Advances in CAR T-Cell Control: Dual Receptor Knockout and Drug-Regulated Switches

Researchers have unveiled multiple engineering strategies to enhance CAR T-cell therapy efficacy and safety, including a dual receptor knockout approach for solid tumors and drug-controlled switches that enable precise regulation of therapeutic activity.

In a groundbreaking advancement for solid tumor treatment, researchers have developed a strategy that dramatically enhances the potency of CAR T-cell therapy through the precise genetic ablation of prostaglandin E2 (PGE2) signaling by knocking out its dual receptors in the engineered immune cells themselves. While CAR T-cell therapy has revolutionized treatment for certain blood cancers, its application to solid tumors has faced significant challenges due to the hostile, immunosuppressive microenvironment these tumors create. PGE2, a bioactive lipid mediator prevalent in the tumor milieu, plays a pivotal role in dampening immune responses by engaging its receptors on immune cells, effectively muffling the anti-tumor activity of CAR T cells.

The dual receptor knockout was engineered using state-of-the-art gene editing tools that ensure both accuracy and durability of receptor ablation within the CAR T-cell genome. This precise gene editing approach was followed by rigorous functional assays to confirm that the modified CAR T cells were not only free from PGE2-mediated signaling but also retained their vital cytotoxic functions and proliferative capacity. The reprogrammed immune cells exhibited a marked resistance to the immunosuppressive forces typically present in the tumor microenvironment.

In preclinical models of notoriously resistant solid tumors, the modified CAR T cells demonstrated a significantly enhanced ability to infiltrate tumor masses and sustain their anti-cancer activity over extended periods. This translated into a profound delay in tumor progression and, in some cases, complete regression, effects seldom seen with conventional CAR T therapies. Beyond their improved efficacy, these receptor-deficient CAR T cells also showed a surprising reduction in systemic inflammatory side effects, a common complication of cellular immunotherapies.

The strategy emerges from an extensive understanding of PGE2's multifaceted role in tumor biology and immune evasion. By disabling two distinct receptors, the approach assures a more comprehensive blockade of PGE2's immunosuppressive signals, which might otherwise bypass single receptor-targeted interventions. Detailed phenotypic analyses revealed that the dual receptor knockout did not impair essential receptor signaling pathways responsible for normal T-cell function and navigation, preserving the therapeutic cells' fitness and resilience in vivo.

Further in-depth molecular profiling illuminated how disabling PGE2 receptors recalibrates the CAR T-cell transcriptome towards a more activated and persistent state, characterized by upregulation of effector molecules and resistance to exhaustion, a chronic dysfunction state that often limits CAR T efficacy.

In parallel developments, researchers have combined rational design and library-based optimization of a protein–protein interaction of human origin to develop venetoclax-controlled drug-regulated off-switch PPI (DROP)-CARs. Chimeric antigen receptor (CAR) T cell therapy is constrained by on-target, off-tumor toxicities and cellular exhaustion because of chronic antigen exposure. DROP-CARs enable dose-dependent release of the tumor-targeting scFv and consequent reduction in T cell binding to the tumor cell.

Most CARs currently used in the clinic are second-generation (2G) and comprise an antigen-binding moiety (typically a single-chain variable fragment, scFv), fused to a hinge, a transmembrane region and the endodomains of one costimulatory receptor and of CD3ζ. Limitations to the 2G-CAR include that chronic antigen exposure can render the engineered T cells exhausted, toxicity can result from on-target reactivity against healthy tissues, and overresponsiveness at high antigen density or tumor burden can trigger adverse events such as cytokine release syndrome (CRS).

On-switch and off-switch CAR designs that allow remote control of T cell activity levels by small-molecule administration represent a promising strategy for balancing function and safety. While there has been remarkable progress in the field, switch designs based on human-derived domains (that is, to minimize immunogenicity) that are responsive to clinically approved small molecules are rare and existing ones have limitations.

The venetoclax-controlled DROP-CAR system was developed through rational protein design and library screening to generate a stable protein–protein interaction of human origin that can be efficiently disrupted by the clinically approved molecule venetoclax. The researchers presented proof of concept for a dual DROP-CAR controlled by different small molecules, as well as for logic-gated synthetic receptors enabling STAT3 signaling. They demonstrated in vitro and in vivo function of DROP-CAR T cells.

Researchers at Texas A&M University have developed two new chemically induced proximity (CIP) systems for controlling gene expression. The team developed a caffeine-inducible system that can alter cancer-specific signaling pathways and, in unpublished research, modulate CRISPR machinery. The other is a modified rapamycin CIP that can turn CRISPR activation (CRISPRa) off to help reduce the chance of off-target effects.

The caffeine-controlled system, dubbed CHASER, was created by inserting an existing CIP system called "COSMO" into the nanobody LaM8 to allow it to allosterically respond to caffeine. Using the CHASER platform, the team was able to use caffeine to induce and change the expression of tyrosine receptor kinases (TRKs) in cell culture. TRKs are integral transmembrane proteins involved in cellular signaling and tumor proliferation and metastasis.

The team can split CRISPR into two separate pieces and attach each piece to caffeine-responsive modules. When caffeine is added, the two parts snap back together, rebuilding the full CRISPR system and turning it on. CRISPR only becomes active when caffeine is present, giving a simple and controllable way to turn gene editing on and off. The paper reports that the team even used diluted tea, coffee, and Red Bull in cell culture to trigger CHASER-mediated gene activation.

The team also transformed and reprogrammed a rapamycin-induced CIP called UniRapR into a genetic 'off' switch called RASER, also based on LaM8 nanobodies. While CHASER induces a tighter conformation of two proteins, RASER brings two domains together in such a way that it disrupts the interaction between the proteins. The team screened a range of RASER nanobody candidates in vitro using fluorescence as a readout and settled on one particular design that led to nearly 70% lower GFP expression in their cell model.