PiggyBac Chimeric Antigen Receptor (CAR) Expression Vector
Utilizing chimeric antigen receptor (CAR) vectors to produce engineered T cells (also known as CAR T cells) that can recognize tumor-associated antigens has emerged as a promising approach in the treatment of cancer. In CAR T-cell therapy, T cells derived from either patients (autologous) or healthy donors (allogeneic) are modified to express CAR, a chimeric construct which combines antigen binding with T cell activation for targeting tumor cells.
Structurally, a CAR consists of four main components: (1) an extracellular antigen recognition domain made up of an antibody-derived single chain variable fragment (scFv) of known specificity. The scFv facilitates antigen binding and is composed of the variable light chain and heavy chain regions of an antigen-specific monoclonal antibody connected by a flexible linker; (2) an extracellular hinge or spacer which connects the scFv with the transmembrane domain and provides flexibility and stability to the CAR structure; (3) a transmembrane domain which anchors the CAR to the plasma membrane and bridges the extracellular hinge as well as antigen binding domain with the intracellular signaling domain. It plays a critical role in enhancing receptor expression and stability; (4) and an intracellular signaling domain which is typically derived from the CD3 zeta chain of the T cell receptor (TCR) and contains immunoreceptor tyrosine-based activation motifs (ITAMs). The ITAMs become phosphorylated and activate downstream signaling upon antigen binding, leading to the subsequent activation of T cells. In addition, the intracellular region may contain one or more costimulatory domains (derived from CD28, CD137 etc.) in tandem with the CD3 zeta signaling domain for improving T cell proliferation and persistence.
The structure of CAR has evolved over the past few years based on modifications to the composition of the intracellular domains. The first-generation CARs consisted of only a single intracellular CD3 zeta-derived signaling domain. While these CARs could activate T cells, they exhibited poor anti-tumor activity in vivo due to the low cytotoxicity and proliferation of T cells expressing such CARs. This led to the advent of the second-generation CARs which included an intracellular costimulatory domain in addition to the CD3 zeta signaling domain leading to a significant improvement of the in vivo proliferation, expansion and persistence of T cells expressing second generation CARs. To further optimize the anti-tumor efficacy of CAR-T cells, third generation CARs were developed which included two intracellular, cis-acting costimulatory domains in addition to CD3 zeta. Thereafter, fourth generation CARs were derived from second-generation CARs by modifying their intracellular domain for inducible or constitutive expression of cytokines. The fifth and the latest generation of CARs are also derived from second-generation CARs by the incorporation of intracellular domains of cytokine receptors.
Our piggyBac CAR expression vector is a highly efficient tool for achieving non-viral, transposon-based delivery of second-generation CAR expression cassettes into T cells. The piggyBac system is derived from the piggyBac transposon, which is originally isolated from the cabbage looper (Trichoplusia ni; a moth species).
The piggyBac system contains two vectors, both engineered as E. coli plasmids. One vector, referred to as the helper PBase plasmid, encodes the transposase. The other vector, referred to as the transposon plasmid, contains two terminal repeats (TRs) bracketing the region to be transposed. The entire CAR expression cassette including the scFv region, the hinge, the transmembrane domain and the intracellular CD3 zeta signaling domain as well as the costimulatory domain is cloned into this region.
When the PBase vector and the piggyBac transposon vector are co-transfected into target cells, the transposase produced from the helper would recognize the two TRs on the transposon, and insert the flanked region including the two TRs into the host genome. Insertion typically occurs at host chromosomal sites that contain the TTAA sequence, which is duplicated on the two flanks of the integrated fragment.
PiggyBac is a class II transposon, meaning that it moves in a cut-and-paste manner, hopping from place to place without leaving copies behind. (In contrast, class I transposons move in a copy-and-paste manner.) Because the helper plasmid is only transiently transfected into host cells, it will get lost over time. With the loss of the helper plasmid, the integration of the transposon in the genome of host cells becomes permanent. If these cells are transfected with the helper plasmid again, the transposon could get excised from the genome of some cells, footprint free.
For further information about this vector system, please refer to the papers below.