PiggyBac Antibody Heavy Chain Gene Expression Vector
Recombinant antibodies have extensive applications in the fields of diagnostics and therapeutic medicine. Currently, there are about 180 monoclonal antibody products approved worldwide targeting specific proteins, such as PD-L1, HER2, IL-17R, and VEGF. The development of genetic engineering has facilitated successful production of these therapeutic antibodies, which have ideal tumor penetration behavior, short retention times, and reduced immunogenicity. These therapeutic recombinant antibodies are mainly produced in mammalian cells in vitro (e.g., CHO cells), since the post-transcriptional modification in this expression system is closest to the in vivo processes in humans.
The antigen-binding site of all antibodies consists of VL+VH dimers where VL and VH represent variable fragment light chains and heavy chains, respectively. Within these fragments, most sequence variation is located in complementarity-determining regions (CDRs), which influences antigen-binding specificities. By customizing sequences encoding the heavy chain variable region, customers may generate vectors to satisfy their personalized need for antigen-binding specificities. In addition to the variable (V) region, the heavy and light chains contain a constant (C) region, which determines the antibody’s isotype. Of the five immunoglobulin (antibody) isotypes (IgG, IgA, IgM, IgD and IgE), IgG is the most abundant in circulation. Moreover, almost all backbones of the approved therapeutic antibodies are IgG. In the vector design process, the heavy chain constant region may either be selected from our popular heavy chain constant region database (containing human and mouse IgG1 and mouse IgG2a and 2b) or be pasted by customers using their own sequence. Additionally, signal peptides (e.g., IL-2 sig) can be added into the vector to increase secretion of the recombinant antibody.
VectorBuilder’s piggyBac antibody heavy chain expression vector system is a highly efficient tool for achieving non-viral, transposon-based delivery of antibody heavy chain expression cassette into mammalian cells. This system contains two vectors, both of which are engineered as E. coli plasmids. The first vector is referred to as the helper PBase plasmid, encoding the transposase. The second vector is referred to as the piggyBac transposon plasmid which contains two terminal repeats (TRs) bracketing the antibody heavy chain expression cassette to be transposed. When the helper PBase plasmid and the piggyBac transposon plasmids are co-transfected into target cells, the transposase from the helper plasmid will recognize the two TRs on the transposon and insert the flanked antibody heavy chain expression cassette 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 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 genes encoding the antibody heavy chain becomes permanent in the genome of host cells.
For further information about this vector system, please refer to the papers below.
|MAbs. 14:2014926 (2022)||Overview of antibody therapeutics|
|Nat Protoc. 13:99 (2018)||Overview of design for antibody expression vectors|
|Protein Expr Purif.118:105-12(2016)||Description of signals which can increase the secretory protein production|
|Cell. 122:473 (2005)||Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice|
Our piggyBac expression vectors are designed to drive high-level permanent antibody heavy chain expression in mammalian cells under a user-selected promoter when used in conjunction with the helper plasmid.
Technical simplicity: Delivering plasmid vectors into cells by conventional transfection is technically straight forward, and far easier than virus-based vectors which require the packaging of live viruses.
Permanent integration of vector DNA: The piggyBac antibody heavy chain expression vector facilitates long-term expression of antibody heavy chains in mammalian cells by allowing integration of the transposon carrying the expression cassettes into the host genome.
Reproducibility and scalability: The recombinant protein is harvested directly from transfected host cells. Therefore, reproducibility of different batches can be easily achieved. Moreover, the amplification of host cells allows for large-scale antibody production.
Limited cell type range: The delivery of piggyBac vectors into cells relies on transfection. The efficiency of transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo. These issues limit the use of the piggyBac system.
Optimal expression ratio of the heavy to light chain difficult to achieve: All antibodies consist of heavy chains and light chains. For successful antibody production, a precise expression ratio of the heavy to light chain is required. This can only be achieved by co-transfecting a second plasmid expressing the antibody light chain. Achieving optimal expression of both heavy chain and light chain may be difficult to control due to expression driven by different promoters in two distinct vectors.
5’ ITR: 5’ inverted terminal repeat. When a DNA sequence is flanked by two ITRs, the piggyBac transposase can recognize them and insert the flanked region including the two ITRs into the host genome.
Promoter: The promoter that drives your gene of interest 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.
IL2-sig: Signal peptide of Homo sapiens interleukin 2. It facilitates the secretion of protein.
Heavy Chain Variable Region (VH): Heavy chain variable region for antigen recognition.
Heavy Chain Constant Region (CH): Heavy Chain constant region encoding isotype.
rBG pA: Rabbit beta-globin polyadenylation signal. It facilitates transcriptional termination of the upstream heavy chain constant region cassette.
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’ ITR: 3’ inverted 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.