Proprietary phagemid vector
Rabbit antibodies routinely reach picomolar affinity where mouse antibodies sit in the nanomolar range — but the dominant rabbit κ1 light chain carries an extra disulfide bond that prevents rabbit Fab from folding in E. coli. Abwiz Bio built a phagemid vector that displays it anyway: no κ1-deficient rabbit strain, no human constant domains, no scFv reformatting. The immune repertoire is panned in the format it came out of the animal.
Four routes around the rabbit Fab disulfide problem — and what each one costs
| Route | What it gives up |
|---|---|
| κ1-deficient rabbit strain | The dominant light-chain isotype, and with it most of the natural repertoire |
| Chimeric Fab with human constant domains | Native heavy/light pairing, and it front-loads a humanization variable before you have a lead |
| scFv reformatting | Reliable ranking — scFv affinity and stability do not carry over to IgG |
| Abwiz Bio proprietary phagemid vector | Nothing. Full repertoire, native Fab format, direct to IgG. |
Comparison of the published workarounds for rabbit Fab expression in E. coli against Abwiz Bio’s vector. Primary references for the three workaround routes are linked in the discussion below.
Unlocking rabbit Fab-phage display for the first time
Rabbit antibodies possess higher CDR sequence and length diversity in both light and heavy chains compared to mouse or human antibodies; rabbit antibodies often possess pM KD binding affinity compared to nM KD for mouse antibodies. This makes rabbit monoclonal antibodies excellent sources of diagnostic or therapeutic lead candidates. However, rabbit Fabs have a unique disulfide architecture that prevents their expression in E. coli. Historically, this has led to the development of sub-optimal systems that do not use the full rabbit immune repertoire, including the use of a rabbit strain that lacks the dominant disulfide-containing K1 isotype, construction of chimeric Fabs containing human constant domains, or significant engineering in scFv format. All these workarounds necessarily limit the diversity of the resulting immune library and fail to access the full power of the rabbit immune repertoire. We have had many discussions with clients and industry peers who have given up on constructing immune phage display libraries from rabbits due to their inability to express rabbit antibody fragments in E. coli
Indeed, our early attempts with rabbit Fab-phage selection at Abwiz Bio using an industry standard phagemid vector resulted in few binders and low sequence diversity from an immune library (left image below). However, determined to unlock rabbit Fab display, we put significant effort into optimizing our vector by incorporating unique elements. When using our proprietary vector with the same immune library, we were able to reliably isolate hundreds of binders with diverse sequences (right image below), including a majority of clones possessing the dominant kappa K1 isotype.
Our highly optimized Fab-phage display vector expresses rabbit Fabs without modification and without sacrificing antibody diversity. For the first time, we at Abwiz have unlocked the power of phage display for use in rabbit antibody discovery.
See how we have used this next-generation phagemid vector for panning of rabbit immune libraries to isolate rare, functional mAbs against GPCRs and ion channels and to make highly-specific companion diagnostic rabbit monoclonal antibodies.
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