Development of Radioligand Therapy-Responsive Syngeneic Prostate Models through Murine Prostate-Specific Membrane Antigen Point Mutation.

Robust, reproducible, and meaningful research requires suitable murine preclinical models that effectively mimic the disease and possess a functional immune system. In the context of metastatic castration-resistant prostate cancer (mCRPC) studies and with all the opportunities offered by theranostics, particularly prostate-specific membrane antigen (PSMA)-targeted therapies, the development of PSMA-positive murine models is essential for evaluating and further exploring these therapies in immunocompetent animals. However, strategies based on the transfection of murine cell lines with the human version of PSMA have led to immune rejection by the host. To preserve an intact immune system, which is indispensable, we focused on a strategy involving the expression of the murine version of PSMA. The core objective of this strategy was to evaluate radiotracer efficacy, using imaging and therapeutic response as complementary endpoints. While the imaging results using [68Ga]-Ga-PSMA-11 were satisfactory, we did not observe any therapeutic response to the radioligand therapy (RLT) using PSMA-617. A deeper analysis of the differences between murine PSMA (mPSMA) and human PSMA (hPSMA), as well as between the ligands PSMA-11 and PSMA-617, revealed significantly lower internalization and overall weaker radiotracer total binding in the mPSMA context, especially pronounced with PSMA-617. We identified a key motif in the amino acid sequence of hPSMA that is involved in internalization but is absent in that of mPSMA. Introducing a single amino acid substitution into the mPSMA sequence was sufficient to restore the internalization motif. We then investigated whether this point-generated mutation in murine cell lines was tolerated by the host immune system and improved sensitivity to RLT through restored internalization. This single-point mutation successfully led to an internalization rate comparable to that of the hPSMA; nonetheless, using PSMA-617 RLT, no therapeutic efficacy was observed. This study highlights the lack of equivalence between PSMA-11 and PSMA-617 ligands. The inability to use the PET imaging ligand (PSMA-11) for therapy prediction and model development hindered progress and complicated the creation of immune-tolerant mPSMA models. Comparative analyses of hPSMA versus mPSMA and PSMA-11 versus PSMA-617 revealed substantial differences in internalization, externalization, and radiotracer behavior despite preserved binding affinityeffects most pronounced with PSMA-617. Molecular docking further suggested that species-specific structural differences in the PSMA binding pocket, particularly steric hindrance introduced by Ser550 in murine PSMA, may limit optimal ligand positioning, independent of chelator chemistry. These findings indicate that both the receptor trafficking and structural features of the PSMA binding pocket may contribute to the observed differences and should be considered when developing translationally relevant murine models. Nevertheless, this study represents a valuable first step toward the development of syngeneic, PSMA-expressing murine models for preclinical theranostic studies.