Tol2 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 that 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, and (4) an intracellular signaling domain that 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 second-generation CARs which included an intracellular costimulatory domain in addition to the CD3 zeta signaling domain leading to a significant improvement in 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 Tol2 CAR expression vector is a highly efficient tool for achieving non-viral, transposon-based delivery of second-generation CAR expression cassettes into T cells. This vector system is derived from the Tol2 transposon, which is originally isolated from the teleost fish, medaka (Oryzias latipes). Based on sequence homology, the Tol2 transposon was found to be closely related to the hAT family of non-autonomous elements found throughout vertebrate genomes.

The Tol2 system comprises two components: the transposon plasmid and the transposase. The transposon plasmid contains two inverted terminal repeats (ITRs) bracketing the region to be transposed. The transposase can be delivered into target cells through two methods. The helper plasmid can be transiently transfected into cells. Alternatively, target cells can be injected with transposase mRNA generated by in vitro transcription from the helper plasmid. When the transposon and helper plasmids are co-transfected into target cells, the transposase produced from the helper plasmid would recognize the two ITRs on the transposon and insert the flanked region including the two ITRs into the host genome. At each insertion site, the Tol2 transposase creates an 8 bp duplication, resulting in identical 8 bp direct repeats flanking each transposon integration site in the genome. Insertion occurs without any significant bias for the insertion site sequence. This is unlike transposon systems which have specific target consensus sites. For example, piggyBac transposons are typically inserted at sites containing the sequence TTAA. Through both methods of delivering transposase, it is expressed for only a short time. Upon the loss of the helper plasmid or degradation of transposase mRNA, the integration of the transposon into the host genome becomes permanent.

Tol2 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.) If Tol2 transposase is reintroduced into the cells, the transposon could get excised from the genome of some cells, resulting in either precise or imprecise excisions with indels created.

For further information about this vector system, please refer to the papers below.

Permanent integration of vector DNA: The Tol2 CAR expression vector helps achieve long-term expression of CAR expression cassettes in T cells by allowing permanent integration of the transposon carrying the CAR cassette into the host cell genome.

Technical simplicity: Tol2-based CAR vectors can be delivered into T cells by electroporation which is technically simpler compared to using virus-based CAR constructs. Viral vectors require the packaging of live virus before they can be transduced into T cells, making the process technically challenging and time-consuming.

Low cost: Manufacturing clinical-grade Tol2 vectors is far less expensive than viral vectors which makes clinical development of Tol2-based CAR T cells much more cost-effective. This offers a significant advantage in the development of CAR T therapies since the clinical efficacy of CAR T cells cannot be accurately predicted by preclinical studies in mice models and should be determined by clinical studies.

Large cargo space: Our Tol2 transposon vector can accommodate ~11 kb of total DNA. The plasmid backbone and transposon-related sequences only occupies about 3 kb, leaving plenty of room to accommodate all components of the CAR expression cassette. 

Delivery method: Tol2-based CAR expression vectors are typically delivered into T cells by electroporation which typically results in a low to medium frequency of cells with successful integration of the CAR expression cassette. Additionally, electroporation can be toxic to T cells which in turn could lead to reduced yield of functional CAR T cells. Therefore, it may be necessary to culture the T cells ex vivo for extended periods to attain the quantities needed for infusion, which over time could alter cell phenotype leading to reduced long-term memory function.

5' ITR: Tol2 5' terminal repeat. When a DNA sequence is flanked by two ITRs, the Tol2 transposase can recognize them, and insert the flanked region including the two ITRs into the host genome.

Promoter: The promoter driving your CAR expression cassette is placed here.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

CD8-leader: Leader signal peptide of T-cell surface glycoprotein CD8 alpha chain. Directs transport and localization of the protein to the T-cell surface.

scFv: Single chain variable fragment derived from a monoclonal antibody of known specificity. Recognizes cells in an antigen-specific manner.

Hinge: Extracellular hinge region of the CAR. Connects scFv with the transmembrane region providing stability and flexibilty for efficient CAR expression and function; enhances efficiency of tumor recognition; improves expansion of CAR-T cells.

Transmembrane domain: Transmembrane domain of the CAR. Anchors the CAR to the plasma membrane and bridges the extracellular hinge as well as antigen recognition domains with the intracellular signaling region; enhances receptor expression and stability.

Costimulatory domain: Intracellular costimulatory domain of the CAR. Improves overall survival, proliferation, and persistence of activated CAR-T cells.

CD3zeta: Intracellular domain of the T cell receptor-CD3ζ chain. Acts as a stimulatory molecule for activating T cell-mediated immune response.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream CAR expression cassette.

3' ITR: Tol2 3' terminal repeat.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.