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.

WWOX, the common fragile site FRA16D gene product, regulates ATM activation and the DNA damage response.

Genomic instability is a hallmark of cancer. The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor spanning the common chromosomal fragile site FRA16D. Here, we report a direct role of WWOX in DNA damage response (DDR) and DNA repair. We show that Wwox deficiency results in reduced activation of the ataxia telangiectasia-mutated (ATM) checkpoint kinase, inefficient induction and maintenance of γ-H2AX foci, and impaired DNA repair. Mechanistically, we show that, upon DNA damage, WWOX accumulates in the cell nucleus, where it interacts with ATM and enhances its activation. Nuclear accumulation of WWOX is regulated by its K63-linked ubiquitination at lysine residue 274, which is mediated by the E3 ubiquitin ligase ITCH. These findings identify a novel role for the tumor suppressor WWOX and show that loss of WWOX expression may drive genomic instability and provide an advantage for clonal expansion of neoplastic cells.

Autophosphorylation and Pin1 binding coordinate DNA damage-induced HIPK2 activation and cell death.

Excessive genome damage activates the apoptosis response. Protein kinase HIPK2 is a key regulator of DNA damage-induced apoptosis. Here, we deciphered the molecular mechanism of HIPK2 activation and show its relevance for DNA damage-induced apoptosis in cellulo and in vivo. HIPK2 autointeracts and site-specifically autophosphorylates upon DNA damage at Thr880/Ser882. Autophosphorylation regulates HIPK2 activity and mutation of the phosphorylation-acceptor sites deregulates p53 Ser46 phosphorylation and apoptosis in cellulo. Moreover, HIPK2 autophosphorylation is conserved between human and zebrafish and is important for DNA damage-induced apoptosis in vivo. Mechanistically, autophosphorylation creates a binding signal for the phospho-specific isomerase Pin1. Pin1 links HIPK2 activation to its stabilization by inhibiting HIPK2 polyubiquitination and modulating Siah-1-HIPK2 interaction. Concordantly, Pin1 is required for DNA damage-induced HIPK2 stabilization and p53 Ser46 phosphorylation and is essential for induction of apotosis both in cellulo and in zebrafish. Our results identify an evolutionary conserved mechanism regulating DNA damage-induced apoptosis.

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.

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.

Identification of CD318, TSPAN8 and CD66c as target candidates for CAR T cell based immunotherapy of pancreatic adenocarcinoma.

A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules.

CRISPR-Mediated Editing of the B Cell Receptor in Primary Human B Cells.

Vaccination approaches have generally focused on the antigen rather than the resultant antibodies generated, which differ greatly in quality and function between individuals. The ability to replace the variable regions of the native B cell receptor (BCR) heavy and light chain loci with defined recombined sequences of a preferred monoclonal antibody could enable curative adoptive cell transfer. We report CRISPR-mediated homologous recombination (HR) into the BCR of primary human B cells. Ribonucleoprotein delivery enabled editing at the model CXCR4 locus, as demonstrated by T7E1 assay, flow cytometry, and TIDE analysis. Insertion via HR was confirmed by sequencing, cross-boundary PCR, and restriction digest. Optimized conditions were used to achieve HR at the BCR variable heavy and light chains. Insertion was confirmed at the DNA level, and transgene expression from the native BCR promoters was observed. Reprogramming the specificity of antibodies in the genomes of B cells could have clinical importance.

Immunometabolic Regulations Mediated by Coinhibitory Receptors and Their Impact on T Cell Immune Responses.

Host immunity provides wide spectrum protection that serves to eradicate pathogens and cancer cells, while maintaining self-tolerance and immunological homeostasis. Ligation of the T cell receptor (TCR) by antigen activates signaling pathways that coordinately induce aerobic glycolysis, mitochondrial activity, anabolic metabolism, and T effector cell differentiation. Activation of PI3K, Akt, and mTOR triggers the switch to anabolic metabolism by inducing transcription factors such as Myc and HIF1, and the glucose transporter Glut1, which is pivotal for the increase of glucose uptake after T cell activation. Activation of MAPK signaling is required for glucose and glutamine utilization, whereas activation of AMPK is critical for energy balance and metabolic fitness of T effector and memory cells. Coinhibitory receptors target TCR-proximal signaling and generation of second messengers. Imbalanced activation of such signaling pathways leads to diminished rates of aerobic glycolysis and impaired mitochondrial function resulting in defective anabolic metabolism and altered T cell differentiation. The coinhibitory receptors mediate distinct and synergistic effects on the activation of signaling pathways thereby modifying metabolic programs of activated T cells and resulting in altered immune functions. Understanding and therapeutic targeting of metabolic programs impacted by coinhibitory receptors might have significant clinical implications for the treatment of chronic infections, cancer, and autoimmune diseases.

The role of metabolic reprogramming in T cell fate and function.

T lymphocytes undergo extensive changes in their metabolic properties during their transition through various differentiation states, from naïve to effector to memory or regulatory roles. The cause and effect relationship between metabolism and differentiation is a field of intense investigation. Many recent studies demonstrate the dependency of T cell functional outcomes on metabolic pathways and the possibility of metabolic intervention to modify these functions. In this review, we describe the basic metabolic features of T cells and new findings on how these correlate with various differentiation fates and functions. We also highlight the latest information regarding the main factors that affect T cell metabolic reprogramming.

Clinical significance of T cell metabolic reprogramming in cancer.

Conversion of normal cells to cancer is accompanied with changes in their metabolism. During this conversion, cell metabolism undergoes a shift from oxidative phosphorylation to aerobic glycolysis, also known as Warburg effect, which is a hallmark for cancer cell metabolism. In cancer cells, glycolysis functions in parallel with the TCA cycle and other metabolic pathways to enhance biosynthetic processes and thus support proliferation and growth. Similar metabolic features are observed in T cells during activation but, in contrast to cancer, metabolic transitions in T cells are part of a physiological process. Currently, there is intense interest in understanding the cause and effect relationship between metabolic reprogramming and T cell differentiation. After the recent success of cancer immunotherapy, the crosstalk between immune system and cancer has come to the forefront of clinical and basic research. One of the key goals is to delineate how metabolic alterations of cancer influence metabolism-regulated function and differentiation of tumor resident T cells and how such effects might be altered by immunotherapy. Here, we review the unique metabolic features of cancer, the implications of cancer metabolism on T cell metabolic reprogramming during antigen encounters, and the translational prospective of harnessing metabolism in cancer and T cells for cancer therapy.