The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity

To improve the efficacy and safety of chimeric antigen receptor (CAR)-expressing T cell therapeutics through enhanced CAR design, we analysed CAR structural factors that affect CAR-T cell function. We studied the effects of disulphide bonding at cysteine residues and glycosylation in the HD on CAR-T function. We used first-generation CAR[V/28/28/3z] and CAR[V/8a/8a/3z], consisting of a mouse vascular endothelial growth factor receptor 2 (VEGFR2)-specific single-chain variable fragment tandemly linked to CD28- or CD8α-derived HD, transmembrane domain (TMD) and a CD3ζ-derived signal transduction domain (STD). We constructed structural variants by substituting cysteine with alanine and asparagine (putative N-linked glycosylation sites) with aspartate. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] formed homodimers, the former through a single HD cysteine residue and the latter through the more TMD-proximal of the two cysteine residues. The absence of disulphide bonds did not affect membrane CAR expression but reduced antigen-specific cytokine production and cytotoxic activity. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] harboured one N-linked glycosylation site, and CAR[V/8a/8a/3z] underwent considerable O-linked glycosylation at an unknown site. Thus, N-linked glycosylation of CAR[V/28/28/3z] promotes stable membrane CAR expression, while having no effect on the expression or CAR-T cell activity of CAR[V/8a/8a/3z]. Our findings demonstrate that post-translational modifications of the CAR HD influence CAR-T cell activity, establishing a basis for future CAR design.     

Cell Therapies in Bladder Cancer Management

Currently, bladder cancer (BC) represents a challenging problem in the field of Oncology. The high incidence, prevalence, and progression of BC have led to the exploration of new avenues in its management, in particular in advanced metastatic stages. The recent inclusion of immune checkpoint blockade inhibitors as a therapeutic option for BC represents an unprecedented advance in BC management. However, although some patients show durable responses, the fraction of patients showing benefit is still limited. Notwithstanding, cell-based therapies, initially developed for the management of hematological cancers by infusing immune or trained immune cells or after the engineering of chimeric antigen receptor (CAR) expressing cells, are promising tools to control, or even cure, solid tumors. In this review, we summarize recent cell-based immunotherapy studies, with a special focus on BC.     

Learning from TCR Signaling and Immunological Synapse Assembly to Build New Chimeric Antigen Receptors (CARs)

Chimeric antigen receptor (CAR) T cell immunotherapy is a revolutionary pillar in cancer treatment. Clinical experience has shown remarkable successes in the treatment of certain hematological malignancies but only limited efficacy against B cell chronic lymphocytic leukemia (CLL) and other cancer types, especially solid tumors. A wide range of engineering strategies have been employed to overcome the limitations of CAR T cell therapy. However, it has become increasingly clear that CARs have unique, unexpected features; hence, a deep understanding of how CARs signal and trigger the formation of a non-conventional immunological synapse (IS), the signaling platform required for T cell activation and execution of effector functions, would lead a shift from empirical testing to the rational design of new CAR constructs. Here, we review current knowledge of CARs, focusing on their structure, signaling and role in CAR T cell IS assembly. We, moreover, discuss the molecular features accounting for poor responses in CLL patients treated with anti-CD19 CAR T cells and propose CLL as a paradigm for diseases connected to IS dysfunctions that could significantly benefit from the development of novel CARs to generate a productive anti-tumor response.     

Chimeric Antigen Receptor-Engineered T Cell Therapy for the Management of Patients with Metastatic Prostate Cancer: A Comprehensive Review

Prostate cancer (PCa) has a vast clinical spectrum from the hormone-sensitive setting to castration-resistant metastatic disease. Thus, chemotherapy regimens and the administration of androgen receptor axis-targeted (ARAT) agents for advanced PCa have shown limited therapeutic efficacy. Scientific advances in the field of molecular medicine and technological developments over the last decade have paved the path for immunotherapy to become an essential clinical modality for the treatment of patients with metastatic PCa. However, several immunotherapeutic agents have shown poor outcomes in patients with advanced disease, possibly due to the low PCa mutational burden. Adoptive cellular approaches utilizing chimeric antigen receptor T cells (CAR-T) targeting cancer-specific antigens would be a solution for circumventing the immune tolerance mechanisms. The immunotherapeutic regimen of CAR-T cell therapy has shown potential in the eradication of hematologic malignancies, and current clinical objectives maintain the equivalent efficacy in the treatment of solid tumors, including PCa. This review will explore the current modalities of CAR-T therapy in the disease spectrum of PCa while describing key limitations of this immunotherapeutic approach and discuss future directions in the application of immunotherapy for the treatment of metastatic PCa and patients with advanced disease.     

T Lymphocyte Antigen 4-Modified Dendritic Cell Therapy for Asthmatic Mice Guided by the CCR7 Chemokine Receptor

The CD80/CD86-CD28 axis is a critical pathway for immuno-corrective therapy, and the cytotoxic T lymphocyte antigen 4 (CTLA4) is a promising immunosuppressor targeting the CD80/CD86-CD28 axis; however, its use for asthma therapy needs further optimization. A human CTLA4 fused with the IgCγ Fc (CTLA4Ig) and mouse CC chemokine receptor type7 (CCR7) coding sequences were inserted into a recombinant adenovirus (rAdV) vector to generate rAdV-CTLA4Ig and rAdV-CCR7. The naive dendritic cells (DCs) were infected with these rAdVs to ensure CCR7 and CTLA4Ig expression. The therapeutic effects of modified DCs were evaluated. rAdV-CTLA4Ig and rAdV-CCR7 infected DCs improved all asthma symptoms. Inflammatory cell infiltration and cytokine analysis showed that rAdV-CTLA4Ig and rAdV-CCR7-modified DC therapy reduced the number of eosinophils and lymphocyte and neutrophil infiltration in the lung. Interestingly, assessment of the humoral immunity showed that the IL-4 and IFNγ levels of the rAdV-CTLA4Ig and rAdV-CCR7-modified DC-treated mice decreased significantly and did not reverse the Th1/Th2 balance. DCs expressing CCR7 displayed guidance ability for DC migration, primarily for DCs in the inflammatory lung. Additionally, the rAdVs caused an inflammatory response by inducing DC differentiation, inflammatory cell infiltration and changes in cytokines; however, mice transplanted with rAdV-green fluorescent protein (GFP)-infected DCs displayed no asthma manifestations. In conclusion, CTLA4Ig-modified DCs exhibited a therapeutic effect on asthma, and CCR7 may guide DC homing. The combination of these two molecules may be a model for precision-guided immunotherapy.     

Tumor-Associated Macrophages (TAMs) in Colorectal Cancer (CRC): From Mechanism to Therapy and Prognosis

Colorectal cancer (CRC) is a malignant tumor in the digestive system whose incidence and mortality is high-ranking among tumors worldwide. The initiation and progression of CRC is a complex process involving genetic alterations in cancer cells and multiple factors from the surrounding tumor cell microenvironment. As accumulating evidence has shown, tumor-associated macrophages (TAMs)—as abundant and active infiltrated inflammatory cells in the tumor microenvironment (TME)—play a crucial role in CRC. This review focuses on the different mechanisms of TAM in CRC, including switching of phenotypical subtypes; promoting tumor proliferation, invasion, and migration; facilitating angiogenesis; mediating immunosuppression; regulating metabolism; and interacting with the microbiota. Although controversy remains in clinical evidence regarding the role of TAMs in CRC, clarifying their significance in therapy and the prognosis of CRC may shed new light on the optimization of TAM-centered approaches in clinical care.     

Circulating Tumor Cells (CTC) and Cell-Free DNA (cfDNA) Workshop 2016: Scientific Opportunities and Logistics for Cancer Clinical Trial Incorporation

Despite the identification of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) as potential blood-based biomarkers capable of providing prognostic and predictive information in cancer, they have not been incorporated into routine clinical practice. This resistance is due in part to technological limitations hampering CTC and cfDNA analysis, as well as a limited understanding of precisely how to interpret emergent biomarkers across various disease stages and tumor types. In recognition of these challenges, a group of researchers and clinicians focused on blood-based biomarker development met at the Canadian Cancer Trials Group (CCTG) Spring Meeting in Toronto, Canada on 29 April 2016 for a workshop discussing novel CTC/cfDNA technologies, interpretation of data obtained from CTCs versus cfDNA, challenges regarding disease evolution and heterogeneity, and logistical considerations for incorporation of CTCs/cfDNA into clinical trials, and ultimately into routine clinical use. The objectives of this workshop included discussion of the current barriers to clinical implementation and recent progress made in the field, as well as fueling meaningful collaborations and partnerships between researchers and clinicians. We anticipate that the considerations highlighted at this workshop will lead to advances in both basic and translational research and will ultimately impact patient management strategies and patient outcomes.     

Efficacy and Feasibility of the Epithelial Cell Adhesion Molecule (EpCAM) Immunomagnetic Cell Sorter for Studies of DNA Methylation in Colorectal Cancer

The aim of this work was to assess the impact on measurements of methylation of a panel of four cancer gene promoters of purifying tumor cells from colorectal tissue samples using the epithelial cell adhesion molecule (EpCAM)-immunomagnetic cell enrichment approach. We observed that, on average, methylation levels were higher in enriched cell fractions than in the whole tissue, but the difference was significant only for one out of four studied genes. In addition, there were strong correlations between methylation values for individual samples of whole tissue and the corresponding enriched cell fractions. Therefore, assays on whole tissue are likely to provide reliable estimates of tumor-specific methylation of cancer genes. However, tumor cell tissue separation using immunomagnetic beads could, in some cases, give a more accurate value of gene promoter methylation than the analysis of the whole cancer tissue, although relatively expensive and time-consuming. The efficacy and feasibility of the immunomagnetic cell sorting for methylation studies are discussed.     

Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers, such as chitosan (CS), appear of particular interest as nonviral vectors due to their capacity to facilitate cellular delivery of bioactive cargoes including peptide nucleic acids (PNAs) or DNA vaccines. We have investigated the ability of a PNA conjugated to different CPPs to inhibit the replication of duck hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its PNA cargo, was able to drastically inhibit late stages of DHBV replication. In the mouse model, conjugation of HBV DNA vaccine to modified CS (Man-CS-Phe) improved cellular and humoral responses to plasmid-encoded antigen. Moreover, other systems for gene delivery were investigated including CPP-modified CS and cationic nanoparticles. The results showed that these nonviral vectors considerably increased plasmid DNA uptake and expression. Collectively promising results obtained in preclinical studies suggest the usefulness of these safe delivery systems for the development of novel therapeutics against chronic hepatitis B.