Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+ metastatic prostate cancer

Abstract

Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial, it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with “on-target off-tumor” activity. Here, we show that the intracellular co-stimulatory signaling domain can determine a CAR's sensitivity for tumor antigen expression. A 4-1BB intracellular co-stimulatory signaling domain in PSCA-CARs confers improved selectivity for higher tumor antigen density, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory signaling domain. PSCA-CARs exhibit robust in vivo anti-tumor activity in patient-derived bone-metastatic prostate cancer xenograft models, and 4-1BB-containing CARs show superior T cell persistence and control of disease compared with CD28-containing CARs. Our study demonstrates the importance of co-stimulation in defining an optimal CAR T cell, and also highlights the significance of clinically relevant models in developing solid cancer CAR T cell therapies.

Immunobiology of chimeric antigen receptor T cells and novel designs

“…87 Similarly, PSCA-directed CARs utilizing a 4-1BB costimulatory domain demonstrated activity only in response to cells expressing high levels of the antigen, whereas CARs utilizing a CD28 costimulatory domain were able to target cells expressing either high or low levels of the PSCA antigen. 37 Interestingly, these findings correlate with the kinetics of CAR T cell responses in vivo which may reflect the relative sensitivity of each CAR. 88 As these findings are currently limited to a few models, further studies are needed to determine if these findings are generalizable to all CAR constructs and how this can be incorporated into CAR T cell therapy to reduce the occurrence of antigen low relapse.…”

“…Each of these costimulatory domains have proven sufficient in promoting effective CAR T cell responses in vivo in various preclinical models and clinical trials, however, the incorporation of the 4-1BB domain has demonstrated a significant impact on the in vivo persistence of CAR T cells. 34,36,37 This has been most notable when comparing CAR T cells targeting CD19-positive leukemia with either the CD28 or the 4-1BB costimulatory domain. In pediatric patients with relapsed/ refractory ALL, treatment with a CD28-CAR resulted in persistence which was limited to approximately 2 months in patients achieving remission.…”

Effective Combination Immunotherapy using Oncolytic Viruses to Deliver CAR Targets to Solid Tumors

“…T-cell isolation, lentivirus production and transduction, and ex vivo expansion of CAR T cells was performed as previously described 23 .…”

“…Flow cytometric analysis was performed as previously described 23 . Tumor cells were identified using Ep-CAM (CD326) (BioLegend, Clone: 9C4).…”

Mathematical deconvolution of CAR T-cell proliferation and exhaustion from real-time killing assay data

“…PBT030 endogenously expresses high level of IL13Ra2. HT1080 and PBT138 do not express IL13Ra2 and were lentivirally engineered to express varied levels based on different promoter strengths to investigate the relationship between killing kinetics and antigen expression level: High (>70% + ) driven by the EF1a promoter, Medium (between 40% + -70% + ) driven by the PGK promoter, Low (<20% + ) driven by the attenuated PGK100 promoter [35,36]. These cell lines are denoted with H, M, L respectively e.x.…”