Characterizing immunocompetent models of PSMA+ prostate cancer to investigate [225Ac]Ac-PSMA-617 efficacy in mice

Introduction: Prostate-specific membrane antigen (PSMA) is a clinically validated target and highly expressed in metastatic castration-resistant prostate cancer (mCRPC).¹ While [⁶⁸Ga]Ga-PSMA-11 is successfully used for PET imaging and monitoring of prostate cancer patients^2,3, [¹⁷⁷Lu]Lu-PSMA-617 (Pluvicto) is the first FDA approved radioligand therapy (RLT) for progressive PSMA-positive mCRPC.⁴ However, many mCRPC patients develop resistance after several cycles of [¹⁷⁷Lu]Lu-PSMA-617 and relapse eventually.⁵ A promising strategy to enhance [¹⁷⁷Lu]Lu-PSMA-617 efficacy is the combination with immunotherapy. Preclinical studies investigating RLT in combination with immunotherapeutic approaches are needed but have been challenging due to the lack of suitable syngeneic models with reliable PSMA expression. In this work we present a new strategy of Abatacept co-inoculations to establish PSMA-positive tumors in immunocompetent mice and its suitability for studying PSMA-RLT efficacy and immunological effects in immunocompetent mice.

Methods: RM1, B6Myc and Myc-CaP cells were transduced with the lentiviral vector pLV-hPSMA, respectively. Male C57BL/6 mice were inoculated either with s.c. 1 x 10⁵ RM1-hPSMA cells or 2 x 10⁶ B6Myc-hPSMA cells, and male FVB mice received 2 x 10⁶ Myc-CaP-hPSMA cells. For all three models, cells were suspended in 100 µl PBS/Matrigel (1:1) containing either none or 1.25mg of the immunomodulator Abatacept (Orencia). Tumor growth was monitored semiweekly by CT. In vivo PSMA expression was followed by [⁶⁸Ga]Ga-PSMA-11 PET/CT up to 12 days post inoculation for RM1-hPSMA and B6Myc-hPSMA tumors, and up to 24 days for Myc-CaP-hPSMA tumors. To test T cell reactivity, splenic CD8 T cells were isolated from mice inoculated either with or without Abatacept (5 mice per group). CD8 T cells from each animal were added to 6-wells containing either no cells, parental or hPSMA+ cells, incubated overnight and stained (CD8, IFNγ, CD69) for flow cytometry. To evaluate RLT efficacy in mice with Abatacept, tumors were randomized into two groups (n=10/group) after reaching an average tumor volume of 100 mm³ and RLT groups were injected intravenously with 30 kBq [²²⁵Ac]Ac-PSMA-617. Therapeutic efficacy was assessed by tumor volumetry (CT) and survival. OsiriX Software was used for PET/CT analysis and CT volumetry. GraphPad Prism was used for statistical analysis.

Results: As shown in Figure 1A, tumors co-inoculated with Abatacept grew out and retained target expression up to day 12 for RM1-hPSMA (SUV_max = 2.39 ± 1.19; n=10) and B6Myc-hPSMA SUV_max = 2.43 ± 0.54; n=3), and up to day 24 for Myc-CaP-hPSMA (SUV_max = 2.46 ± 0.56; n=11). When splenic CD8 T cells isolated from mice bearing RM1-hPSMA tumors (either with or without Abatacept) were co-cultured without cells or with parental RM1 cells no changes in IFN&gamma; or CD69 expression were observed (Fig.1B). However, when challenged with RM1-hPSMA cells, the percentage of IFN&gamma;+ T cells increased to 0.329 ± 0.175% (p=0.0188) from mice without Abatacept compared to 0.099 ± 0.019% for Abatacept co-inoculated animals. A significant increase was also observed in CD69+ T cell populations with 8.41 ± 1.74% (p=0.0184) from mice without Abatacept compared to 5.19 ± 1.72% for Abatacept co-inoculated animals. All three tumor models were used for efficacy studies with [²²⁵Ac]Ac-PSMA-617 and showed strong tumor growth delay compared to control groups (Fig.1C), resulting in significantly improved median survival of 21d (RLT) versus 9d (control) for RM1-hPSMA (p<0.0001), 18d versus 6d for B6Myc-hPSMA (p=0.0002) and 28d versus 21d for Myc-CaP-hPSMA (p<0.0001).

Conclusions: Abatacept co-inoculations promote immune tolerance of hPSMA+ cells in immunocompetent mice to ensure stable PSMA expression without losing responsiveness to RLT. This new strategy for establishing immunocompetent models with reliable target expression can be a useful tool for future studies investigating the immunological consequences of RLT in mice.

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