One of the biggest challenges in engineering CAR T-cells to target solid tumors is that the CAR T-cells are unable to penetrate the vascular endothelium. This prevents the CAR T-cells from trafficking and infiltrating solid tumors. Furthermore, solid tumors can have antigen heterogeneity, which results in the poor recognition of cancer cells in the tumor environment. Increasing the specificity of CAR T-cells to multiple antigens might increase the on-target and off-target effects. However, the cancer cells can downregulate the targeted antigen, evading the therapy and decreasing the effectiveness of the CAR T-cells [3].

To tackle the challenge of on-target off-tumor toxicity and solid tumor targeting, the iGEM team in Munich decided to focus on improving the specificity of the CAR T-cell activation. To achieve this we developed two approaches focused on making use of unique characteristics of the tumor microenvironment: pH and cell clustering.

Our second approach to increase CAR T-cell therapy’s specificity also relies on certain characteristics of the tumor microenvironment, specifically its immuno-architecture. Usually, an increased density of lymphocytes infiltrating the tumor environment can be expected. Cluster formation of CD8+ T-cells is particularly interesting: t is driven by the positive feedback loop, in which T-cells recruit further T-cells to the site of the tumor. IHC imaging, performed to determine cluster morphometrics, shows that the median density of cells within such clusters in the tumor microenvironment ranges between 100–1,000 mm−2 across different patients and cancer types [7]. We use the tendency of T-cell clustering in our project by introducing a proximity-based induction, where CAR T-cells are activated by neighboring cells of the cluster, thus localizing their activity and toxicity to the tumor site.

Figure 2: Schematic representation of our quorum sensing loop system.

1. MESA dimerization: A ligand triggers two nanobodies to dimerize. As a result, the transcription factor tTA is cleaved by a protease.
2. tTA dimerization: Two cleaved tTA dimerize.
3. DNA binding: The dimerized tTA binds to the Tet Response Element (TRE). This activates ligand and CAR expression.
4a. Ligand: The ligand is expressed and secreted.
4b. CAR: The Chimeric Antigen Receptor is expressed and transported to the cell membrane.

Figure 2: Schematic representation of our quorum sensing loop system.

1. MESA dimerization: A ligand triggers two nanobodies to dimerize. As a result, the transcription factor tTA is cleaved by a protease. 2. tTA dimerization: Two cleaved tTA dimerize. 3. DNA binding: The dimerized tTA binds to the Tet Response Element (TRE). This activates ligand and CAR expression. 4a. Ligand: The ligand is expressed and secreted. 4b. CAR: The Chimeric Antigen Receptor is expressed and transported to the cell membrane.

Our approach involves a quorum sensing system in CAR T-cells. The activation of CAR T-cells in a cluster occurs through signaling from the neighboring cells, which secrete a specific non-immunogenic ligand. This ligand can bind onto an engineered cell surface receptor, triggering an orthogonal signal within the cells. The receptor we chose is MESA – Modular Extracellular Sensor Architecture. Due to its modular structure this is a highly programmable solution comparable in its versatility to synNotch. Unlike the synNotch receptor however, MESA is able to bind and recognize soluble antigens and produces a user-dictated transcriptional output [8].

The detection is carried out by a single-chain variable domain, (scFv) or in our case with a nanobody located extracellularly. For the detection of soluble ligands, a MESA construct relies on dimerisation upon binding. As the result of dimerization, a tobacco-etch virus protease (TEV), which is located on the protease chain of the MESA dimer, is brought into proximity with its target sequence, which is located on the target chain. This results in a trans-cleavage event that liberates the transcription factor [9].

In our project we used a tetracycline-inducible expression system (TeT), where Tetracycline transcription activator (tTA) is released upon cleavage. The transcription factor then translocates to the nucleus and regulates expression of our target genes. For this, two tTA dimerize and the resulting dimer binds to the Tet Response Element (TRE) in the promoter region. As a result, the transcription factor activates the production of the chimeric antigen receptors as well as the soluble ligands. This ligand is transported to the cell membrane and secreted after expression and is then able to bind to the MESA receptor activating the feedback loop.

The CAR T-cells in the cluster are thus able to recognize the tumor antigens and release cytokines to combat cancer. The peak cytotoxicity is linked to the number of CAR present on the cell surface, thus the on-target off-tumor response is minimized due to the lack of clustering in the healthy tissue.

This approach is extremely versatile, as a certain number of parameters, including binding strength between the ligand and the MESA receptor or the expression rate of the chosen expression system, can be selected according to the specific needs. This allows the modulation of the critical concentration of cells required to initiate a CAR T-cell response, allowing one to make the system suitable for its application in different types of cancers and patients exhibiting varying T-cell cluster density.

Furthermore, our approach is novel and innovative. Although CAR T-cells engineered to secrete a specific ligand or to (de)activate depending on the presence of a small molecule switch have already been implemented, these processes remain largely ineffective and the methods were not yet combined to create a quorum sensing system [10].