^ “CAR-T cells developed by TALEN enable precise attack on solid tumors”

“The idea was to program a circuit with gates”Shipra Das

To overcome the challenges, her team developed TALEN-edited allogeneic CAR-T cells that express a logical IF/THEN system. This is a dual-control mechanism designed to activate CAR-T cells only in specific tumor conditions, increasing precision and reducing off-target side effects. Shipra Das explains:

»The idea was to program a circuit with gates. The first tumor-specific signal would serve as input, be processed by the gate, and then signal the expression of a CAR that can target a more commonly expressed tumor antigen that might also be found in normal tissues.”

TALEN was chosen over CRISPR

The system works in two steps. First, a constitutively expressed CAR targets FAP+ CAFs in the tumor microenvironment (the IF state). This intervention triggers the expression of a second CAR that targets mesothelin (a TAA) on tumor cells (the DANN response).

The dual-sensing approach ensures activation of CAR-T cells only in the presence of CAFs and tumor cells, improving specificity and minimizing damage to healthy tissue (see Figure 1).

“TALEN is particularly advantageous in terms of specificity because it binds to a longer recognition sequence – 32 nucleotides compared to 20-24 for CRISPR”Laurent Poirot

PDCD1 encodes the immune checkpoint protein PD-1, which tumors often use to suppress T cell activity. By knocking out PDCD1the manipulated CAR T cells become resistant to this immunosuppression and thus increase their antitumor activity.

To knock out the two genes, TALEN-mediated gene editing was preferred over CRISPR, as Laurent Poirot explains:

»Both CRISPR and TALEN are highly efficient. However, clinical-grade nucleases depend on activity, specificity and precision. TALEN is particularly advantageous in terms of specificity because it binds to a longer recognition sequence – 32 nucleotides compared to 20-24 for CRISPR. The longer recognition sequence means fewer off-target effects, which is crucial for avoiding genomic toxicity.«

One CAR activates the other

The CARs were then inserted using different techniques. The FAP CAR (Targeting CAFs) was introduced by lentiviral transduction, which stably integrates the CAR gene into T cells.

The mesothelin CAR (Targeting Tumor Cells) was integrated PDCD1 Locus via AAV-mediated homologous recombination. This precise integration enabled control of the mesothelin CAR by the same regulatory elements that would normally regulate PD-1, ensuring inducible expression based on the logic-driven IF/THEN system.

“Targeting FAP-expressing CAFs, which play a key role in excluding T cells from tumors, allows CAR-T cells to further infiltrate the tumor”Shipra Das

Specifically, when the FAP CAR is activated, it initiates a TCR signaling cascade. Since then PDCD1 If the site where the mesothelin CAR is integrated is regulated by this pathway, FAP-CAR activation leads to surface expression of the mesothelin CAR. The FAP-CAR thus serves as a gatekeeper to trigger the expression of the mesothelin-CAR in solid tumors, but also serves another purpose.

»Targeting FAP-expressing CAFs, which play a key role in excluding T cells from tumors, allows CAR-T cells to further infiltrate the tumor. CAFs also modulate the immunosuppressive microenvironment and disrupt the immune suppression in the tumor,” says Shipra Das.

When these dual CAR-T cells come into contact with their respective targets in the tumor environment, they trigger an immune response by releasing cytotoxic molecules (such as perforin and granzymes) that kill the CAFs and mesothelin-expressing tumor cells.

The survival rate is significantly increased

The dual CAR-T cells were enriched using magnetically activated cell sorting (MACS) and comprehensively tested through various assays, including cytotoxicity, gene expression, and T cell infiltration in 3D tumor spheroids. Over and beyond, in vivo Experiments in mice showed that TALEN-engineered dual CAR-T cells effectively target solid tumors while ensuring safety.

Two mouse models were used. A bilateral tumor model used mesothelin-positive (ML+) cells implanted in the left flank and a mixture of FAP+ CAFs and ML+ cells in the right flank. Another orthotopic model for triple-negative breast cancer (TNBC) used TNBC cells and patient-derived CAFs implanted into the mammary fat pad.

In the bilateral model, dual CAR T cells accumulated specifically in the right flank (FAP+ and ML+), where they activated the mesothelin CAR, demonstrating the effectiveness of the logical IF/THEN system. Tumor growth and weight were significantly reduced in the right flank, while no tumor reduction was observed in the left flank (ML+ but FAP−), demonstrating strong target specificity and the absence of off-tumor effects.

“Everything here was built with the goal of moving to the clinic, but it’s a gradual process. Regulatory authorities require very strict evidence of safety and effectiveness before clinical trials”Laurent Poirot

The mice in the study did not achieve complete healing, and according to Shipra Das, this was to be expected due to the limitations of the model systems used. While the researchers attempted to mimic human tumors as closely as possible by incorporating human CAFs and tumor cells, the injected human CAR-T cells may not have had the appropriate supporting environment in the murine environment.

Although the results are encouraging as a proof of concept, future efforts will focus on testing in more physiologically relevant models and optimizing the strategy to better address the complexity of solid tumors.

»Everything here was set up with the aim of moving into the clinic, but it is a gradual process. Regulatory authorities require very strict evidence of safety and effectiveness before clinical trials. We have to validate everything preclinically before we start a clinical program, which can take between two and five years,” says Laurent Poirot.

Link to original article in Molecular Therapy:

TALEN-edited allogeneic inducible dual CAR T cells enable effective targeting of solid tumors while mitigating off-tumor toxicity