Lentivirus 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.

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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 were capable of activating 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 lentivirus CAR expression vector is a highly efficient viral vector tool for delivering second generation CAR expression cassettes into T cells. The lentivirus 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 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, lentiviral 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 lentiviral 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.

References	Topic
Br J Cancer. 120:26 (2019)	Review on next-generation CAR T cells
Mol Ther Oncolytics. 3:16014 (2016)	Review on CAR models
Sci Transl Med. 3: 95ra73 (2011)	Lentivirus-mediated CAR expression for treating chronic lymphocytic leukemia
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

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Highlights

Our lentivirus CAR expression vector is derived from the third-generation lentiviral vector system and 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.

Experimental validation

Validation of CAR-induced T-cell

flow cytometry analysis for CAR CAR Jurkat cell activation

Figure 1. Validation of CAR-induced T-cell activation through detection of surface CD69 upregulation using CAR-expressing Jurkat cells and CD19-expressing target cells (Ramos cells). (A) Jurkat cells were transduced with lentivirus encoding CD19-targeting CAR and co-cultured with CD19-expressing Ramos cells. Upon binding of CD19, Jurkat cells are activated, as shown by production of CD69 surface antigen. (B) Following incubation, flow cytometry analysis was performed to assess CAR Jurkat cell activation. The surface expression of CD69 on CAR Jurkat cells drastically increased upon co-culturing with CD19+ Ramos cells (blue), signifying the robust CAR-mediated activation of Jurkat cells.

Advantages

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, lentiviral transduction can deliver genes permanently into host cells due to the integration of the viral vector into the host genome, thereby allowing long-term expression of CAR cassettes in T cells.

High viral titer: Our lentiviral vector can be packaged into high titer virus. When lentivirus is obtained through our virus packaging service, titer can reach >10⁸ transducing unit per ml (TU/ml). At this titer, transduction efficiency for cultured mammalian cells can approach 100% when an adequate amount of viral is used.

High transduction efficiency: Lentiviral vectors exhibit a high transduction efficiency in T cells. As a result, for developing lentivirus-based CAR T cells, a relatively small number of patient-derived T cells can be easily transduced and expanded to achieve the dosage amount required for a clinical infusion.

Customizable internal promoter: Our vector is designed to self-inactivate the promoter activity in its 5' LTR upon integration into the genome. As a result, users can put in their own promoter to drive their CAR expression cassette within the vector. This is a distinct advantage over our MMLV retrovirus vectors, which rely on the promoter function of 5' LTR to drive the ubiquitous expression of the CAR expression cassette.

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 two features. One is the partition of genes required for viral packaging and transduction into several helper plasmids; the other is self-inactivation of the promoter activity in the 5' LTR upon vector integration. As a result, it is essentially impossible for replication competent virus to emerge during packaging and transduction. The health risk of working with our vector is therefore minimal.

Disadvantages

Limited cargo space: The wildtype lentiviral genome is ~9.2 kb. In our vector, the components necessary for viral packaging and transduction occupy ~2.8 kb, which leaves ~6.4 kb to accommodate the user's DNA of interest. When the vector goes beyond this size limit, viral titer can be severely reduced. Our vector is routinely used for inserting several functional elements besides the CAR, such as promoter and drug resistance. A CAR combined with these additional elements could exceed 6.4 kb, which could result in compromised viral production.

Risk of insertional mutagenesis: Due to their ability to permanently integrate into the host genome, lentivirus-based CAR expression vectors pose the risk of insertional mutagenesis. However, the risk of insertional mutagenesis with lentiviral vectors is less in comparison to retroviral vectors, which unlike lentiviral vectors have an inherent tendency to integrate near gene transcription start sites and proto-oncogenes.

Technical complexity: The use of lentiviral 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 lentiviral vectors is significantly higher compared to non-viral vectors and therefore, is a major limitation associated with the clinical development of lentivirus-based CAR T therapies.

Key components

RSV promoter: Rous sarcoma virus promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.

5' LTR-ΔU3: A deleted version of the HIV-1 5' long terminal repeat. In wildtype lentivirus, 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 in wildtype virus, 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. On our vector, 5' LTR-ΔU3 is deleted for a region that is required for the LTR's promoter activity normally facilitated by the viral transcription factor Tat. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the RSV promoter engineered upstream of Δ5' LTR.

Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.

RRE: HIV-1 Rev response element. It allows the nuclear export of viral RNA by the viral Rev protein during viral packaging.

cPPT: HIV-1 central polypurine tract. It creates a "DNA flap" that increases nuclear importation of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.

Promoter: The promoter driving your downstream 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 flexibility 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.

WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances viral RNA stability in packaging cells, leading to higher titer of packaged virus.

mPGK promoter: Mouse phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression 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.

3' LTR-ΔU3: A truncated version of the HIV-1 3' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (due to the fact that 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in 3' LTR-ΔU3 serves to terminates all upstream transcripts produced both during viral packaging and after viral integration into the host genome.

SV40 early pA: Simian virus 40 early polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during viral RNA transcription during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.

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.

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