Crystal Mackall, MD, director of the Stanford Center for Cancer Cell Therapy, gave a presentation on "Synthetic Biology" as part of AACR2022's main symposium, "CAR T Cells in Solid Tumors: Bullish or Bearish." Dr. Mackall shared how her team developed a novel strategy to better control the activity of CAR T cells in vivo, which may improve the effectiveness of solid tumor therapy.

Contrary to the remarkable outcomes of CAR T cell therapy in liquid tumors (i.e., hematologic malignancies), solid tumors have proved to be a greater challenge. Several factors intrinsic to solid tumors limit CAR T’s therapeutic potential, such as deficits in tumor-specific antigens and heterogeneous antigen expression. In fact, most CAR T cells are designed to target solid tumor-associated antigens, such as CEA, ERBB2, EGFR, GD2, mesothelin, MUC1, and PSMA, which are highly expressed in tumor tissue but barely present or even undetected in normal tissue. Nevertheless, targeting tumor-associated antigens (TAAs) may still lead to adverse effects, ultimately limiting CAR T’s efficacy (Marofi et al. 2021).

Regulating CAR T cell activity
To develop CAR T cell therapies that are both efficacious and safe, Mackall’s team has envisioned a new regulatory switch that would enable turning-on CAR T cell activity. They have dubbed this system a Signal Neutralization by an Inhibitable Protease (SNIP) CAR T cells, in which the activity of the antigen receptor is controlled by a co-expressed protease (Labanieh et al., 2022). Two main modifications enable receptor control in SNIP CAR T cells, including the expression of a protease, NS3 from the hepatitis C virus (HCV), and an NS3 cleavage site incorporated within the CAR sequence. Together, these modifications provide a mechanism to maintain CAR signaling silenced unless a drug, grazoprevir, is supplied to inhibit NS3 protease activity, thus preserving the CAR intact and enabling signaling.

A similar approach was implemented previously, where the HCV NS3 sequence was incorporated into a CAR construct, enabling control by the protease inhibitor drug, Asunaprevir (Juillerat et al. 2019). In that setting, the NS3 cutting sequence was incorporated together with that of the protease/degron component into the CAR construct (i.e., switch-off CAR or Swiff CAR), such that drug dosing leads to CAR degradation, inhibiting subsequent CAR surface expression and signaling.

SWIFF CAR T cell regulation strategy. In the SWIFF CAR construct, the addition of sequences for a protease cutting site, protease, and degron enable regulating CAR receptor’s expression at the cell surface. In this system, in the absence of a protease inhibitor drug, cleavage at the NS3 cutting site enables the expression of the functional CAR at the cell surface. Supplying a protease inhibitor leads to the degradation and thus off-state of CAR signaling. Retrieved from Juillerat et al. 2019. (https://creativecommons.org/licenses/by/4.0/)

SNIP CAR T cells: A potential therapy for solid tumors

The clear advantage of the system developed by Mackall’s group lies in its full silenced state in the absence of drug in vivo, which they achieved by optimizing the NS3 cutting sequence. In contrast, other strategies aiming at regulating CAR activity with small molecules are unable to induce complete silencing (Labanieh et al., 2022). No leakiness helps SNIP CAR T cells to achieve potent effector function upon drug dosing, even more so than unregulated CAR T cells. Complete silencing of SNIP CAR T cells translates to a potentially greater capacity to clear solid tumors, as demonstrated in orthotopic solid tumor models by the Mackall team. Additionally, in a receptor tyrosine kinase-like orphan receptor 1-specific (ROR1) toxicity model, SNIP CAR T cells enacted efficient anti-tumor activity with better survival and reduced toxicity than conventional CAR T cells.

In their recent Cell publication, Mackall’s team reported that, upon short periods of activation with grazoprevir, SNIP CAR T cells were more active than their unregulated CAR T cell counterparts based on cytokine release (i.e., Il2). They also found that a key benefit of SNIP CAR T cells is their ability to recover from exhaustion through a silencing period. Transcriptional hallmarks confirmed the greater expression of exhaustion markers in unregulated CAR T cells compared to the respective SNIP CAR T cells. Additionally, SNIP CAR T cells showed greater expansion capacity and ability to generate memory and effector subsets.

Overall, the main benefit of strategies such as SNIP CARs is clearly having a tunable system and safety switches in place to manage and contra rest potential on-target but off-tumor effects, thereby extending CAR T cell therapeutic potential.

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