PiggyBac Antibody Heavy and Light Chain Coexpression 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.
All antibodies contain heavy and light chains, both of which consist of variable (V) and constant (C) regions. The antigen-binding site consists of VH+VL dimers where VH and VL represent variable fragment heavy chains and light chains, respectively. Within these fragments, most sequence variation is located in complementarity determining regions (CDRs), which influences antigen-binding specificity. In mammals, antibodies are divided into five isotypes (IgG, IgA, IgM, IgD and IgE) based on the heavy chain C region sequence. Of the five immunoglobulin (antibody) isotypes, IgG is the most abundant in circulation. Moreover, almost all backbones of the approved therapeutic antibodies are IgG. An antibody of a given isotype either has lambda (λ) or kappa (κ) light chains which are encoded by the light chain C region. While the heavy chain is typically associated with target specificity, the light chain is associated with preventing self-reactivity and enhancing receptor activation. The ratio of the two types of light chains varies from species to species.
VectorBuilder’s piggyBac antibody heavy and light chain coexpression vector system is a highly efficient tool for achieving non-viral, transposon-based delivery of antibody heavy and light chain expression cassettes 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 and light chain expression cassettes to be transposed. The coding regions for heavy and light chains are separated by a polyadenylation signal. The transcription of the heavy and light chains is respectively driven by separate promoters. In the vector design process, by customizing sequences encoding the heavy chain and light chain variable regions, customers may generate vectors to satisfy their personalized need for antigen-binding specificities. The heavy and light chain constant regions may either be selected from our popular constant region database (containing human and mouse IgG1 and mouse IgG2a and 2b heavy chains; and containing human and mouse κ and human and mouse λ2 and mouse λ1 light chains) 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.
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 expression cassettes 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 and light chains 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|
Our piggyBac expression vector is optimized for high-efficiency transfection and high yield of monoclonal antibody heavy and light chains in mammalian cells. By customizing sequences encoding the variable heavy and light chains into the vector, customers may obtain antibodies with high-affinity to target antigens.
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, transfection of mammalian cells with the piggyBac transposon plasmid along with the helper plasmid can deliver genes carried on the transposon permanently into host cells due to the integration of the transposon into the host genome.
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. Moreover, presenting coding regions for both heavy and light chains on a single plasmid further simplifies the transfection procedure.
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 and light chains. For successful antibody production, a precise expression ratio of the heavy to light chain is required. Though both coding regions for the heavy and light chains are present on a single vector, it cannot be guaranteed that the optimal expression of the heavy and light chains can be achieved due to mechanisms such as promoter occlusion. Specifically, the coding region arranged near the downstream promoter may be transcribed at a lower level than the coding region arranged near the upstream promoter.
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 the protein.
Heavy Chain Variable Region (VH): Heavy chain variable region for antigen recognition.
Heavy Chain Constant Region (CH): Heavy chain constant region encoding isotype.
BGH pA: Bovine growth hormone polyadenylation signal. It allows transcription termination and polyadenylation of mRNA transcribed by Pol ll RNA polymerase.
Light Chain Variable Region (VL): Light chain variable region for antigen recognition
Light Chain Constant Region (CL): Light chain constant region
rBG pA: Rabbit beta-globin polyadenylation signal. It allows transcription termination and polyadenylation of mRNA transcribed by Pol ll RNA polymerase.
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.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.