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Mammalian Antibody Heavy and Light Chain Coexpression Vector

Overview

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 and light chain coexpression vector has been designed specifically for high-yield production of monoclonal antibody heavy and light chains. 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.

In this vector system, the coding regions for heavy and light chains are included within a single plasmid 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.

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

References Topic
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

Highlights

Our expression vector is optimized for high-efficiency transfection and high yield of monoclonal heavy and light chain antibodies 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.

Advantages

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.

Disadvantages

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 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 chain 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.

Key components

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.

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.

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.

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