Generation of human parallel chimeric antigen receptor (pCAR) T cells to achieve synergistic T cell co-stimulation

Dual co-stimulation may be harnessed using parallel chimeric antigen receptors (pCARs) in which two distinct co-stimulatory units are adjacently localized on the plasma membrane. This protocol summarizes construct design, human T cell isolation, retroviral transduction, tissue culture expansion, and preclinical testing of pCAR T cells, exemplified by receptors that co-target avb6 integrin and ErbB dimers. For complete details on the use and execution of this protocol, please refer to Muliaditan et al. (2021)

Anti-Folate Receptor alpha-directed Antibody Therapies Restrict the Growth of Triple Negative Breast Cancer

PURPOSE: Highly-aggressive triple negative breast cancers (TNBCs) lack validated therapeutic targets and have high risk of metastatic disease. Folate Receptor alpha (FRα) is a central mediator of cell growth regulation that could serve as an important target for cancer therapy. EXPERIMENTAL DESIGN: We evaluated FRα expression in breast cancers by genomic (N = 3414) and immunohistochemical (N = 323) analyses and its association with clinical parameters and outcomes. We measured the functional contributions of FRα in TNBC biology by RNA interference and the anti-tumor functions of an antibody recognizing FRα (MOv18-IgG1), in vitro and in human TNBC xenograft models. RESULTS: FRα is overexpressed in significant proportions of aggressive basal like/TNBC tumors, and in post-neoadjuvant chemotherapy-residual disease associated with a high risk of relapse. Expression is associated with worse overall survival. TNBCs show dysregulated expression of thymidylate synthase, folate hydrolase 1 and methylenetetrahydrofolate reductase, involved in folate metabolism. RNA interference to deplete FRα decreased Src and ERK signaling and resulted in reduction of cell growth. An anti-FRα antibody (MOv18-IgG1) conjugated with a Src inhibitor significantly restricted TNBC xenograft growth. Moreover, MOv18-IgG1 triggered immune-dependent cancer cell death in vitro by human volunteer and breast cancer patient immune cells, and significantly restricted orthotopic and patient-derived xenograft growth. CONCLUSIONS: FRα is overexpressed in high-grade TNBC and post-chemotherapy residual tumors. It participates in cancer cell signaling and presents a promising target for therapeutic strategies such as antibody-drug conjugates, or passive immunotherapy priming Fc-mediated anti-tumor immune cell responses

Hypoxia-sensing CAR T cells provide safety and efficacy in treating solid tumors

Utilizing T cells expressing chimeric antigen receptors (CARs) to identify and attack solid tumors has proven challenging, in large part because of the lack of tumor-specific targets to direct CAR binding. Tumor selectivity is crucial because on-target, off-tumor activation of CAR T cells can result in potentially lethal toxicities. This study presents a stringent hypoxia-sensing CAR T cell system that achieves selective expression of a pan-ErbB-targeted CAR within a solid tumor, a microenvironment characterized by inadequate oxygen supply. Using murine xenograft models, we demonstrate that, despite widespread expression of ErbB receptors in healthy organs, the approach provides anti-tumor efficacy without off-tumor toxicity. This dynamic on/off oxygen-sensing safety switch has the potential to facilitate unlimited expansion of the CAR T cell target repertoire for treating solid malignancies

Synergistic T cell signaling by 41BB and CD28 is optimally achieved by membrane proximal positioning within parallel chimeric antigen receptors

Second generation (2G) chimeric antigen receptors (CARs) contain a CD28 or 41BB co-stimulatory endodomain and elicit remarkable efficacy in hematological malignancies. Third generation (3G) CARs extend this linear blueprint by fusing both co-stimulatory units in series. However, clinical impact has been muted despite compelling evidence that co-signaling by CD28 and 41BB can powerfully amplify natural immune responses. We postulate that effective dual co-stimulation requires juxta-membrane positioning of endodomain components within separate synthetic receptors. Consequently, we designed parallel (p)CARs in which a 2G (CD28+CD3ζ) CAR is co-expressed with a 41BB-containing chimeric co-stimulatory receptor. We demonstrate that the pCAR platform optimally harnesses synergistic and tumor-dependent co-stimulation to resist T cell exhaustion and senescence, sustaining proliferation, cytokine release, cytokine signaling, and metabolic fitness upon repeated stimulation. When engineered using targeting moieties of diverse composition, affinity, and specificity, pCAR T cells consistently elicit superior anti-tumor activity compared with T cells that express traditional linear CARs

Synergistic T cell signaling by 41BB and CD28 is optimally achieved by membrane proximal positioning within parallel chimeric antigen receptors

Second generation (2G) chimeric antigen receptors (CARs) contain a CD28 or 41BB co-stimulatory endodomain and elicit remarkable efficacy in hematological malignancies. Third generation (3G) CARs extend this linear blueprint by fusing both co-stimulatory units in series. However, clinical impact has been muted despite compelling evidence that co-signaling by CD28 and 41BB can powerfully amplify natural immune responses. We postulate that effective dual co-stimulation requires juxta-membrane positioning of endodomain components within separate synthetic receptors. Consequently, we designed parallel (p)CARs in which a 2G (CD28+CD3ζ) CAR is co-expressed with a 41BB-containing chimeric co-stimulatory receptor. We demonstrate that the pCAR platform optimally harnesses synergistic and tumor-dependent co-stimulation to resist T cell exhaustion and senescence, sustaining proliferation, cytokine release, cytokine signaling, and metabolic fitness upon repeated stimulation. When engineered using targeting moieties of diverse composition, affinity, and specificity, pCAR T cells consistently elicit superior anti-tumor activity compared with T cells that express traditional linear CARs