A tool kit for rapid cloning and expression of recombinant antibodies

Abstract

Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

“…Expi293F cells were transfected with pVitro1-hygro-mcs antibody constructs using the ExpiFectamine293 Transfection kit (Thermo Scientific) as per manufacturer's instructions. The anti-human epidermal growth factor receptor 2 (HER2) and the melanomaassociated antigen-specific chondroitin sulfate proteoglycan (CSPG4) antibody constructs were previously described (25,26).…”

“…Heavy and light chain variable regions were cloned in pVitrohygro-1 (Invivogen) using polymerase incomplete primer extension (PIPE) cloning as previously described (25…”

“…RNA released from single cells was converted to cDNA using reverse transcription followed by a semi-nested RT-PCR with Ig specific primers. Matched variable H and L chains were sequenced and cloned into a single expression vector containing the H and L constant IgG1 region sequences as previously reported (25,26). The full antibody was then expressed in a human expression host.…”

Sequencing and cloning of antigen-specific antibodies from mouse memory B cells

“…The most commonly used method to obtain matched immunoglobulin heavy and light chains from single cells involves cloning of the variable domains into vectors encoding the heavy-and/or light-chain constant domains 6,[24][25][26][27] . Transient transfection of these vectors in mammalian HEK293 cells allows antibody expression in a relatively short time.…”

Adding Functions to Biomaterial Surfaces through Protein Incorporation

“…Recombinantly expressed whole antibody molecules can be produced more quickly and much less expensively than animal-derived monoclonal antibodies (mAbs) [130] and with higher and more consistent yields. Ease of access to their gene sequences also enables relatively simple modification of their affinity, specificity or stability but technical complexities associated with their expression -and the inessential nature of nonantigen-binding domains in many applications -mean that most researchers avoid constant domains and express instead small, typically monovalent fragments that retain the ligand-binding pocket of the parent molecule for binding studies.…”