Rabbit immunization + Fab-phage display
Why rabbit antibodies?
Rabbit recombinant monoclonal antibodies represent a novel source of untapped potential and make great tools for use as research reagents or for high sensitivity immuno-detection in diagnostic assays. Rabbit antibodies are emerging as a unique source of lead candidates for therapeutic applications. Abwiz Bio has partnered with experts in rabbit antibody humanization, and our recombinant monoclonal antibody development platform is ideal for engineering humanized variants after the initial lead identification.
Recombinant rabbit monoclonal antibodies have historically remained elusive due to intellectual property and technological barriers. Rabbit antibodies have unique advantages that make them ideal for research or diagnostic uses or as sources of therapeutic lead candidates. Due to higher diversity within antigen binding regions (CDRs), rabbit antibodies created from immunized sources have higher affinity compared to mouse antibodies, and rabbits can respond to antigens that are non-immunogenic in mice. Rabbit antibodies typically possess picomolar KD binding affinities, making affinity maturation unnecessary.
How are rabbit antibodies typically made?
Rabbit monoclonal antibodies are typically derived from hybridomas, which are commonly poorly characterized and can exhibit high batch-to-batch variability. Hybridoma library screening is limited in size (104-105), often missing rare clones that recognize important, less immunodominant epitopes. B-cell cloning methods suffer from similar limitations in library size and can lose additional clones due to poor gene recovery. Abwiz Bio's proprietary recombinant rabbit monoclonal antibody development platform, RabWizTM combines the power of the rabbit immune response with the large library sizes (109-1011) of phage libraries.
But I thought rabbit Fabs do not perform well in phage display format?
Rabbit Fabs have a unique disulfide architecture that can cause expression problems in E. coli. Historically, this lead to the development of sub-optimal systems that do not use the full rabbit immune repertoire. This review article from 2017 recommends using chimeric rabbit/human Fab format-based phage display to avoid the expression issue. However, by using a novel patented antibody library construction method, "WizAmpTM" (US 9,890,414), and a proprietary phage vector, the RabWizTM platform has been used to develop functional antibodies for hundreds of targets, including peptide, protein and cell-based antigens, using a fully rabbit Fab-phage display platform. See some of the unique characteristics and advantages of RabWizTM below.
Unique Advantages of RabWizTM
A proprietary phage display vector compatible with unmodified rabbit Fabs
The unique disulfide architecture of rabbit Fabs is known to cause toxicity in E. coli, which degrades the quality of phage libraries. Others have used sub-optimal systems that restrict usage of the full rabbit immune repertoire. Our highly optimized Fab-phage display vector expresses rabbit Fabs without modification.
A primer set that covers maximal VH gene diversity
Through iterative improvement after creating dozens of rabbit libraries, we have created a primer set that covers the highest reported rabbit VH FR1 sequence diversity to date by including many FR1 sequences that are not currently in publicly available databases. Only by amplifying all antibody genes can you ensure that the entire rabbit immune repertoire is isolated during library construction.
WizAmpTM cloning (US 9,890,414)
In our patented WizAmp™ procedure (US 9,890,414), a single non-gene-specific primer is used to amplify antibody genes prior to digestion and cloning into the library vector. This enables uniform amplification of antibody genes, allowing creation of unbiased, high quality libraries.
Project Development Timeline
| Steps | Deliverables | Time | |
|---|---|---|---|
| Immunization | •Antigen preparation •Rabbit immunization | - | ~2 months |
| Library construction | Using robust WizAmpTM protocol | - | 3 weeks |
| Selection | •Phage panning •Fab screening •Sequencing/H3 grouping | •Fab ELISA data •VH/VL sequence data •Affinity measurement (optional) | 2-3 weeks |
| IgG production | •IgG cloning •IgG expression/purification •IgG ELISA | •IgG ELISA data •~1mg antibody •Affinity measurement (optional) | 4 weeks |
| From rabbit immunization to validated IgG | ~5 months |
RabWizTM has proven success
71+ targets for in-house projects
Phospho/peptide-binding antibody products sold in our catalog
Dozens more targets currently in our development pipeline
30+ targets for client projects projects
Including diverse targets for diagnostic and therapeutic lead candidates
100+ total successful targets with RabWizTM
We have developed our RabWizTM through iterative improvement of each step in our protocols to create a robust platform that has been successfully used to create commercial reagents and diagnostic and therapeutic lead candidates.
RabWizTM vs Alternative Strategies
In rabbit monoclonal antibody development, B-cell cloning is generally preferred over hybridoma technology as it overcomes the issue with poor antibody production from rabbit hybridoma cells. B-cell cloning is a technology that directly amplifies antibody genes from single B-cells to clone light and heavy chains from immune cells. There may be an initial stage of target specific selection of B-cells with surface IgG prior to the isolation of single B-cells.
However, whether using B-cell cloning or hybridoma approach, the number of cells that can be covered is limited (generally 104 to 105 cells) and there are no down-stream selection strategies that could efficiently remove cross-reactive clones or isolate binders to various epitopes and antigen types such as peptide, protein, and cells. Therefore, isolated antibodies are likely against dominant epitope(s) that are more immunogenic and may not show favorable specificity and function as these binders are often against less immunogenic and/or minor epitope(s). Furthermore, single cell PCR involved in the B-cell cloning may lose some of the clones and could have cross-contamination between the wells as it will require a very sensitive amplification from a small number of cells.
Compared to B-cell cloning, Abwiz Bio's patented antibody library cloning method WizAmp™ efficiently covers the maximum diversity from immune cells including antibody producing plasma cells to ensure the isolation of those clones against minor and rare but important epitope(s). Though Abwiz Bio’s proprietary Needle-in-a-Haystack™ phage display technology, this combines conventional animal immunization protocols with the robustness of phage display. These together enable the generation of antibodies with affinities typically attained only through hybridoma development, but doing so with the speed and diversity of phage display technology. The process allows for efficient, high throughput identification of rare binders with distinctive characteristics required for a specific application.
High affinity rabbit antibodies can be created in less than 6 months from the date of rabbit immunization. Abwiz Bio’s monoclonal antibody development platform is coupled with our friendly business terms (no royalties and minimal upfront fee).
| RabWizTM | Rabbit hybridoma/B-cell cloning | |
|---|---|---|
| Library size | 109-1011 | 104-105 |
| Select mAbs using cell-based, peptide or protein antigens | ✅ | ✅ |
| Recover mAbs against dominant epitopes | ✅ | ✅ |
| Recover mAbs against rare epitopes | ✅ | ❌ |
| Recover mAb genes from all immune cells | ✅ | ❌ |
| Re-use libraries in downstream selection strategiesA | ✅ | ❌ |
AA huge advantage to RabWizTM libraries is that they are archived upon creation. Hybridoma and B-cell libraries cannot be archived in long-term storage without loss of cell viability, leading to loss of rare mAb clones. RabWizTM; libraries are stored as frozen DNA, allowing for selection strategies that require repeated selections, such as epitope masking panning, which enables isolation of rare clones recognizing neutralizing epitopes after initial selection of non-neutralizing antibodies recognizing dominant epitopes.
Pricing
Antibody development STANDARD package for maximum antibody diversity
USD 55,000~ (Research use only, royalty needed for commercialization)
USD 70,000~ (Fee for service, commercialization rights and sequence IP belong to client)
3 rabbit animal immunizations
Serum evaluation (using a single antigen provided by the customer)
2 antibody library constructions from 2 organ tissues (bone marrow and spleen)
Panning of 2 libraries (phage display selection using a single antigen provided by the customer)
Live cell panning available (option)
Screenings (190 clones) including ELISA analysis and flow cytometry (option)
Sequence analysis (full length)
Delivery of 500 µL of crude Fab
Recombinant IgG development available on request
Antibody development STARTER package
USD 39,000~ (Research use only, royalty needed for commercialization)
USD 45,000~ (Fee for service, commercialization rights and sequence IP belong to client)
3 rabbit animal immunizations
Serum evaluation (using a single antigen provided by the customer)
1 antibody library constructions from one organ tissues (bone marrow recommended)
Panning of 1 library (phage display selection using a single antigen provided by the customer)
Live cell panning available (option)
Screenings (95 clones) including ELISA analysis and flow cytometry (option)
Sequence analysis (full length)
Delivery of 500 µL of crude Fab
Recombinant IgG development available on request
Academic collaboration
Joint research with academia for therapeutic purposes is also welcome. We can develop antibodies in collaboration after evaluating the target potential. We can jointly apply for NIH SBIR and STTR grants for resources.
Difference between standard and starter packages
In our long experience, we have found that the diversity of antibodies that can be obtained depends on the organ used for library construction. Specifically, spleen, bone marrow, PBMCs (peripheral blood mononuclear cells) and lymph nodes, and GALT (gut-associated lymphoid tissue) are sources of antibodies. Our experience has shown that we can obtain a much greater variety of antibodies using spleen and bone marrow than using PBMC or GALT. In many cases, we have developed high performance antibodies from bone marrow. Antibodies can also be produced from the spleen, but they are often less diverse compared to antibodies derived from bone marrow. If cost is the limiting factor, we recommend bone marrow as the source of the antibody library. However, if you are aiming to develop antibodies against difficult targets such as GPCRs or specific epitopes with desired functions, we strongly recommend that you generate antibodies from both spleen and bone marrow.
*It is also possible to generate additional libraries from spleen after using the simple package (we archive the spleen as a backup organ), to perform library construction and screening later.
Partnership Opportunities
Abwiz Bio is pleased to perform contract work and to engage in co-development partnerships with a variety of research institutions and pharmaceutical companies. Abwiz Bio offers a variety of services that leverage our world-class, proprietary technology platform. Our partnership structure is highly flexible, and we are excited and interested in pursuing partnerships with pharmaceutical companies, research institutes or individual academic researchers. Call or e-mail us today to see how we can help you achieve your goals.
Recombinant mAb development contracts
Co-development projects
Fee-for-service arrangements
