Universal CARs, universal T cells, and universal CAR T cells

Abstract

Currently, the two approved T cell products with chimeric antigen receptors (CAR) are from autologous T cells. These CAR T cells approved for clinical use must be generated on a custom-made basis. This case-by-case autologous T cell production platform remains a significant limiting factor for large-scale clinical application due to the costly and lengthy production process. There is also an inherent risk of production failure. The individualized, custom-made autologous CAR T cell production process also posts constriction on the wide application on diverse tumor types. Therefore, universal allogeneic T cells are needed for the preparation of universal CAR T cells that can serve as the "off-the-shelf" ready-to-use therapeutic agents for large-scale clinical applications. Genome-editing technologies including ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), and CRISPR-Cas9 are being used to generate the universal third-party T cells. In addition, split, universal, and programmable (SUPRA) CARs are being developed to enhance the flexibility and controllability of CAR T cells. The engineered universal T cells and universal CARs are paving the road for a totally new generation of CAR T cells capable of targeting multiple antigens and/ or being delivered to multiple recipients without re-editing of T cells. This may escalate to a new wave of revolution in cancer immunotherapy. This review summarized the latest advances on designs and development of universal CARs, universal T cells, and clinical application of universal CAR T cells.

Fig. 1

Structures of chimeric antigen receptors (CAR). First generation of CARs contains the single chain variable region (scFv) of a monoclonal antibody, T cell receptor transmembrane domain, and an intracellular signaling domain of CD3 zeta chain. The second generation of CARs contains a single co-stimulatory domain (CD28 or 4-1BB), whereas the third generation of CARs may have two or more co-stimulatory domains (CD27, CD28, 4-1BB or OX40). The fourth generation CARs contain a controllable on-off switch or a molecule (additional element) to enhance T cell function, enrichment, and minimize senescence

Fig. 2

The structure of BBIR CAR. In the BBIR CAR, avidin serves as the extracellular ligand binding domain and is linked to the transmembrane and intracellular signaling domains. The biotinylated antigen-specific molecules can be full-length antibodies, scFvs, or other tumor-specific ligands. Through the binding of biotin to avidin, any extracellular signal linked to the biotin can activate the T cells. Therefore, the BBIR CAR remains constant and can serve as a universal CAR. (adapted from Urbanska, K, et al.; Cancer Res. 2012; 72(7); 1844–1852)

Fig. 3

The structure of SUPRA CAR. A SUPRA CAR system consists of a zipCAR and a zipFv. A zipFv has a scFv linked to a leucine zipper (AZip). A zipCAR has a cognate leucine zipper (BZip) that can bind to the AZip. Through the binding between A- and B-leucine zippers, any extracellular signal linked to the AZip can activate the T cells. Therefore, the SUPRA CAR remains constant and can serve as a universal CAR. The affinity between the A- and B-leucine zippers can be adjusted so that the signaling strength and activity can be dialed up or down as desired. When AZip is linked to a null antigen, the signaling is quenched, and T cells become inactive. (adapted from Choi, JH, et al., Cell 2018; 173: 1–13)

Fig. 4

Production of universal TCR⁻/HLA⁻ T cells using ZFN (zinc finger nuclease). a The T cells were obtained from healthy adult donors. ZFN mRNA pairs were delivered to the T cells by electroporation. ZFN pairs designed to target TCR αor βconstant regions and HLA-A lead to DNA double-strand breaks at the given sites and cause deletion or insertion of nucleotides, resulting in permanent loss of gene expression. These TCR⁻/HLA⁻ T cells made from allogeneic healthy donors can be used as universal T cells for preparation of “off-the-shelf” CAR T cells. b Structure of the ZFN pair. One ZFN pair was designed to bind exon 1 of the TCRα constant region (TRAC), and the underlined nucleotide sequences represent the targeted binding sequences. The red blocks represent coding regions and the black block represents a noncoding region (adapted from Torikai H, et al. Blood 2012; 119(24):5697–5705)

Fig. 5

Production of universal CD52⁻/TCR⁻ T cells using TALEN (transcription activator-like effector nuclease). a The T cells were obtained from healthy adult donors. TALEN mRNAs targeting specific sites of CD52 and the constant region of the T cell receptor α chain (TRAC) were electro-transferred into the T cells at the same time leading to permanent loss of gene expression. These CD52⁻/TCR⁻ T cells can be used for the preparation of “off-the-shelf” CAR T cells. b Diagram of TALEN-targeted sites. The blue band represents the chromosome. Scissors represent TALENs. The red blocks represent the targeted sites of TALENs. (adapted from Poirot, L, et al., Cancer Res. 2015; 75(18); 3853–3864)

Fig. 6

Production of universal TCR⁻/HLA⁻ T cells using the CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat /CRISPR- associated protein 9). The T cells were obtained from healthy adult donors. Cas9 mRNA and gRNAs targeting the constant region of the T cell receptor αor βchain (TRAC or TRBC) and B₂M were transferred to T cells by electroporation. The Cas9 endonuclease is capable of cleaving the TRAC, TRBC, and B₂M under the guidance of corresponding gRNAs and inducing indels at pre-validated sites. These universal TCR⁻/HLA⁻ T cells with reduced alloreactivity can be used for the preparation of “off-the-shelf” CAR T cells. (adapted from Ren, J, et al. Clin Cancer Res. 2016; 23(9); 2255–2266)