Synthesis and Comparative In Vivo Evaluation of Site-Specifically Labeled Radioimmunoconjugates for DLL3-Targeted ImmunoPET

Abstract

Delta-like ligand 3 (DLL3) is a therapeutic target for the treatment of small cell lung cancer, neuroendocrine prostate cancer, and isocitrate dehydrogenase mutant glioma. In the clinic, DLL3-targeted 89Zr-immunoPET has the potential to aid in the assessment of disease burden and facilitate the selection of patients suitable for therapies that target the antigen. The overwhelming majority of 89Zr-labeled radioimmunoconjugates are synthesized via the random conjugation of desferrioxamine (DFO) to lysine residues within the immunoglobulin. While this approach is admittedly facile, it can produce heterogeneous constructs with suboptimal in vitro and in vivo behavior. In an effort to circumvent these issues, we report the development and preclinical evaluation of site-specifically labeled radioimmunoconjugates for DLL3-targeted immunoPET. To this end, we modified a cysteine-engineered variant of the DLL3-targeting antibody SC16-MB1 with two thiol-reactive variants of DFO: one bearing a maleimide moiety (Mal-DFO) and the other containing a phenyloxadiazolyl methyl sulfone group (PODS-DFO). In an effort to obtain immunoconjugates with a DFO-to-antibody ratio (DAR) of 2, we explored both the reduction of the antibody with tris­(2-carboxyethyl) phosphine (TCEP) as well as the use of a combination of glutathione and arginine as reducing and stabilizing agents, respectively. While exerting control over the DAR of the immunoconjugate proved cumbersome using TCEP, the use of glutathione and arginine enabled the selective reduction of the engineered cysteines and thus the formation of homogeneous immunoconjugates. A head-to-head comparison of the resulting 89Zr-radioimmunoconjugates in mice bearing DLL3-expressing H82 xenografts revealed no significant differences in tumoral uptake and showed comparable radioactivity concentrations in most healthy nontarget organs. However, 89Zr-DFOPODS-DAR2SC16-MB1 produced 30% lower uptake (3.3 ± 0.5 %ID/g) in the kidneys compared to 89Zr-DFOMal-DAR2SC16-MB1 (4.7 ± 0.5 %ID/g). In addition, H82-bearing mice injected with a 89Zr-labeled isotype-control radioimmunoconjugate synthesized using PODS exhibited ∼40% lower radioactivity in the kidneys compared to mice administered its maleimide-based counterpart. Taken together, these results demonstrate the improved in vivo performance of the PODS-based radioimmunoconjugate and suggest that a stable, well-defined DAR2 radiopharmaceutical may be suitable for the clinical immunoPET of DLL3-expressing cancers.

Site-Specific Radiohalogenation of a HER2-Targeted Single-Domain Antibody Fragment Using a Novel Residualizing Prosthetic Agent

“…5 To obviate the need for these tactics and take advantage of the benefits of site-specific labeling, we sought to build on the encouraging results we obtained labeling proteins with radiometals using a PODS moiety. [21][22][23]31 Among the attractive features we hoped to extend to these radiohalogens include efficient and rapid conjugation at biologically relevant pH and improved in vitro and in vivo stability compared with corresponding conjugates labeled using maleimide chemistry. 21,23 These results encouraged us to combine PODS with previously validated residualizing prosthetic agents�iso-[ 131 I]-SGMIB 29 and iso-[ 211 At]SAGMB 6 �to derive iso-[ 131 I]GMIB-PODS and iso-[ 211 At]AGMB-PODS, respectively.…”

“…While this results in a much lower number of potential conjugation sites than are available for lysine-targeted reagents, multiple cysteines can nonetheless still be modified. This can be ameliorated somewhat by genetically inserting capped thiols into the IgG light chain; however, in this case, the reaction conditions must be carefully tuned for each mAb to avoid reduction of its native disulfides. sdAbs, in contrast, have an ideal structure for truly site-specific thiol modification, as they generally have only one conserved disulfide linkage that is stable under most reducing conditions. , Because the C-terminus of sdAbs points away from its CDR regions, it represents an excellent position for modification with minimal chance of disrupting antigen binding affinity.…”

Degradable ZnS-Supported Bioorthogonal Nanozymes with Enhanced Catalytic Activity for Intracellular Activation of Therapeutics

“…The release of thiols was detected by following the previous report using Ellman’s reagent. , Briefly, DTNB was dissolved in 0.1 M phosphate buffer containing 1 mM EDTA to obtain the stock solution. Then, 100 μL solution containing 20 nM nanoparticles and 0.1 mM DTNB were added into a 96-well clear plate and tracked the absorbance at 412 nm at 37 °C using a Molecular Devices SpectraMax M2 plate reader for 1 h.…”

PET Imaging of Lung Cancers in Precision Medicine: Current Landscape and Future Perspective

“…102 Furthermore, based on 89 Zr-labeled SC16, they developed a PODS-based radioimmunoconjugate DFO(PODS)-(DAR2)-SC16-MB1 that exhibited 30−40% lower radioactivity in the kidney. 103…”

“…Most importantly, DLL3 immunoPET proved a rank-order correlation for response to SC16LD6.5 therapy in SCLC patient-derived xenograft models . Furthermore, based on 89 Zr-labeled SC16, they developed a PODS-based radioimmunoconjugate DFO­(PODS)-(DAR2)­SC16-MB1 that exhibited 30–40% lower radioactivity in the kidney …”