Fine-tuning the antigen sensitivity of CAR T cells: emerging strategies and current challenges

Chimeric antigen receptor (CAR) T cells are “living drugs” that specifically recognize their target antigen through an antibody-derived binding domain resulting in T cell activation, expansion, and destruction of cognate target cells. The FDA/EMA approval of CAR T cells for the treatment of B cell malignancies established CAR T cell therapy as an emerging pillar of modern immunotherapy. However, nearly every second patient undergoing CAR T cell therapy is suffering from disease relapse within the first two years which is thought to be due to downregulation or loss of the CAR target antigen on cancer cells, along with decreased functional capacities known as T cell exhaustion. Antigen downregulation below CAR activation threshold leaves the T cell silent, rendering CAR T cell therapy ineffective. With the application of CAR T cells for the treatment of a growing number of malignant diseases, particularly solid tumors, there is a need for augmenting CAR sensitivity to target antigen present at low densities on cancer cells. Here, we discuss upcoming strategies and current challenges in designing CARs for recognition of antigen low cancer cells, aiming at augmenting sensitivity and finally therapeutic efficacy while reducing the risk of tumor relapse

Magnetic CAR T cell purification using an anti-G4S linker antibody

Chimeric antigen receptor (CAR) redirected T cells are successfully employed in the combat against several hematological malignancies, however, are often compromised by low transduction rates making refinement of the CAR T cell products necessary. Here, we report a broadly applicable enrichment protocol relying on marking CAR T cells with an anti-glycine4-serine (G4S) linker antibody followed by magnetic activated cell sorting (MACS). The protocol is broadly applicable since the G4S peptide is an integral part of the vast majority of CARs as it links the VH and VL recognition domains. We demonstrate the feasibility by using the canonical second generation CARs specific for CEA and Her2, respectively, obtaining highly purified CAR T cell products in a one-step procedure without impairing cell viability. The protocol is also applicable to a dual specific CAR (tandem CAR). Except for CD39, T cell activation/exhaustion markers were not upregulated after separation. Purified CAR T cells retained their functionality with respect to antigen-specific cytokine secretion, cytotoxicity, and the capacity to proliferate and eliminate cognate tumor cells upon repetitive stimulation. Collectively, the one-step protocol for purifying CAR T cells extends the toolbox for preclinical research and specifically for clinical CAR T cell manufacturing

CAR Triggered Release of Type-1 Interferon Limits CAR T-Cell Activities by an Artificial Negative Autocrine Loop

The advent of chimeric antigen receptor (CAR) T cells expedited the field of cancer immunotherapy enabling durable remissions in patients with refractory hematological malignancies. T cells redirected for universal cytokine-mediated killing (TRUCKs), commonly referred to as “fourth generation” CAR T-cells, are designed to release engineered payloads upon CAR-induced T-cell activation. Building on the TRUCK technology, we aimed to generate CAR T-cells with a CAR-inducible artificial, self-limiting autocrine loop. To this end, we engineered CAR T-cells with CAR triggered secretion of type-1 interferons (IFNs). At baseline, IFNα and IFNβ CAR T-cells showed similar capacities in cytotoxicity and cytokine secretion compared to conventional CAR T-cells. However, under “stress” conditions of repetitive rounds of antigen stimulation using BxPC-3 pancreas carcinoma cells as targets, anti-tumor activity faded in later rounds while being fully active in destructing carcinoma cells during first rounds of stimulation. Mechanistically, the decline in activity was primarily based on type-1 IFN augmented CAR T-cell apoptosis, which was far less the case for CAR T-cells without IFN release. Such autocrine self-limiting loops can be used for applications where transient CAR T-cell activity and persistence upon target recognition is desired to avoid lastin

CopR, a Global Regulator of Transcription to Maintain Copper Homeostasis in Pyrococcus furiosus

Although copper is in many cases an essential micronutrient for cellular life, higher concentrations are toxic. Therefore, all living cells have developed strategies to maintain copper homeostasis. In this manuscript, we have analyzed the transcriptome-wide response of Pyrococcus furiosus to increased copper concentrations and described the essential role of the putative copper-sensing metalloregulator CopR in the detoxification process. To this end, we employed biochemical and biophysical methods to characterize the role of CopR. Additionally, a copR knockout strain revealed an amplified sensitivity in comparison to the parental strain towards increased copper levels, which designates an essential role of CopR for copper homeostasis. To learn more about the CopR-regulated gene network, we performed differential gene expression and ChIP-seq analysis under normal and 20 μM copper-shock conditions. By integrating the transcriptome and genome-wide binding data, we found that CopR binds to the upstream regions of many copper-induced genes. Negative-stain transmission electron microscopy and 2D class averaging revealed an octameric assembly formed from a tetramer of dimers for CopR, similar to published crystal structures from the Lrp family. In conclusion, we propose a model for CopR-regulated transcription and highlight the regulatory network that enables Pyrococcus to respond to increased copper concentrations

Designed Ankyrin Repeat Protein (DARPin) to target chimeric antigen receptor (CAR)-redirected T cells towards CD4(+)T cells to reduce the latent HIV(+)cell reservoir

Chimeric Antigen Receptor (CAR)-redirected T cells show great efficacy in the patient-specific therapy of hematologic malignancies. Here, we demonstrate that a DARPin with specificity for CD4 specifically redirects and triggers the activation of CAR engineered T cells resulting in the depletion of CD4(+)target cells aiming for elimination of the human immunodeficiency virus (HIV) reservoir