In Situ Programming of CAR T Cells

Abstract

Gene therapy makes it possible to engineer chimeric antigen receptors (CARs) to create T cells that target specific diseases. However, current approaches require elaborate and expensive protocols to manufacture engineered T cells ex vivo, putting this therapy beyond the reach of many patients who might benefit. A solution could be to program T cells in vivo. Here, we evaluate the clinical need for in situ CAR T cell programming, compare competing technologies, review current progress, and provide a perspective on the long-term impact of this emerging and rapidly flourishing biotechnology field.

Keywords: CAR T cell therapy; gene therapy; nanoparticle; off-the-shelf T cell therapy.

Figure 1

Viral vectors (T cell targeting/DNA integrating). (a) Production of chimeric antigen receptor (CAR)–encoding lentiviral vector. To target lymphocytes, vectors are pseudotyped with engineered glycoproteins that recognize lymphocyte surface markers as entry receptors. (b) Systemic infusion of lentivirus results in the transduction of a small number of circulating T cells, which subsequently undergo clonal proliferation and differentiation into effector cells following antigen encounters.

Figure 2

Nonviral vectors (T cell targeting/DNA integrating). (a) Schematic of the T cell–targeted DNA nanocarrier. Also depicted are the two plasmids that were encapsulated into the nanoparticles; these encode the CAR and the hyperactive iPB7 transposase. (b) Schematic illustrating how to reprogram T cells in situ to express tumor-specific CARs using genes carried by polymeric nanoparticles. These particles are coated with ligands that target them to cytotoxic T cells, so once they are infused into the patient’s circulation they can transfer the genes they carry into the lymphocytes and program them to express the tumor-targeting CARs on their surfaces. Abbreviations: AMP, ampicillin resistance gene; BGHPA, bovine growth hormone polyadenylation signal; CAR, chimeric antigen receptor; DTS, DNA-targeting sequences; EF1A, eukaryotic translation elongation factor 1 alpha 1; iPB7, hyperactive piggyBac transposase; ORI, origin of replication; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element.

Figure 3

Nonviral vectors (nonintegrating). (a) Lymphocyte-targeted nanoparticles. (i) Shown is a nanocarrier loaded with in vitro–transcribed mRNA for the transient in situ reprogramming of T cells with disease-specific CARs. Surface-anchored targeting ligands selectively bind the nanoparticles to T cells and initiate rapid receptor-induced endocytosis to internalize them. (ii) Following infusion, these nanoreagents (yellow dots) quickly and specifically program antigen-recognizing capabilities into circulating T cells. In contrast to DNA nanocarriers, synthetic mRNA molecules are directly translated into therapeutic target proteins without the need to enter the nucleus, ensuring high transfection rates and rapid therapeutic effects. (b, i) Nontargeted nanoparticles. (ii) Due to the lack of a targeting ligand on their surface, these particles (cyan dots) nonspecifically transfect circulating blood cells, including T cells, B cells, monocytes, eosinophils, neutrophils, and granulocytes. Transient CAR expression reprograms these cells into new functions and phenotypes. Specific microRNA targeting sequences could be included in the synthetic mRNA construct to ensure translation is limited to therapeutically desirable target cells. Abbreviations: ArcA, antireverse cap analog; B, B cell; CAR, chimeric antigen receptor; M, monocyte; mRNA, messenger RNA; T, T cell.