Safer CAR Engineering Platform for CRS Toxicity Management

All products and services are For Research Use Only and CANNOT be used in the treatment or diagnosis of disease.

With the increasing use of CARTs, it is important to understand their unique side effects. The key challenge for CARTs' successes is that potent systemic immune activation drives the life-threatening toxicity of cytokine release syndrome (CRS). CAR is composed of modular components, and the optimal molecular design of CAR can be achieved through many changes in the constituent protein domains. Therefore, it is necessary to develop CAR design beyond conventional structures to overcome these limitations and expand the use of CAR-T cells to a wider range of malignancies.

Creative Biolabs provides a comprehensive review of the most promising CAR-T cell safety strategies and is committed to providing novel CAR design services, and CAR-T cell modification services to support CRS toxicity management. We provide a variety of safety strategies to achieve limited CRS toxicity, including but not limit to integrating external components (such as OFF and ON switch CAR) or combining OR-, AND- and NOT-gate CAR-T cells to target multiple antigens, or introducing small molecules adaptors to CAR-modified T cells to control excessive activation. These next-generation CARs have laid the foundation for developing novel therapeutic CAR-T cells.

Highlighted CAR Design Strategies for Better CRS Toxicity Management

Suicide Gene-based OFF-Switch CARs

Inducible caspase-9 (iCasp9) is a novel suicide gene model and has now been integrated into CAR-T cell therapy. In this novel model, iCasp9 is merged to the transduced CAR-T cell as a homodimer and shows a high affinity for a chemical inducer of dimerization (CID) AP1903. AP1903 can be administered following CAR-T cell administration. Once the CID has been injected, it causes iCasp9 dimerization and thus activates the caspase and induces the apoptosis of the CAR-T cells.

Activation of iCasp9 induces the death of transduced cells.

Fig.1 Activation of iCasp9 induces the death of transduced cells.

ON-Switch CARs

Another way to overcome potential toxicity is to develop therapeutic lymphocytes expressing an "ON-switch". Instead of eliminating overreacting CAR-T cells that cause life-threatening conditions, the use of an activating "switch-on" molecule enables the precise remote control of these cells in terms of timing, site of action, and dosage. Adaptor switchable CARs are introduced to the ON-Switch CARs and designed to improve the flexibility, tumor specificity, and controllability of CAR-T cells. Creative Biolabs provides diverse adaptor CAR platforms including but not limited to: Tag-specific adaptor CARs, bispecific antibody (bsAb)-binding adaptor CARs, and constant fragment (Fc)-binding adaptor CARs

Schematic representation of switchable adaptors

Fig.2 Schematic representation of switchable adaptors.

Small-molecule-assisted shutoff (SMASh) CARs

Another strategy is Small Molecule Assisted shutoff (SMASh), which provides reversible control of CAR production by turning off the cytolytic properties of genetically engineered lymphocytes. The CAR construct, also known as the switch-off CAR (SWIFF-CAR), binds to the drug-sensitive viral protease and degron (protein destabilizing component) of the CAR. In the absence of specific small-molecule drugs, degulon is excised from the CAR, thereby exposing the antigen-targeted scFv on the surface of CAR-T cells. When the drug was added to the culture medium, the cleavage of degeron catalyzed by the viral protease was inhibited. Therefore, the SWIFF-CAR system allows CAR to be degraded through the proteolytic pathway of T cells without depleting the cells. Their reversibility, continuous control, and cost-effectiveness are attractive features.

Principle of the SWIFF-CAR.

Fig.3 Principle of the SWIFF-CAR.

"AND", "OR", and "NOT" Gate CARs

Multi-antigen-targeted "AND", "OR", and "NOT" Gate CARs are novel strategies for eliminating on-target off-tumor toxicity. Creative Biolabs provides various types of gate CARs based on the signal transmission pattern: dual CAR-T cells, tandem CAR-T cells, trivalent CAR-T cells, and pooled CAR-T cells.

Schematic representation of

Fig.4 Schematic representation of "AND", "OR", and "NOT" gate CARs.

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Reference

Andrea, A. E., et al. (2020). Engineering Next-Generation CAR-T Cells for Better Toxicity Management. International Journal of Molecular Sciences, 21(22), 8620.