Anthony Park

Regional Delivery of Chimeric Antigen Receptor–Engineered T Cells Effectively Targets HER2+ Breast Cancer Metastasis to the Brain

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.

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 CARs 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.

Novel oncolytic chimeric orthopoxvirus causes regression of pancreatic cancer xenografts and exhibits abscopal effect at a single low dose

BackgroundPancreatic ductal adenocarcinoma (PDAC) has been increasing by 0.5% per year in the United States. PDAC portends a dismal prognosis and novel therapies are needed. This study describes the generation and characterization of a novel oncolytic chimeric orthopoxvirus for the treatment of pancreatic cancer.MethodsAfter chimerization and high-throughput screening, CF33 was chosen from 100 new chimeric orthopoxvirus isolates for its ability to kill pancreatic cancer cells. In vitro cytotoxicity was assayed in six pancreatic cancer cell lines. In vivo efficacy and toxicity were evaluated in PANC-1 and MIA PaCa-2 xenograft models.ResultsCF33 caused rapid killing of six pancreatic cancer cells lines in vitro, releasing damage-associated molecular patterns, and regression of PANC-1 injected and non-injected distant xenografts in vivo after a single low intratumoral dose of 103 plaque-forming units. Using luciferase imaging, CF33 was noted to preferentially replicate in tumors which corresponds to the low viral titers found in solid organs.ConclusionThe low dose of CF33 required to treat pancreatic cancer in this preclinical study may ease the manufacturing and dosing challenges currently facing oncolytic viral therapy.

Development and optimization of PSCA-specific CAR T cells for the treatment of bone metastatic prostate cancer.

Meeting abstractsnnProstate Cancer (PCa) is the third most common cancer type in the United States, with over 200,000 new cases projected to be diagnosed this year. In approximately 80% of PCa patients, tumor phenotype includes overexpression of prostate stem cell antigen, or PSCA. Furthermore, PSCA

A Novel Oncolytic Chimeric Orthopoxvirus Encoding Luciferase Enables Real-Time View of Colorectal Cancer Cell Infection

This study hypothesizes that a novel oncolytic chimeric orthopoxvirus CF33-Fluc is imageable and targets colorectal cancer cells (CRCs). A novel chimeric orthopoxvirus (CF33) was constructed. The thymidine kinase locus was replaced with firefly luciferase (Fluc) to yield a recombinant virus—CF33-Fluc. In vitro cytotoxicity and viral replication assays were performed. In vivo CRC flank xenografts received single doses of intratumoral or intravenous CF33-Fluc. Viral biodistribution was analyzed via luciferase imaging and organ titers. CF33-Fluc infects, replicates in, and kills CRCs in vitro in a dose-dependent manner. CF33 has superior secretion of extracellular-enveloped virus versus all but one parental strain. Rapid tumor regression or stabilization occurred in vivo at a low dose over a short time period, regardless of the viral delivery method in the HCT-116 colorectal tumor xenograft model. Rapid luciferase expression in virus-infected tumor cells was associated with treatment response. CRC death occurs via necroptotic pathways. CF33-Fluc replicates in and kills colorectal cancer cells in vitro and in vivo regardless of delivery method. Expression of luciferase enables real-time tracking of viral replication. Despite the chimerism, CRC death occurs via standard poxvirus-induced mechanisms. Further studies are warranted in immunocompetent models.

406. Development and Optimization of PSCA-Specific CAR T Cells for the Treatment of Bone Metastatic Prostate Cancer

Prostate Cancer (PCa) is the third most common cancer type in the United States, with over 200,000 new cases projected to be diagnosed this year. In approximately 80% of PCa patients, tumor phenotype includes overexpression of prostate stem cell antigen, or PSCA. Furthermore, PSCA is expressed on nearly 100% of bone metastatic prostate cancers, making it an attractive immunotherapeutic target. We have genetically engineered T cells to express chimeric antigen receptors (CARs) which specifically target PSCA. Recent clinical trials with CARs targeting CD19 for B-cell malignancies have demonstrated impressive results, yet replicating this success with other antigen targets remains elusive. Immunotherapy against solid tumors poses a more difficult tumor challenge because of the immunosuppressive microenvironment that can significantly hinder CAR efficacy. Additionally, there have been instances of on-target, off-tumor toxicity due to low levels of antigen expression on normal tissue.In the current project we have modified various components of our CAR constructs to improve specificity and overall therapeutic efficacy. Through various in vitro functional assays and in vivo xenograft models, we have evaluated and optimized a PSCA-targeting CAR. We first compared two single-chain variable fragments with different paratopes. While both show comparable potency, one of the scFvs showed nonspecific activity against PSCA-negative tumor lines. Similarly, our data suggest that the 28ζ-costimulatory domain, regardless of linker length, also shows non-specific activation and killing of PSCA-negative tumor lines as compared to the 4-1BB costimulatory domain. Finally, we have demonstrated differences between long, middle, and short linker lengths in intracellular cytokine production, activation, and killing capacities in vitro and in vivo. By modifying both the ectodomain and intracellular region, we are able to improve the specificity and functionality of our PSCA-CARs, which is essential to developing effective immunotherapies for this advanced disease.

204. HER2-Specific Chimeric Antigen Receptor T Cells for the Treatment of Breast-to-Brain Metastasis

Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells has demonstrated robust and durable clinical efficacy in patients with CD19+ B-cell malignancies. Broader application of this approach to brain and other advanced solid tumors is an immediate goal for the field and is presently under intense investigation. In 2014, 40,000 individuals in the U. S. alone succumbed to breast cancer, primarily as a result of metastatic disease. Approximately 30 percent of breast cancer patients carry an amplification of the HER2 gene and/or HER2 over-expression, which confers a particularly poor prognosis. Among the most common sites of HER2-positive breast cancer metastases is the brain. For patients with breast cancer that has metastasized to the brain, the 1-year survival rate is a dismal 20 percent. Despite clinical successes in both preventing relapse and treating systemic disease with HER2-targeted therapies, the currently available agents are only modestly effective in managing brain metastasis. Our group has demonstrated safety and transient anti-tumor responses in two U. S. FDA-authorized phase I clinical trials evaluating local intracranial adoptive transfer of CAR T cells in glioma patients.Our current project builds on this clinical experience with locally administered CAR T cells that specifically target HER2 for the treatment of breast-to-brain metastasis. Our lab has initiated design and testing of CAR T cell therapy targeting HER2 based on an scFv derived from trastuzumab. We anticipate that local intracranial delivery will enhance therapeutic response, while reducing the likelihood of off-tumor systemic toxicities as previously observed. Importantly, HER2 expression in normal brain tissue is limited, supporting HER2 as a therapeutic target in brain metastasis. Our innovative CAR T cell platform focuses on engineering central memory T cells (Tcm) for therapeutic application, with the intent of improving persistence of T cells after infusion, a critical parameter correlated with ultimate therapeutic success. Herein, we have evaluated several HER2-CAR constructs incorporating either the CD28 or 4-1BB co-stimulatory domain using both in vitro T cell functional assays as well as orthotopic patient-derived xenograft models of breast-to-brain metastasis. While HER2-28ζ and HER2-BBζ CAR T cells similarly kill HER2+ breast cancer cells in vitro, BBζ CARs demonstrate lower induction of the exhaustion marker PD-1 and proliferate better compare with 28ζ CARs. Both HER2-CARs similarly lead to tumor eradication and prolonged survival. Based on these data, we have successfully developed HER2-specific CAR T cells, and plan to clinically develop these CARs for the treatment of HER2+ metastatic disease.

Development of chimeric antigen receptor (CAR) T-cell immunotherapy for glioblastoma targeting epidermal growth factor receptor variant III (EGFRvIII)

Glioblastoma multiforme (GBM) is the most common brain tumor, with a poor prognosis of less than 14 months after initial diagnosis. Gene amplification and mutation of epidermal growth factor receptor (EGFR) are frequently observed in primary GBM. The most common variant of EGFR, known as EGFRvIII, is expressed in approximately 30% of GBM patients, but is absent on normal cells, making it a desirable target for cancer immunotherapy. In addition to the surge of adoptive cell immunotherapies for the treatment of advanced cancers, chimeric antigen receptors (CARs) have come to the forefront as a promising therapeutic strategy. We have engineered genetically modified T cells to express CARs that specifically target tumor cells expressing EGFRvIII and/or amplified EGFR for the treatment of malignant gliomas. n nOur studies have focused on optimizing the EGFRvIII-CAR structural design, including the antigen-targeting domain (scFv), extracellular non-signaling linker, and intracellular co-stimulatory signaling domains, to improve overall CAR T cell specificity and efficacy. Thus, we have compared five different scFvs for targeting EGFRvIII, and four different linkers with varying lengths. CAR activity was evaluated using a variety of in vitro functional assays, including CD107a degranulation, IFN-γ production, PD-1 induction, T cell proliferation and tumor killing assays. Using these assays, we have defined CARs capable of mediating highly-specific activation and killing of target tumor cells expressing EGFRvIII and/or amplified EGFR, with minimal targeting of endogenous EGFR-expressing cells. We further validated our EGFRvIII-specific CAR T cells using preclinical human xenograft models of GBM. Our in vivo tumor studies show dose-dependent killing by locally administered EGFRvIII-CAR T cells against subcutaneous tumors expressing EGFRvIII. Combined, these studies demonstrate potent anti-tumor activity of EGFRvIII-specific CAR T cells.

Development of HER2-specific chimeric antigen receptor T cells for the treatment of breast-to-brain metastasis

Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells has demonstrated robust and durable clinical efficacy in patients with CD19+ B cell malignancies. Broader application of this approach to brain and other advanced solid tumors is an immediate goal for the field and is presently under intense investigation. In 2014, 40,000 individuals in the U.S. alone succumbed to breast cancer, primarily as a result of metastatic disease. Approximately 30 percent of breast cancer patients carry an amplification of the HER2 gene and/or HER2 over-expression, which confers a particularly poor prognosis. Among the most common sites of HER2-positive breast cancer metastases is the brain. For patients with breast cancer that has metastasized to the brain, the 1-year survival rate is a dismal 20 percent. Despite clinical successes in both preventing relapse and treating systemic disease with HER2-targeted therapies, the currently available agents are only modestly effective in managing brain metastasis. Our group has demonstrated safety and transient anti-tumor responses in two U.S. FDA-authorized Phase I clinical trials evaluating local intracranial adoptive transfer of CAR T cells in glioma patients. Our current project builds on this clinical experience with locally administered CAR T cells that specifically target HER2 for the treatment of breast-to-brain metastasis. n nOur lab has initiated design and testing of CAR T cell therapy targeting HER2 based on an scFv derived from trastuzumab. We anticipate that local intracranial delivery will enhance therapeutic response, while reducing the likelihood of off-tumor systemic toxicities as previously observed. Importantly, HER2 expression in normal brain tissue is limited, supporting HER2 as a therapeutic target in brain metastasis. Our innovative CAR T cell platform focuses on engineering central memory T cells (Tcm) for therapeutic application, with the intent of improving persistence of T cells after infusion, a critical parameter correlated with ultimate therapeutic success. Herein, we have evaluated several HER2-CAR constructs incorporating either the CD28 or 4-1BB co-stimulatory domain using both in vitro T cell functional assays as well as orthotopic patient-derived xenograft models of breast-to-brain metastasis. While HER2-28z and HER2-BBz CAR T cells similarly kill HER2+ breast cancer cells in vitro, BBz CARs demonstrate lower induction of the exhaustion marker PD-1 and proliferate better compare with 28z CARs. Both HER2-CARs similarly lead to tumor eradication and prolonged survival. Based on these data, we have successfully developed HER2-specific CAR T cells, and plan to clinically develop these CARs for the treatment of HER2+ metastatic disease.