Golden Gate assembly with a bi-directional promoter (GBid): A simple, scalable method for phage display Fab library creation

Abstract

Fabs offer an attractive platform for monoclonal antibody discovery/engineering, but library construction can be cumbersome. We report a simple method -Golden Gate assembly with a bidirectional promoter (GBid) -for constructing phage display fab libraries. in GBid, the constant domains of the Fabs are located in the backbone of the phagemid vector and the library insert comprises only the variable regions of the antibodies and a central bi-directional promoter. This vector design reduces the process of Fab library construction to "scFv-like" simplicity and the double promoter ensures robust expression of both constituent chains. To maximize the library size, the 3 fragments comprising the insert -two variable chains and one bi-directional promoter -are assembled via a 3-fragment overlap extension PCR and the insert is incorporated into the vector via a high-efficiency one-fragment, one-pot Golden Gate assembly. the reaction setup requires minimal preparatory work and enzyme quantities, making GBid highly scalable. Using GBid, we constructed a chimeric chicken-human fab phage display library comprising 10 10 variants targeting the multi-transmembrane protein human CD20 (hCD20). Selection/counter-selection on transfected whole cells yielded hCD20-specific antibodies in four rounds of panning. The simplicity and scalability of GBid makes it a powerful tool for the discovery/engineering of fabs and igGs.The discovery and engineering of monoclonal antibodies (mAbs) using phage display usually utilizes one of two simplified display formats. Single-chain variable fragments (scFvs) represent the simplest format for antibody phage display in which only the domains directly involved in interaction with the antigen -the variable heavy and variable light chains -are represented. Antibody-binding fragments (Fabs) more closely resemble the antigen-binding "business end" of IgG molecules and incorporate both the variable regions and their immediately neighboring constant subdomain -typically CH1 for the heavy chain and CLκ or CLλ for the light chain. Under oxidizing conditions, the constant domains in Fabs are covalently linked via a disulfide bond.The attractiveness of the scFv as a medium for IgG discovery lies in its overall simplicity. The presence of only a single chain generally gives rise to higher expression, and cloning of scFvs into phagemid vectors is relatively straightforward, typically requiring only a single overlap extension PCR reaction that links the two variable domains via a short flexible linker 1-3 . Unfortunately, the binding affinity of scFvs can change significantly upon reformatting to Fabs or IgGs, creating a need to verify the binding/activity of the molecules in the more complex formats in applications where Fabs or IgGs are the desired final product 4-8 .Fabs more closely resemble full-length IgGs and are generally more stable than scFvs 6,9 , making these molecules a more reliable indicator of full-length IgG behavior. Unfortunately, the construction of phage display Fab libraries is less straight...

Chicken‐derived CD20 antibodies with potent B‐cell depletion activity

“…Importantly, chickens do not have a CD20 homologue, thus eliminating species‐induced constraints on hCD20 epitope accessibility. In this study we characterize four novel anti‐hCD20 Abs discovered from the immune repertoire of chickens 27 . We show that all four chicken‐derived Abs are superior to RTX in both Fc‐mediated mechanisms of action—ADCC (≥10‐fold) and CDC (4–8‐fold).…”

“…Anti‐hCD20 Abs diluted in Dulbecco's phosphate‐buffered saline (DPBS)/1% bovine serum albumin (BSA) were incubated with 6 × 10 5 Raji cells in V‐bottom 96‐well plates at 4°C for 1 h in a final volume of 50 μl. The cells were washed with cold DPBS/1% BSA and incubated at 4°C for 1 h with 50 μg/ml goat anti‐human Fcγ (Jackson ImmunoResearch) labelled with Atto 488 (A488) N‐hydroxysuccinimide (NHS) ester as previously described 27 . Binding of mAbs to live single cells gated by light scatter characteristics was measured using a BD LSRFortessa X‐20 flow cytometer.…”

“…We previously reported the use of chicken immunization coupled with whole-cell panning of a Fab display phage library to identify two novel anti-hCD20 mAbs-AC1 and AC11. 27 We subsequently identified two additional mAbs-ACR7 and ACR8-that were sparsely present in the same selected library, through next-generation sequencing (NGS) analysis of the enriched Fab heavy chains. In this report we describe the characterization of these four chicken-human chimaeric Abs and the humanization of one Ab-AC11.…”

Ultra high diversity factorizable libraries for efficient therapeutic discovery

“…H(S p , S s ) can be thought of as roughly being the number of bits needed to write down every sequence in the factorizable library using an optimal encoding multiplied by ln (2). The encoding differs depending on the position along the sequence and the lengths of the prefix and suffix used to generate the sequence it is encoding.…”

“…SAPS iteratively improves segment libraries with respect to an objective function that evaluates the result of their concatenation into a full length factorizable library. After the synthesis of segment library DNA oligonucleotides, segment libraries can be joined with a combination of overhang and blunt end ligation similar to Golden Gate Assembly to create a factorizable library [2, 3].…”