Purpose: Metastasis to the brain from breast cancer remains a significant clinical challenge, and may be targeted with CAR-based immunotherapy. CAR design optimization for solid tumors is crucial due to the absence of truly restricted antigen expression and potential safety concerns with “on-target off-tumor” activity. Here, we have optimized HER2-CAR T cells for the treatment of breast to brain metastases, and determined optimal second-generation CAR design and route of administration for xenograft mouse models of breast metastatic brain tumors, including multifocal and leptomeningeal disease. Experimental Design: HER2-CAR constructs containing either CD28 or 4-1BB intracellular costimulatory signaling domains were compared for functional activity in vitro by measuring cytokine production, T-cell proliferation, and tumor killing capacity. We also evaluated HER2-CAR T cells delivered by intravenous, local intratumoral, or regional intraventricular routes of administration using in vivo human xenograft models of breast cancer that have metastasized to the brain. Results: Here, we have shown that HER2-CARs containing the 4-1BB costimulatory domain confer improved tumor targeting with reduced T-cell exhaustion phenotype and enhanced proliferative capacity compared with HER2-CARs containing the CD28 costimulatory domain. Local intracranial delivery of HER2-CARs showed potent in vivo antitumor activity in orthotopic xenograft models. Importantly, we demonstrated robust antitumor efficacy following regional intraventricular delivery of HER2-CAR T cells for the treatment of multifocal brain metastases and leptomeningeal disease. Conclusions: Our study shows the importance of CAR design in defining an optimized CAR T cell, and highlights intraventricular delivery of HER2-CAR T cells for treating multifocal brain metastases. Clin Cancer Res; 24(1); 95–105. ©2017 AACR.

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.

Chimeric antigen receptor (CAR)-engineered T cell therapy for solid tumors is limited by the lack of tumor-restricted and homogeneous expression of tumor antigens1,2. Therefore, we engineered an oncolytic virus to express a non-signaling, truncated CD19 (CD19t) protein for tumor-selective delivery, enabling targeting by CD19-specific CAR T cells. Infecting tumor cells with a chimeric oncolytic vaccinia virus coding for CD19t (OV19t) produced de novo CD19 surface-antigen expression prior to virus-mediated tumor lysis. Co-cultured CD19-CAR T cells secreted cytokines and elicited potent cytolytic activity against infected tumors. Using multiple mouse tumor models, intratumoral delivery of OV19t induced tumor expression of CD19t and improved tumor control following CD19-CAR T cell administration. CAR T cell–mediated tumor killing also promoted release of virus from dying tumor cells, which propogated tumor expression of CD19t. These data demonstrate a novel immunotherapy approach utilizing oncolytic viruses to promote de novo CAR T cell targeting of solid tumors.One Sentence SummaryWe describe a novel and effective combination immunotherapy utilizing oncolytic viruses to deliver de novo cell surface expression of CD19 antigen promoting CD19-CAR T cell anti-tumor responses against solid tumors.

Chimeric antigen receptor (CAR) T cell therapy has led to impressive clinical responses in patients with hematological malignancies; however, its effectiveness in patients with solid tumors has been limited. While CAR T cells for the treatment of advanced prostate and pancreas cancer, including those targeting prostate stem cell antigen (PSCA), are being clinically evaluated and are anticipated to show bioactivity, their safety and the impact of the immunosuppressive tumor microenvironment (TME) have not been faithfully explored preclinically. Using a novel human PSCA knockin (hPSCA-KI) immunocompetent mouse model, we evaluated the safety and therapeutic efficacy of PSCA-CAR T cells. We demonstrated that cyclophosphamide (Cy) pre-conditioning significantly modified the immunosuppressive TME and was required to uncover the efficacy of PSCA-CAR T cells in metastatic prostate and pancreas cancer models, with no observed toxicities in normal tissues with endogenous expression of PSCA. This combination dampened the immunosuppressive TME, generated pro-inflammatory myeloid and T cell signatures in tumors, and enhanced the recruitment of antigen-presenting cells, as well as endogenous and adoptively transferred T cells, resulting in long-term anti-tumor immunity.

Chimeric antigen receptor (CAR) T cell therapy has led to impressive clinical responses in patients with hematological malignancies; however, its utility in patients with solid tumors has been limited. While CAR T cells for the treatment of advanced prostate cancer are being clinically evaluated and are anticipated to show bioactivity, their safety and the impact of the immunosuppressive tumor microenvironment (TME) have not been faithfully explored preclinically. Using a novel human prostate stem cell antigen knock-in (hPSCA-KI) immunocompetent mouse model and syngeneic murine PSCA CAR T cells, we performed analyses of normal and tumor tissues by flow cytometry, immunohistochemistry, and/or RNA sequencing. We further assessed the beneficial impact of cyclophosphamide (Cy) pre-conditioning on modifications to the immunosuppressive TME and impact on PSCA-CAR T cell safety and efficacy. We observed an in vivo requirement of Cy pre-conditioning in uncovering the efficacy of PSCA-CAR T cells in prostate and pancreas cancer models, with no observed toxicities in normal tissues with endogenous PSCA expression. This combination also dampened the immunosuppressive TME, generated pro-inflammatory myeloid and T cell signatures in tumors, and enhanced the recruitment of antigen-presenting cells, and endogenous as well as adoptively-transferred CAR T cells, resulting in long-term anti-tumor immunity.

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<div>Abstract<p><b>Purpose:</b> Metastasis to the brain from breast cancer remains a significant clinical challenge, and may be targeted with CAR-based immunotherapy. CAR design optimization for solid tumors is crucial due to the absence of truly restricted antigen expression and potential safety concerns with “on-target off-tumor” activity. Here, we have optimized HER2-CAR T cells for the treatment of breast to brain metastases, and determined optimal second-generation CAR design and route of administration for xenograft mouse models of breast metastatic brain tumors, including multifocal and leptomeningeal disease.</p><p><b>Experimental Design:</b> HER2-CAR constructs containing either CD28 or 4-1BB intracellular costimulatory signaling domains were compared for functional activity <i>in vitro</i> by measuring cytokine production, T-cell proliferation, and tumor killing capacity. We also evaluated HER2-CAR T cells delivered by intravenous, local intratumoral, or regional intraventricular routes of administration using <i>in vivo</i> human xenograft models of breast cancer that have metastasized to the brain.</p><p><b>Results:</b> Here, we have shown that HER2-CARs containing the 4-1BB costimulatory domain confer improved tumor targeting with reduced T-cell exhaustion phenotype and enhanced proliferative capacity compared with HER2-CARs containing the CD28 costimulatory domain. Local intracranial delivery of HER2-CARs showed potent <i>in vivo</i> antitumor activity in orthotopic xenograft models. Importantly, we demonstrated robust antitumor efficacy following regional intraventricular delivery of HER2-CAR T cells for the treatment of multifocal brain metastases and leptomeningeal disease.</p><p><b>Conclusions:</b> Our study shows the importance of CAR design in defining an optimized CAR T cell, and highlights intraventricular delivery of HER2-CAR T cells for treating multifocal brain metastases. <i>Clin Cancer Res; 24(1); 95–105. ©2017 AACR</i>.</p></div>