Mammalian 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.
VectorBuilder’s mammalian antibody heavy chain gene expression vector has been designed specifically for high-yield production of monoclonal antibody heavy chains. 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 specificity. 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.
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 expression vector is optimized for high-efficiency transfection and high yield of monoclonal heavy chains in mammalian cells. By customizing sequences encoding the variable heavy chains into the vector, customers may obtain antibodies with high-affinity to target antigens.
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.
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.
Non-integration of vector DNA: Conventional transfection of 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 (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.
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.
Promoter: The promoter driving 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.
SV40 late PA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
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.
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.