Radioligand therapy (RLT) with a ligand binding the prostate specific antigen (PSMA; ¹⁷⁷Lu-PSMA-617) is a promising therapeutic option for patients with metastatic castration-resistant prostate cancer (mCRPC). However, approx. 50 % of patients do not respond to RLT. Thus, there is an urgent need to improve RLT efficacy through rationally designed combination therapies. In this work three approaches to enhance the efficacy of RLT were explored: 1. To improve tumor targeting by using ARB to enhance PSMA expression levels; 2. To exploit tumor vulnerabilities induced by ¹⁷⁷Lu-PSMA-617 (combination ¹⁷⁷Lu-PSMA-617 with DDR inhibition); and 3. To use ¹⁷⁷Lu-PSMA-617 as an in situ vaccine to enhance PCa immunogenicity (combination ¹⁷⁷Lu-PSMA-617 and ICB).

1. Effect of ARB on PSMA expression was assessed in human PCa cell lines 22Rv1, C4-2, and LNCaP. Immunohistochemistry and flow cytometry revealed low (22Rv1) and high (C4-2 and LNCaP) basal PSMA expression. ARB increased PSMA levels in 22Rv1, C4-2, and LNCaP (2.2/2.3/2.6-fold, p = 0.0005/0.03/0.046) after one week. 22Rv1 xenografts were treated with ARB for two weeks. Positron emission tomography /computed tomography (PET/CT) demonstrated higher tumor uptake of ⁶⁸Ga-PSMA-11 after ARB (p = 0.004). Thus, ARB enhances PSMA expression which might allow for improved targeting with RLT.

2. RLT induces DNA damage leading to activation of DDR signaling. DDR activation might mitigate RLT-induced cytotoxicity and, vice versa, inhibition of DDR signaling might sensitize tumors to RLT. Here, a combination regimen of Ataxia telangiectasia and Rad3 related (ATR) and/or Poly(ADP-ribose)-Polymerase (PARP) inhibitors and ionizing radiation (IR/RLT) was investigated in 22Rv1, C4-2, and LNCaP in vitro and in the 22Rv1 xenograft model. Combined IR and DDRi impaired cell growth and enhanced cell death as demonstrated by a modified colony formation assay and flow cytometry. In vivo, RLT/DDRi combinations did not enhance anti-tumor efficacy; the RLT/ATRi combination resulted in a slightly but non-significantly prolonged time to progression. Future studies will focus on evaluating the lack of efficacy of combined RLT and DDRi in vivo and how to overcome it.

3. Hypothesizing that RLT may enhance anti-prostate cancer immunity by inducing immunogenic cell death, synergy of RLT with PD-1 immune checkpoint blockade (ICB) was investigated. In vitro, stimulation of murine RM1-PGLS cells with IFN-γ phosphorylated STAT1 leading to upregulation of PD-L1 and MHC class I; additionally, IR enhanced PD-L1 and MHC class I expression. This provided the rationale for testing the RLT/ICB combination in vivo. While monotherapies did not improve outcome significantly, the RLT/ICB regiment significantly increased median time-to-progression compared to NT (p < 0.0001 vs NT). Future studies, using PCa mouse models and patient samples, will explore the mechanistic basis for these observation with the goal to identify the most promising RLT/immunotherapy combination(s) for inducing durable responses in PCa

Together, PSMA-RLT has become a game-changer in the management of PCa. To exploit its full potential the field needs to understand what constitutes an optimal radiotherapeutic strategy, and which tumor cell-intrinsic resistance mechanisms and protective tumor-microenvironment interactions need to be targeted, in order to develop more effective RLT-combination therapies. The design of and treatment with combination therapies is very complex and challenging. Data presented in this work shed light on this complexity and first encouraging results show that RLT efficacy can be enhanced by rational combinatorial approaches.