Preclinical Evaluation of Novel Folate Receptor 1-Directed CAR T Cells for Ovarian Cancer.

Treatment options for ovarian cancer patients are limited, and a high unmet clinical need remains for targeted and long-lasting, efficient drugs. Genetically modified T cells expressing chimeric antigen receptors (CAR), are promising new drugs that can be directed towards a defined target and have shown efficient, as well as persisting, anti-tumor responses in many patients. We sought to develop novel CAR T cells targeting ovarian cancer and to assess these candidates preclinically. First, we identified potential CAR targets on ovarian cancer samples. We confirmed high and consistent expressions of the tumor-associated antigen FOLR1 on primary ovarian cancer samples. Subsequently, we designed a series of CAR T cell candidates against the identified target and demonstrated their functionality against ovarian cancer cell lines in vitro and in an in vivo xenograft model. Finally, we performed additional in vitro assays recapitulating immune suppressive mechanisms present in solid tumors and developed a process for the automated manufacturing of our CAR T cell candidate. These findings demonstrate the feasibility of anti-FOLR1 CAR T cells for ovarian cancer and potentially other FOLR1-expressing tumors.

Targeting Stage-Specific Embryonic Antigen 4 (SSEA-4) in Triple Negative Breast Cancer by CAR T Cells Results in Unexpected on Target/off Tumor Toxicities in Mice.

Due to the paucity of targetable antigens, triple-negative breast cancer (TNBC) remains a challenging subtype of breast cancer to treat. In this study, we developed and evaluated a chimeric antigen receptor (CAR) T cell-based treatment modality for TNBC by targeting stage-specific embryonic antigen 4 (SSEA-4), a glycolipid whose overexpression in TNBC has been correlated with metastasis and chemoresistance. To delineate the optimal CAR configuration, a panel of SSEA-4-specific CARs containing alternative extracellular spacer domains was constructed. The different CAR constructs mediated antigen-specific T cell activation characterized by degranulation of T cells, secretion of inflammatory cytokines, and killing of SSEA-4-expressing target cells, but the extent of this activation differed depending on the length of the spacer region. Adoptive transfer of the CAR-engineered T cells into mice with subcutaneous TNBC xenografts mediated a limited antitumor effect but induced severe toxicity symptoms in the cohort receiving the most bioactive CAR variant. We found that progenitor cells in the lung and bone marrow express SSEA-4 and are likely co-targeted by the CAR T cells. Thus, this study has revealed serious adverse effects that raise safety concerns for SSEA-4-directed CAR therapies because of the risk of eliminating vital cells with stem cell properties.

MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors.

Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.

α-Hederin inhibits G protein-coupled receptor kinase 2-mediated phosphorylation of ß2-adrenergic receptors.

BACKGROUND: Recently is has been shown that α- and ß-hederin increase the ß2-adrenergic responsiveness of alveolar type II cells (A549) and human airway smooth muscle cells (HASM), respectively, by inhibiting the internalization of ß2-adrenergic receptors (ß2AR) under stimulating conditions. Internalization of ß2AR is initiated by phosphorylations of certain serines and threonines by cAMP dependent protein kinase A (PKA) and G protein-coupled receptor kinases (GRK). PURPOSE: To evaluate the effect of α-hederin on PKA and GRK2 mediated phosphorylation of GFP-tagged ß2AR. STUDY DESIGN: To study this process we performed In-Cell Western using isoprenaline stimulated HEK293 cells overexpressing ß2AR as GFP fusion protein and specific antibodies against PKA (Ser345/346) and GRK2 (Ser355/356) phosphorylation sites. RESULTS: There was no effect found on the PKA mediated phosphorylation (n = 14) but we could show that α-hederin (1 µM, 12 h) significantly inhibits GRK2 mediated phosphorylation at Ser355/356 by 11 ± 5% (n ≥ 29, p ≤ 0.01) under stimulating conditions compared to the positive control. In Förster resonance energy transfer (FRET) experiments using the isolated kinases in solution α-hederin did not show any influence neither to GRK2 nor to PKA. CONCLUSION: Taken together, these results indicate that α-hederin acts as an indirect GRK2 inhibitor leading to a reduced homologous desensitization of ß2AR-GFP in HEK293 cells.