁹Zr]Zr-^SSKDFO-pertuzumab, or [⁸⁹Zr]Zr-^malDFO-pertuzumab [3.7 −3.9 MBq (100-105 μCi), 20-21 μg, in 100 μL of PBS] to athymic nude mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts (n = 5). The images on the left are coronal slices, and those on the right are maximum intensity projections (MIPs); (B) biodistribution data from the mice used for PET imaging collected after the terminal imaging timepoint at 144 h (n = 5).