Background Early studies targeting anti-CTLA4 in metastatic castration resistant prostate cancer (mCRPC) offered hope for immunotherapy (IO) in these patients (NCT00861614). However, follow-up studies of anti-PD1 and/or anti-CTLA4 (e.g. CheckMate 650, KEYNOTE-921) have not moved the needle forward beyond the current taxane or novel androgen therapies. One novel mechanism proposes that targeting of androgen receptor (AR) in T-cells may increase their effector function and improve IO outcomes.¹ Translational data and analysis of the mCRPC TME to inform this novel hypothesis and the limited benefit of IO are needed.

Methods We employed a cohort of 69 commercially procured primary and metastatic tissue samples from patients treated with standard of care therapy (taxane, androgen targeting). All patients had confirmed M1 metastasis before tissue collection. We deployed IHC (CD8, MHCI), multiplex immunofluorescence (mIF) to investigate CTLA4 pathway and T regulatory cell prevalence and a 22-protein antibody panel using multiplex ion beam imaging (MIBI) for LAG-3/myeloid phenotype interrogation. Images were segmented into single cell resolution and marker prevalence and co-expression analyzed.

Results Despite it’s ‘cold’ tumor designation, T-cell presence in mCRPC by mIF is abundant (mean value of 396 CD3+ T-cells/mm²N=49 patients). However, these T-cells reside in the tumor periphery and their expression of PD-1 and CTLA4 is minimal (mean of 20cells/mm² CD3+CTLA4+, 35.8 cells/mm² for CD3+PD1+). T-regs in these treatment experienced samples are largely absent (average 5.7 cells/mm²). Distal metastases have 2.6 fold less T-cells than prostate tissues overall, yet retain an appreciable T-cell presence (mean 150 cells/mm²). IHC evaluation revealed that 83% of patients, and 100% of distal metastases, had deep loss of tumor cell MHCI expression (<25% tumor cell positivity). MIBI revealed a cell cluster defined by AR-expression in T-cells in only 13% of patients. We also observed an enrichment of LAG-3 in PD1+ tumor associated macrophages rather than in T-cells.

Conclusions Although the TME of mCRPC has appreciable T-cell presence, they lack checkpoint expression of CTLA4/PD1/LAG3. This may be due to the loss of tumor MHCI expression rendering these T-cells unable to engage with tumor antigens. These observations, as well as low Treg presence in the TME, may partially elucidate the mCRPC limited clinical benefit to anti-CTLA4/anti-PD1 therapy. The expression of AR in CD8 T-cells is only present in a minority of patients and may not be a universal mechanism of IO sensitization with AR-targeted therapy. Our data suggest that strategies that increase tumor antigen presentation may increase IO benefit in mCRPC.

Acknowledgements We acknowledge the Contributions of the following collaborators:

1. Jimena Trillo-Tinoco for pathological expertise and Evisa Gjini for study design contributions prior to their departure from Bristol Myers Squibb

2. The members of the Tissue Biomarker Laboratory at the Dana Farber Cancer institute Center for Immuno-Onclogy for multiplex Immunofluorescence staining and analysis (Scott Rodig, Stephen Hodi, Emma Welsh, Kathleen Pfaff)

3. Aliri Bioanalysis for performing MIBI staining, imaging and analysis. (Corinne Ramos for pathology expertise and analysis, Richard Ruez for data analysis and project execution, Erika Pfaunmiller for project execution, Amandine Gerstenberg and Martha Baydoun for computational analysis)

Reference

1. Guan X, Polesso F, Wang C, Sehrawat A, Hawkins R, Murray S, Thomas G, Caruso B, Thompson R, Wood M, Hipfinger C, Hammond S, Graff J, Xia Z, Moran A. Androgen receptor activity in T cells limits checkpoint blockade efficacy. Nature. 2022;606:791–796.

Consent Samples were commercially obtained from vendors in compliance with informed consent