Pretargeted Trop-2 ImmunoPET for Rapid, Selective Detection of Pancreatic Tumors

Abstract

Purpose: Recent clinical advances with the approval of antibody-drug conjugates targeting trophoblast cell-surface antigen 2 (Trop-2), such as sacituzumab govitecan and datopotamab deruxtecan, have garnered tremendous interest for their therapeutic efficacy in numerous tumor types, including breast and lung cancers. ImmunoPET can stratify tumor avidity, clarifying patient eligibility for antibody-drug conjugate therapy as well as a diagnostic companion during therapy. Slow antibody circulation requires days to reach optimal imaging timepoints. To overcome this shortfall, bioorthogonal click chemistry for pretargeting can be employed, decoupling antibody circulation time and the delivery of the radionuclide.

Experimental design: Here, we report the characterization of a new full-length Trop-2.2 antibody showing high affinity for Trop-2-positive cancers and leverage different site-selective labeling and pretargeting radionuclide combinations to yield rapid imaging with minimal radionuclide footprint after imaging. Three pretargeting strategies for fluorine-18, copper-64, and zirconium-89 were explored in addition to site-specific bioconjugation.

Results: We found that pretargeting with [64Cu]Cu-sarcophagine-tetrazine yielded the best images, identifying Trop-2-positive tumors with optimal tumor-to-background ratios. Intriguingly, we found that the full-length antibody, when directly conjugated, yielded rapid accumulation, starting at 3 hours after injection, and led to more than 50% injected activity per gram in the tumor before 24 hours.

Conclusions: [89Zr]Zr-deferoxamine-Trop-2 and pretargeting with [64Cu]Cu-sarcophagine-tetrazine are viable imaging strategies to quickly identify Trop-2-positive tumors for subsequent Trop-2 therapies. See related commentary by Mori et al., p. 2547.

Figure 1.. Characterization of Trop-2.2 as an antibody for imaging Trop-2 in vitro and in vivo.

A) Surface plasmon resonance titrations of Trop-2.2 and hRS7 show Trop-2.2 has a slightly increased affinity for binding Trop-2 over hRS7. B) bead binding of Trop-2 functionalized beads show high-capacity binding for [⁸⁹Zr]Zr-DFO-Lys-Trop-2.2 and that C) radiolabeling does not abolish Trop-2 binding as fraction bound closely matches SPR results. D) PC3, BxPC-3, and Calu6 cell and tumor lysates were confirmed by western blot to be slightly positive, very positive, and negative for Trop-2 expression, respectively. E) Confirmation of Trop-2 expression is membrane bound by cell uptake of [⁸⁹Zr]Zr-DFO-Lys-Trop-2.2 with PC3, BxPC-3, and Calu6 cells where CTL = no cells, TBF = total bound fraction, and B = Trop-2 blocked. F) Cell internalization assay shows no major difference after 2 hours of incubating [⁸⁹Zr]Zr-DFO-Lys-Trop-2.2, which both could be blocked to levels comparable with that of no cells (green bar). Longer incubations show much greater uptake at 4° C (Supplemental Figure 1B). N=3 technical replicates in B, C, E, and F. Bar = SEM

Figure 2.. In vivo accumulation of Trop-2.2 shows high-capacity uptake in BxPC-3 tumors.

A) PET/CT maximum intensity projections of representative mice 24-, 48-, 72-, and 144-hours post injection of [⁸⁹Zr]Zr-DFO-Lys-Trop-2.2 shows high accumulation in BxPC-3 tumors, with no major accumulation in healthy tumor free nude mice. B) Terminal biodistribution confirms high uptake starting at 24 hours. N=5 mice per timepoint and imaging group. Bar = SEM.

Figure 3.. Rapid accumulation of Trop-2.2 in BxPC-3 tumors allows 24-hour pretargeting.

A) Representative PET imaging at 1-, 3-, 6-, 9-, 24-hours post injection of a mouse bearing a BxPC-3 flank tumor with rapid accumulation of [⁸⁹Zr]Zr-DFO-Lys-Trop-2.2 in the tumor starting at 3 hours post injection. B) Biorthogonal click chemistry analog to A) with pretargeted or pre clicked DFO-PEG₇-Tz results in poor antibody distribution and tumor targeting due to the chelator-linker pair. C) By changing to a [⁶⁴Cu]Cu-Sar-Tz based pretargeting strategy, clearer tumor accumulation is observed with minimal to no residual uptake in other organs. An [¹⁸F]F-Al-NOTA-PEG₇-Tz approach was used with TCO- _SSTrop-2.2 in (Supplemental Figure 4A), though gastrointestinal clearance was still prevalent after 4 hours. N=4 mice per imaging group. Red dotted line is tumor boundary. White bar in A) by mouse head is a metal ear tag.