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 MMLV retrovirus CAR expression vector is derived from the Moloney murine leukemia virus, which is a member of the retrovirus family and is highly suitable for retrovirus-mediated delivery of second-generation CAR expression cassettes into T cells. Wildtype MMLV virus has a plus-strand linear RNA genome.

The MMLV retrovirus CAR expression vector is first constructed as a plasmid in E. coli where 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 in between the two MMLV long terminal repeats (LTRs). It is then transfected into packaging cells along with several helper plasmids. Inside the packaging cells, vector DNA located between the two LTRs is transcribed into RNA, and viral proteins expressed by the helper plasmids further package the RNA into virus. Live virus is then released into the supernatant, which can be used to infect target cells directly or after concentration.

When the virus is added to target cells, the RNA cargo is shuttled into cells where it is reverse transcribed into DNA and randomly integrated into the host genome. Any gene(s) that were placed in-between the two LTRs during vector cloning are permanently inserted into host DNA alongside the rest of viral genome.

By design, MMLV retroviral vectors lack the genes required for viral packaging and transduction (these genes are instead carried by helper plasmids used during virus packaging). As a result, virus produced from MMLV retroviral vectors has the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).

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

Our MMLV retrovirus CAR expression vector can be used for the expression of second-generation CARs. It is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient viral transduction of a wide range of cells, efficient vector integration into the host genome, and high-level transgene expression.

Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, retroviral transduction can deliver genes permanently into host cells due to integration of the viral vector into the host genome, thereby enabling long-term expression of CAR expression cassettes.

Large cargo space: The wildtype MMLV retroviral genome is ~8 kb. In our vector, the components necessary for viral packaging and transduction occupy ~2.5 kb, which leaves ~5.5 kb to accommodate the user's DNA of interest. Since our vector is designed for the insertion of only the CAR, this cargo space is sufficient for the expression of all necessary CAR components.

High-level expression: The MMLV 5' LTR contains a strong ubiquitous promoter that drives high-level expression of the user's CAR of interest.

Relative uniformity of gene delivery: Generally, viral transduction can deliver vectors into cells in a relatively uniform manner. In contrast, conventional transfection of plasmid vectors can be highly non-uniform, with some cells receiving a lot of copies while other cells receiving few copies or none.

Effectiveness in vitro and in vivo: While our vector is mostly used for in vitro transduction of cultured cells, it can also be used to transduce cells in live animals.

Safety: The safety of our vector is ensured by partitioning genes required for viral packaging and transduction into several helper plasmids or integrating them into packaging cells. As a result, live virus produced from our vector is replication incompetent.

Dependence on 5' LTR promoter: Expression of the CAR cassette in this vector is driven by the ubiquitous promoter function in the 5' LTR. This is a distinct disadvantage as compared to our lentiviral vectors which allow the user to put in their own promoter to drive their CAR expression cassette. 

Moderate viral titer: Viral titer from our vector reach ~10⁷ TU/ml in the supernatant of packaging cells without further concentration. This is about an order of magnitude lower than our lentiviral vectors.

Risk of Insertional mutagenesis: Gamma retroviral vectors have an intrinsic tendency of integrating close to gene transcription start sites and proto-oncogenes. This increases the chances of insertional mutagenesis and can be a major concern for the clinical application of retrovirus-based CAR vectors. 

Technical complexity: The use of MMLV retroviral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technically demanding and time consuming relative to conventional plasmid transfection.

High manufacturing costs: The cost of producing GMP-grade retroviral vectors is significantly higher compared to non-viral vectors and therefore, is a major limitation associated with the clinical development of retrovirus-based CAR T therapies.

MMLV 5' LTR: MMLV retrovirus 5' long terminal repeat. In wildtype MMLV retrovirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript.

Ψ plus pack2: MMLV retrovirus packaging signal required for the packaging of viral RNA into virus.

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.

MMLV 3' LTR: MMLV retrovirus 3' long terminal repeat. The polyadenylation signal contained in 3' LTR serves to terminate the transcript from the upstream CAR.

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.