Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is a cell surface antigen for therapeutic targeting in prostate cancer. Here, we report broad expression of STEAP1 relative to prostate-specific membrane antigen (PSMA) in lethal metastatic prostate cancers and the development of a STEAP1-directed chimeric antigen receptor (CAR) T cell therapy. STEAP1 CAR T cells demonstrate reactivity in low antigen density, antitumor activity across metastatic prostate cancer models, and safety in a human STEAP1 knock-in mouse model. STEAP1 antigen escape is a recurrent mechanism of treatment resistance and is associated with diminished tumor antigen processing and presentation. The application of tumor-localized interleukin-12 (IL-12) therapy in the form of a collagen binding domain (CBD)-IL-12 fusion protein combined with STEAP1 CAR T cell therapy enhances antitumor efficacy by remodeling the immunologically cold tumor microenvironment of prostate cancer and combating STEAP1 antigen escape through the engagement of host immunity and epitope spreading.
© 2023. The Author(s).

Persistence of the human immunodeficiency virus type-1 (HIV-1) latent reservoir in infected individuals remains a problem despite fully suppressive antiretroviral therapy (ART). While reservoir formation begins during acute infection, the mechanisms responsible for its establishment remain unclear. CD8+ T cells are important during the initial control of viral replication. Here we examined the effect of CD8+ T cells on formation of the latent reservoir in simian immunodeficiency virus (SIV)-infected macaques by performing experimental CD8+ depletion either before infection or before early (that is, day 14 post-infection) ART initiation. We found that CD8+ depletion resulted in slower decline of viremia, indicating that CD8+ lymphocytes reduce the average lifespan of productively infected cells during acute infection and early ART, presumably through SIV-specific cytotoxic T lymphocyte (CTL) activity. However, CD8+ depletion did not change the frequency of infected CD4+ T cells in the blood or lymph node as measured by the total cell-associated viral DNA or intact provirus DNA assay. In addition, the size of the persistent reservoir remained the same when measuring the kinetics of virus rebound after ART interruption. These data indicate that during early SIV infection, the viral reservoir that persists under ART is established largely independent of CTL control.
© 2023. The Author(s).

h4>Summary/h4> Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is a compelling tumor-associated cell surface antigen for therapeutic targeting in solid tumors. We identified broad expression of STEAP1 (87% positive) in lethal metastatic prostate cancer, even more so than prostate-specific membrane antigen (PSMA, 60% positive) which is a clinically established diagnostic and therapeutic target. Second-generation chimeric antigen receptor (CAR) T cells were engineered for reactivity against STEAP1 and demonstrated substantial antitumor activity in metastatic human prostate cancer models in immunodeficient mice. Adoptive transfer of STEAP1 CAR T cells was associated with prolonged peripheral persistence and either disease eradication or substantial tumor growth inhibition with progressive disease demonstrating antigen loss. As STEAP1 CAR T cells were also highly active in antigen density conditions as low as ∼1,500 molecules/cell, we generated a human STEAP1 (hSTEAP1) knock-in (KI) mouse to evaluate the potential for on-target off-tumor toxicities. hSTEAP1-KI mice demonstrated a pattern of systemic hSTEAP1 expression akin to that observed in humans with the greatest expression found in the prostate gland. Mouse-in-mouse studies of STEAP1 CAR T cell therapy in immunocompetent hSTEAP1-KI mice engrafted with disseminated mouse prostate cancer showed preliminary safety without evidence of gross toxicity, cytokine storm, or architectural disruption and increased T cell infiltration at sites of systemic hSTEAP1 expression. Tumor responses and extension of survival were appreciated but antigen loss was identified in recurrent and progressive disease. In summary, we report the extent of STEAP1 expression in treatment-refractory metastatic prostate cancer, the generation of a STEAP1 CAR T cell therapy with promising potency and safety in preclinical studies of advanced prostate cancer, and antigen escape as a mechanism of resistance to effective STEAP1 CAR T cell therapy.

h4>ABSTRACT/h4> Chimeric antigen receptor (CAR) costimulatory domains steer the phenotypic output of therapeutic T cells. In most cases these domains are derived from native immune receptors, composed of signaling motif combinations selected by evolution. To explore if non-natural combinations of signaling motifs could drive novel cell fates of interest, we constructed a library of CARs containing ∼2,300 synthetic costimulatory domains, built from combinations of 13 peptide signaling motifs. The library produced CARs driving diverse fate outputs, which were sensitive to motif combinations and configurations. Neural networks trained to decode the combinatorial grammar of CAR signaling motifs allowed extraction of key design rules. For example, the non-native combination of TRAF- and PLCγ1-binding motifs was found to simultaneously enhance cytotoxicity and stemness, a clinically desirable phenotype associated with effective and durable tumor killing. The neural network accurately predicts that addition of PLCγ1-binding motifs improves this phenotype when combined with TRAF-binding motifs, but not when combined with other immune signaling motifs (e.g. PI3K-or Grb2-binding motifs). This work shows how libraries built from the minimal building blocks of signaling, combined with machine learning, can efficiently guide engineering of receptors with desired phenotypes. h4>Graphical Abstract/h4>