Regular Plasmid Chimeric Antigen Receptor (CAR) Expression Vector

Overview

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 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 regular plasmid CAR expression vector is highly suitable for non-viral, transfection-based delivery of second-generation CAR expression cassettes into T cells. 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 downstream of a user selected promoter. A key feature of transfection with regular plasmid vectors is that it is transient, with only a very low fraction of cells stably integrating the plasmid in the genome (typically less than 1%).

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

References Topic
Br J Cancer. 120:26 (2019) Review on next-generation CAR T cells
BMC Biotechnol. 18:4 (2018) Optimizing DNA electroporation for producing engineered CAR T cells
Mol Ther Oncolytics. 3:16014 (2016) Review on CAR models
J Immunother. 32:689 (2009) Construction and pre-clinical evaluation of an anti-CD19 CAR
Mol Ther. 17:1453 (2009) In vivo characterization of chimeric receptors containing CD137 signal transduction domains

Highlights

Our regular plasmid CAR expression vector is suitable for the expression of second-generation CARs and is optimized for high copy number replication in E. coli and high-efficiency transfection. Cells transfected with the vector can be selected and/or visualized based on the marker gene expression selected by the user.

Advantages

Technical simplicity: Regular plasmid CAR expression 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.

Safety: Regular plasmid CAR expression vectors are typically delivered into T cells by electroporation which results in the CAR to be expressed only transiently without being integrated into the host genome. This significantly reduces the risk of insertional mutagenesis, which is a major safety concern associated with lentivirus or retrovirus-based CAR vectors.

Large cargo space: Our vector can accommodate ~30 kb of total DNA. The plasmid backbone only occupies about 3 kb, leaving plenty of room to accommodate the CAR expression cassette along with other components including the promoter, marker, and additional transgenes of interest.

Low cost: Manufacturing clinical-grade regular plasmid vectors is far less expensive than viral vectors which makes clinical development of regular plasmid-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.

Disadvantages

Delivery method: Regular plasmid CAR expression vectors are typically delivered into T cells by electroporation which results in a low to medium frequency of cells with successful incorporation 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 cells ex vivo for extended periods to attain the quantities required for infusion, which over time could alter cell phenotype leading to reduced long-term memory function.

Non-integration of vector DNA: Transfection of target cells with plasmid vectors is also referred to as transient transfection because the vector stays mostly as episomal DNA in cells without integration. However, plasmid DNA can integrate permanently into the host genome at a very low frequency (one per 102 to 106 cells depending on cell type). If a drug resistance or fluorescence marker is incorporated into the plasmid, cells stably integrating the plasmid can be derived by drug selection or cell sorting after extended culture.

Key components

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.

CMV promoter: Human cytomegalovirus immediate early promoter. It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

BGH pA: Bovine growth hormone polyadenylation. It facilitates transcriptional termination of the upstream ORF.

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.