Chimeric antigen receptor (CAR)-expressing T cells represent a promising approach to cancer therapy, but safety and efficacy are still major hurdles. Scientists at Boston University and Massachusetts Institute of Technology (MIT) have now developed a new CAR technology, called split, universal, and programmable (SUPRA) CAR, which they claim represents the “Swiss army knife of CAR” and can simultaneously address tumor resistance, prevent immune system overactivation, and enhance specificity.
The SUPRA CAR system comprises a universal receptor expressed on T cells, to which a tumor-targeting single-chain variable antibody fragment (scFv), or adaptor molecule, is attached. “Instead of thinking about CAR-T as engineering cells that kill cancer, the way I think about it is as an antibody that drags a killer T cell with it,” says Boston University’s Wilson Wong, Ph.D. Describing the technology in a paper in Cell, Wong and Jang Hwan Cho, Ph.D., also at Boston University, and MIT’s James J. Collins Ph.D., claim the SUPRA CAR technology simultaneously encompasses “multiple, critical upgrades,” including the ability to switch targets without the need to re-engineer the T cells, as well as fine-tune T-cell activation strength, and sense and respond to multiple antigens. In effect the system can fine-tune T-cell activation strength to mitigate toxicity, sense and “logically respond” to multiple antigens to combat relapse, and inducibly control cell-type-specific signaling. The team reports on in vitro and in vivo characterization and evaluation of the technology in a paper entitled “Universal Chimeric Antigen Receptors for Multiplexed and Logical Control of T Cell Responses.”
There is an “urgent need” for a CAR-T system that can more finely tune T-cell activation, boost tumor specificity, and independently control different signaling pathways and cell types, the researchers state. However, existing CAR T cells tested in the clinic typically have what they call “a rigid design that is difficult to alter without re-engineering the T cells.”
Comparing traditional CAR T-cell designs with the new SUPRA CAR system is like comparing a phone charger that can only accommodate a single phone that has the right connecter with two AC adapters that can handle multiple attachments. In contrast with conventional CAR-T systems, the SUPRA CAR technology allows the antigen-targeting molecules to be changed at will, while the system can also be turned on and off and finely tuned to control the level of immune response and so prevent overwhelming immune system activation that can occur with exiting CAR T-cell therapies.
The design hinges on the SUPRA CAR split design. Current CAR designs are based on fixed antigen-specific scFv molecule and intracellular signaling domains on the T cell. Fixed design restricts the antigen specificity and affinity, and when this invariate antigen-specific CAR binds to the target antigen, the signaling domains are activated simultaneously at a predetermined level. This means the level of response can’t be turned up or down.
Research has led to the development of different receptor designs that can address issues such as controllability, flexibility, and specificity, Dr. Wong and colleagues acknowledge, while scientists have also generated split CARs, in which the antigen recognition and signaling parts can be changed independently. However, a CAR system hasn’t yet been developed that incorporates all the desired features and flexibility in one system. The new SUPRA CAR technology is designed to address all key issues by providing the target flexibility, allowing fine controllability to limit immune overactivation, respond to multiple antigens, and allow deactivation, all in one engineered T cell.
The two-component receptor system is composed of a T-cell-expressed universal receptor, or zipCAR, and a tumor-targeting scFv adaptor, or zipFv. The ZipCAR universal receptor is generated by fusing the intracellular signaling domains to a leucine zipper as the extracellular domain. The zipFv adaptor molecule is generated by fusing an appropriate leucine zipper and a scFv. “The scFv of the zipFv binds to the tumor antigen, and the leucine zipper binds and activates the zipCAR on the T cells,” the team explains. “A unique feature of the split CAR design is that it has multiple tunable variables, such as (1) the affinity between leucine zipper pairs, (2) the affinity between tumor antigen and scFv, (3) the concentration of zipFv, and (4) the expression level of zipCAR, that can be used to modulate the T cell response.”
In vitro tests with SUPRA CAR constructs demonstrated the potential of the system to increase tumor specificity and reduce CAR T-cell therapy toxicity by controlling response. In vivo tests demonstrated use of SUPRA CARs to reduce tumor burden in a mouse breast cancer xenograft model and in a blood tumor model. “The observed robust activity of the SUPRA system against different xenograft models demonstrates the potential of the SUPRA CAR system to combat many cancers,” the team claims. Further in vivo studies then confirmed that the SUPRA CAR system can be fine-tuned using multiple approaches to regulate cytokine release, offering the potential to manage toxicities, such as severe cytokine release syndrome that can occur with conventional CAR T-cell therapies.
As proof of concept, the researchers also also engineered two separate CD4+ and CD8+ cell types to show that two orthogonal SUPRA receptors can independently regulate two cell types. “While it is possible to engineer two cell types with conventional fixed CARs that target different antigens, our SUPRA system enables the first orthogonal inducible control of two cell types simultaneously,” they state.
The team hopes that their technology will pave the way to developing a CAR T-cell system that can be used widely as frontline anticancer therapy. “The way I imagine it is a tricked-out cell,” Dr. Wong comments. “We want something off the shelf so we wouldn't have to make it for every patient, which would bring the cost down, with the capability to switch it on or off. We'd also want it to be able to make certain proteins it might need—proteins that chew up solid-tumor boundaries for example. Lots of sensors, lots of switches, all these bells and whistles that make a very smart cell.”