Preclinical evaluation of prostate-specific membrane antigen (PSMA)-based radiotheranostics for metastatic renal cell carcinoma

Introduction: Tumor-associated neovascular endothelial cells often exhibit PSMA overexpression in metastatic clear cell renal cell carcinoma (ccRCC), the most common and deadliest subtype of kidney cancer. Despite clinical evidence of the ability to image ccRCC with PSMA-based imaging agents, preclinical investigation using a PSMA-based imaging and therapeutic approach (radiotheranostics) has been lacking. Here, we aim to address that gap through preclinical validation of our PSMA-based radiotheranostics in experimental models of human and mouse RCC. We hypothesize that PSMA-based alpha-particle-emitting radiopharmaceutical therapy will effectively kill tumor-associated neovascular endothelial cells directly and tumor cells indirectly.

Methods: PSMA-based radiotheranostics (⁶⁸Ga-L1/¹⁷⁷Lu-L1/²²⁵Ac-L1) and a related optical agent were synthesized based on structure-activity relationships we developed for prostate cancer. An autoradiography-based saturation binding assay was developed to evaluate PSMA levels using freshly collected nephrectomy specimens. We developed PSMA-expressing human (PSMA+ 786-O) and mouse RCC (PSMA+ RENCA) cell lines and tumor models for this study. The cell uptake and biodistribution of ²²⁵Ac-L1 were conducted following an established protocol. Murine PSMA+ RENCA xenografts (n=10) were randomized after reaching an average tumor volume of 100 mm³, and the groups were injected intravenously with saline, 1×37 kBq, and 2×37 kBq (7 d), respectively, to evaluate efficacy by tumor volume measurement and survival. To assess the mechanism of in vivo cell kill, at 8 d post-treatment, three mice from each group were removed for immunohistochemistry to evaluate PSMA, CD31 (neovasculature), γ-H2AX (DNA damage) and to determine tumor-associated T cell populations by flow cytometry. PSMA+ 786-O tumors (n=10) with variable PSMA levels (0, 80/20, 40/60, 20/80 PSMA+/PSMA-) were also studied using a single-dose of ²²⁵Ac-L1 (37 kBq).

Results: In vitro autoradiographic assays of human RCC specimens (n=14) revealed high and specific binding of ¹⁷⁷Lu-L1 (K_d, ~2-30 nM) in these tissues. ⁶⁸Ga-L1/²²⁵Ac-L1 demonstrated specific and high cell uptake in PSMA+ RENCA and 786-O cells up to 24 h. In PET imaging, ⁶⁸Ga-L1 exhibited substantial tumor targeting in PSMA+ 786-O and RENCA tumor xenografts. In the RENCA model, tumor and kidney uptake were 4.17±1.34 %ID/g and 7.97±1.86 %ID/g, respectively, at 2 h after injection, and displayed fast clearance, ≤1 %ID/g after 24 h from the tumors and all studied organs. ²²⁵Ac-L1 was particularly effective in the PSMA+ RENCA xenograft model; three and four mice from the treatment groups displayed complete remissions up to 90 d post-treatment. Immunohistochemistry of the tumors revealed significantly low PSMA levels and CD31+ blood vessels and high DNA damage associated with the treated group. Flow cytometry revealed an increased proportion of CD3+CD8+ T cells in the treatment-responsive tumors compared to untreated control tumors, suggesting a concurrent immunomodulatory effect. In 786-O models, tumor growth control of ²²⁵Ac-L1 was correlated to PSMA expression levels of the tumor models tested.

Conclusions: PSMA-based radiotheranostics are promising in RCC, encouraging clinical adaptation to this new indication.

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