Background Chimeric antigen receptor (CAR) T-cell therapies have shown remarkable success in treating hematological malignancies. However, demonstrations of efficacy in solid cancer indications have been limited so far, in part due to antigen heterogeneity. Prostate-specific membrane antigen (PSMA) has an elevated expression in metastastic castration resistant prostate cancer (mCRPC), making it a promising tumor-associated antigen for immune therapy. Although PSMA directed radiotheurapeutics have demonstrated clinical activity, PSMA expression in mCRPC is heterogeneous across different metastatic sites and molecular subtypes of mCRPC. Hence, design of multispecific CAR T-cell, targeting several cancer antigens simultaneously, might be envisaged as one of the best options to overcome antigen heterogeneity in solid cancers. NKG2D ligands (NKG2DL), are a group of 8 stress-induced ligands expressed by virtually all cancer types and absent from normal healthy cells. Therefore, these represent an attractive target for multispecific CAR T-cells across a wide range of cancer indications, including prostate cancer.

Methods To improve CAR T-cell efficacy against prostate cancer, we designed a PSMA/NKG2DL targeting tandem CAR T-cell candidate that encompasses the extracellular domain of the natural NKG2D receptor fused to an anti-PSMA scFv. Functionality of the αPSMA/NKG2DL tandem CAR T-cells was evaluated in comparison to single-targeting CAR T-cells in vitro in cocultures with cells expressing either both PSMA and NKG2DL or NKG2DL only, and in vivo in a mixed prostate PC3-WT/PC3-PSMA+ xenograft model.

Results We show here that αPSMA/NKG2DL tandem CAR T-cells (CAR-Ts) were highly responsive against prostate cancer cell lines expressing either both PSMA and NKG2DL (LNCAP cells and PC-3 PSMA+ cells) or NKG2DL only (PC-3 WT cells). Upon chronic antigen stimulation, αPSMA CAR-Ts and αPSMA/NKG2DL tandem CAR Ts were able to continually lyse PSMA+ tumor cells with no discernible difference between the single and tandem CAR-Ts. Furthermore, PSMA negative prostate cancer cells were targeted by the NKG2D expressing CAR-Ts (single and tandem) but not by the αPSMA CAR-Ts. These results will be further evaluated in an aggressive in vivo xenograft model containing mixed PC3-WT/PC3-PSMA prostate cancer cells.

Conclusions Multispecific CAR-Ts are a promising strategy to overcome antigen heterogeneity and improve anti-tumor efficacy in solid tumors, such as prostate cancer. We have designed a PSMA/NKG2DL targeting tandem CAR T-cell candidate that is highly responsive against both PSMA and NKG2DL expressing and NKGD2L only expressing cells. In addition, by targeting stress-induced targets rather than lineage specific targets, loss of efficacy due to antigenic heterogeneity/modulation may be avoided.

Ethics Approval Animal housing and experimental procedures will be conducted according to the French and European Regulations and the National Research Council Guide for the Care and Use of Laboratory Animals . The animal facility is authorized by the French authorities (Dijon: Agreement N° C 21 231 011 EA). All animal procedures (including surgery, anesthesia and euthanasia as applicable) used in the current study (Ethical protocol: #36462-2022040816229184 v6 ONCO-SC) will be submitted to the Institutional Animal Care and Use Committee of Oncodesign Services (Oncomet) approved by French authorities [CNREEA agreement N° 91 (Oncodesign Services)].