Introduction: Incomplete resection of prostate tumors and failure to detect metastatic tumors in draining lymph nodes can lead to tumor recurrence. This emphasizes the importance of real-time intraoperative visualization of both primary malignant prostate tumors and lymph node metastases during prostatectomy. [FS1] [MF2] Single-chain variable fragment-Fc (scFv-Fc) antibody fragments demonstrate potential for in vivo imaging due to their enhanced tissue penetration and faster blood clearance compared to intact antibodies. Developing double point mutations in the Fc region of scFv-Fc fragments can modulate the binding to neonatal Fc receptor and further shorten the blood circulation which can result in higher tumor:blood ratio and better image contrast. In this study, a NIRF probe based on pharmacokinetically-modulated anti-human prostate stem cell antigen (hPSCA), A2scFv-Fc2 double mutant (A2DM), was developed aiming for guiding prostate cancer surgery through next-day real-time NIRF imaging.

Methods: The anti-hPSCA A2DM was conjugated with IRDye800CW (a NIR fluorophore, Exmax: 775 nm, Emmax: 794 nm) using random (NHS ester, A2DM-NHS800) or site-specific (maleimide, A2DM-mal800) chemistry.[AW3] [MF4]  Successful conjugation and structural integrity were assessed by SDS-PAGE and size exclusion chromatography, respectively. Nanodrop was used to measure dye:protein ratio (DPR). The specific binding of A2DM-NHS800 and A2DM-mal800 to recombinant antigen (hPSCA-mFc) and hPSCA+/- RM9 prostate cancer cells was evaluated by ELISA and flow cytometry, respectively. The fluorescent probes were conjugated with p-SCN-DFO and radiolabeled with zirconium-89 for surrogate immunoPET to profile pharmacokinetics and ex vivo biodistribution in hPSCA[AW5]  knock-in C57BL/6 mice implanted with subcutaneous syngeneic hPSCA+/- RM9 tumors (50 µCi/10 µg). The same mouse model was employed to evaluate A2DM-NHS800 and A2DM-mal800 for NIRF imaging at 24 and 48 h p.i. (n ≥ 3 per group; male and female).

Results: A2DM was successfully conjugated with IRDye800CW via random and site-specific (by mild reduction and incorporation into the hinge disulfides) methods with DPR of 0.5, without impacting dimeric conformation and[AW6] [MF7]  binding affinity to recombinant hPSCA. A2DM-NHS800 and A2DM-mal800 demonstrated antigen-specific binding to RM9-hPSCA cells with negligible binding to RM9 cells. In vivo surrogate immunoPET imaging confirmed hPSCA-specific tumor uptake of 89Zr-A2DM-NHS800 and 89Zr-A2DM-mal800. The IRDye800-conjugated variants showed higher liver retention and faster blood clearance compared to unconjugated A2DM[FS8] [MF9] , resulting in a higher tumor:blood ratio of 89Zr-A2DM-NHS800 (20.3 ± 0.8) and 89Zr-A2DM-mal800 (14.6 ± 0.6) compared to 89Zr-A2DM (11.3 ± 2) at 48 h p.i. Based on NIRF imaging results, both A2DM-NHS800 and A2DM-mal800 clearly delineated hPSCA+ tumors with high contrast to surrounding tissue and no visible uptake in hPSCA- tumors in both male and female mice. A2DM-NHS800 exclusively cleared to the liver in both male and female mice resulting in an unobstructed view of the pelvic region (no background in kidneys or bladder), crucial for prostate cancer detection. However, some of male mice injected with A2DM-mal800 showed noticeable fluorescence signal in kidneys and bladder, likely due to metabolism and cleaved-off IRDye800.  Dose escalation of A2DM-NHS800 showed saturation of hPSCA+ tumors at protein doses of 30 µg or higher and an increase of non-specific accumulations at 24 h p.i.; therefore, the optimal injection dose of A2DM-NHS800 falls within 10-20 µg.

Conclusion: IRDye800 was successfully conjugated with A2DM without impacting binding affinity or specificity to hPSCA. A2DM-NHS800 and A2DM-mal800 showed specific in vivo tumor targeting and high tumor:blood ratio at early time points, which would facilitate next-day image-guided surgery. A2DM-NHS800 and A2DM-mal800 enabled high-contrast NIRF visualization of hPSCA+ tumors as early as 24 h p.i. A2DM-NHS800 showed higher in vivo stability and higher hPSCA+ tumor uptake at 24 h p.i. and the optimal protein dose was determined to be 10-20 µg. A2DM-NHS800 was chosen as the lead candidate for further evaluation in orthotopic and metastasized prostate cancer models.

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Image/Figure Caption:

⁸⁹zr-labled A2DM-NHS800 and A2DM-mal800 showed hPSCA-specific tumor accumulation as early as 24 h p.i. ImmunoPET/CT and NIRF imaging of RM9-hPSCA and RM9 tumor-bearing hPSCA knock-in male mice injected with ⁸⁹zr-labled A2DM-NHS800 (A), and A2DM-mal800 (B) at 24 and 48 h p.i. The injected activity was 1.85 MBq (50 µCi)/10 µg. “+” and “-“ indicate hPSCA⁺ and hPSCA^– tumors.

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