Custom Antibody Services

Custom Rabbit mAb Development

Accelerate the development of diagnostic and therapeutic lead candidates with RabwizTM—our platform for custom rabbit monoclonal antibody production. Combining the power of the rabbit immune response with the large library sizes and power of phage display, RabwizTM delivers antibodies with higher affinity and specificity, even for difficult targets.

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Custom Rabbit mAb Development

Overview

Rabbit recombinant monoclonal antibodies (mAbs) represent a novel source of untapped potential. 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.

Together, this makes rabbit mAbs great tools for use as research reagents, for high sensitivity immuno-detection in diagnostic assays, and as sources of therapeutic lead candidates. However, they have historically remained elusive due to intellectual property and technological barriers.

With RabwizTM—our fully optimized solution for custom rabbit monoclonal antibody production—we can deliver high quality rabbit mAbs with the affinity, specity, and functionality you need, even for your most difficult targets.

Here’s how we do it:

  1. Antigen design and preparation — We have extensive experience using recombinant protein, peptide, small-molecule, and cell-based antigens.
  2. Rabbit immunization and serum analysis — optimized with functional serum assays early in the process to ensure that antibodies with the desired function are present in the rabbit before library construction begins.
  3. Library construction — using our proprietary cloning method (WizAmpTM) that produces library sizes of 109-1011 and captures higher diversity compared to other methods and an engineered phagemid vector that ensures optimized expression of Fabs displayed on the surface of phage.
  4. Selection — identifying the most promising clones via phage panning for desired specificity, affinity, Fab screening, and sequencing. Selection and screening can be performed with small molecule, peptides, recombinant or cell-based antigens.
  5. IgG production — cloning and transfection in HEK293T followed by purification
  6. Validation — using IgG ELISA or flow cytometry and (optional) affinity measurements to validate mAb function

High affinity rabbit antibodies can be created in less than 6 months from the date of rabbit immunization. What’s more, Abwiz Bio’s monoclonal antibody development platform is coupled with our friendly business terms—royalty-free and minimal upfront fees.

Need humanized antibodies or further affinity maturation? With our flexible end-to-end antibody development services, you can get fully validated and optimized humanized mAbs.

Explore our services for Antibody Humanization and Optimization and Antibody Affinity Maturation.

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Benefits

Your success is our success, which is why we’ve optimized every step of our process to deliver high-quality custom rabbit mabs—for every project and every application.

Flexibility

Higher affinity and specificity

Rabbits develop antibodies with high affinity and specificity to various epitopes on a given antigen, and they tend to respond better to targets that are difficult in mice.

No royalty payments

Larger Diversity, More Options

We can uniformly amplify all Ab genes and build large libraries of 5 x 109- 2 x 1010, so you gain access to rare, functional clones that could be your ticket to success.

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Validated quality mAbs

We use stringent validation criteria to QC each antibody before listing it as a product. As a result, we see many of our antibodies out-perform our competitors’ clones.

Proven technology, amazing results

We have optimized and validated custom rabbit monoclonal antibody production over years of effort, iteratively improving each protocol to ensure success. RabwizTM has successfully generated commercial reagents and diagnostic and therapeutic lead candidates.

  • 110+ total successful projects with RabwizTM, including small molecule, peptide, protein and cell-based antigens
  • 82+ rabbit mAbs developed and sold by Abwiz for research reagent use and dozens more in our development pipeline
  • 30+ rabbit mAbs successfully developed as diagnostic or therapeutic lead candidates for clients with diverse targets (e.g., ion channels, small molecules, GPCRs)
  • 400+ immunized rabbit libraries created in total

The RabwizTM Difference

How others develop rabbit mAbs:

In rabbit monoclonal antibody production, B-cell cloning and hybridoma technology are common, with B-cell cloning generally preferred as it overcomes the issue of poor antibody production from rabbit hybridoma cells.

However, whether using B-cell cloning or the hybridoma approach, the number of cells that can be covered is limited (generally 104 to 105 cells) and there are no downstream 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.

Traditionally, if a functional antibody is not obtained in the first attempt, there is no way to efficiently repeat the process because B-cells and hybridomas cannot be re-screened or archived.

How does Rabwiz differ?

The unique and optimized characteristics of Rabwiz enable efficient, high throughput identification of rare binders with distinctive characteristics required for a specific application, making custom rabbit monoclonal antibody production faster and better than ever before.

  • WizAmpTM— our proprietary cloning method—efficiently covers the maximum diversity from immune cells including antibody producing plasma cells so you can obtain clones against minor and rare but important epitope(s).
  • Our proprietary phage display vector paired with rabbit immunization enables the generation of antibodies with affinities typically attained only through hybridoma development, but doing so with the speed and diversity of phage display technology.
  • Unlike hybridoma and B-cell libraries, Rabwiz libraries are archived upon creation—retaining rare mAb clones—so you can perform selection strategies that require repeated selections, such as epitope masking panning.
RabwizTM Rabbit hybridoma/B-cell cloning
Library size (covered diversity) 5x109-1011 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 strategies

Optimized characteristics of RabwizTM

A proprietary phage display vector compatible with unmodified rabbit Fabs delivers better expression

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.

Before engineering → Traditional phagemid vector

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Maximal VH gene diversity to expand your antibody options

Only by amplifying all antibody genes can you ensure that the entire rabbit immune repertoire is isolated during library construction. Through iterative improvement after creating dozens of rabbit libraries, we have optimized our mAb development process to cover the highest reported rabbit VH framework sequence diversity to date by including many FR1 sequences that are not currently in publicly available databases.

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WizAmpTM cloning builds high-quality libraries

WizAmpTM (US 9,890,414) creates unbiased libraries and captures full IgG diversity by using a patented cloning protocol that employs a single non-gene-specific primer that amplifies antibody genes prior to digestion and cloning into the library vector. This enables uniform amplification of antibody genes, allowing the creation of unbiased, high-quality libraries.

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Timeline

Steps Deliverables Time
Immunization •Antigen preparation
•Rabbit immunization
•Serum Analysis
•Serum screening data confirming presence of functional antibodies ~2 months
Library construction •Using robust WizAmpTM gene cloning protocol
•Clone into phagemid vector engineered for optimized expression of rabbit Fab in bacteria
•QC report confirming high quality libraries 3-4 weeks
Selection •Phage panning
•Fab screening
•Sequencing/H3 grouping
•Fab ELISA/flow cytometry data
•VH/VL sequence data
2-3 weeks
IgG production •IgG cloning
•Transfection in HEK293T
•IgG expression/purification
•IgG ELISA/flow cytometry
•IgG validation data
•~1mg antibody
•Affinity measurement (optional)
4 weeks
From rabbit immunization to validated IgG ~5 months

Pricing

Antibody Development Packages

  • STANDARD package for maximum antibody diversity (recommended for therapeutic antibody development)

  • USD 70,000~

  • 3 rabbits immunized
  • Serum evaluation
  • 2 antibody library constructions from 2 organ tissues (bone marrow and spleen)
  • Panning of 2 libraries
  • Live cell panning available (option)
  • Screening including ELISA analysis and flow cytometry (option)
  • Sequence analysis (full length)
  • Delivery of 500 µL of crude Fab
  • Recombinant IgG development available on request
  • STARTER package

  • USD 45,000~

  • 3 rabbits immunized
  • Serum evaluation
  • 1 antibody library constructed from 1 organ tissues (bone marrow recommended)
  • Panning of 1 library
  • Live cell panning available (option)
  • Screening 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 collaborations

Abwiz Bio was founded to advance healthcare technologies and basic research for the betterment of society. Whether you are looking to develop novel therapeutic mAbs or advance immunology studies, Abwiz welcomes collaboration. We are open to developing antibodies in collaboration after evaluating the target potential, and we can jointly apply for NIH SBIR and STTR grants for resources.

FAQs

Rabbit antibodies are emerging as a unique source of lead candidates for therapeutic applications and as tools for diagnostic assays and for use as research reagents for many reasons.

  • Higher diversity within antigen binding regions
  • Rabbit antibodies created from immunized sources have higher affinity compared to mouse antibodies (rabbit antibodies typically possess picomolar KD binding affinities)
  • Rabbits can respond to antigens that are non-immunogenic in mice
  • Humanization of rabbits is rather straightforward, as they predominantly use a single VH and VK family that we have experience humanizing

If you are struggling to get the antibody characteristics you need (e.g., specificity, affinity, etc.) consider using rabbit mAbs—they are particularly useful for difficult targets!

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. Additionally, these approaches are ‘one-shot’ strategies—if they fail on the first attempt, it is impossible to return to the same immune source to obtain new clones.Rabbit hybridoma cells are also known to express poorly.

Recently, single B-cell cloning and deep sequencing approaches are used for rabbit monoclonal antibody development. However, even these methods are lacking the selection of antibodies with desired affinity and specificity and they have to be recombinantly made one by one to test their bindings and functions.

In contrast, Abwiz Bio's proprietary recombinant rabbit monoclonal antibody development platform, RabWizTM platform combines the power of the rabbit immune response with the large library sizes (5x109- 2 x 1010) of phage libraries. Our libraries are archived indefinitely as DNA after library construction, allowing for unique selection strategies including epitope masking with dominant (non-functional) clones to obtain rare neutralizing antibodies. Once the candidate clones are selected and converted to IgG, they tend to express very well unlike hybridoma cells.

Yes! Rabbit Fabs have a unique disulfide architecture that historically has caused expression problems in E. coli, leading to the development of sub-optimal systems that do not use the full rabbit immune repertoire. However, we are able to bypass this barrier with our RabWizTM platform. Our proprietary phage vector has been highly optimized specifically for rabbit Fabs, allowing us to express the dominant Ckappa1 clones that cause expression problems in traditional phage systems. We have successfully used this platform to develop functional rabbit antibodies for hundreds of targets, including peptide, protein and cell-based antigens.

In our experience, the level of antibody diversity 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. We can obtain a much greater variety of high-performance antibodies using spleen and bone marrow than using PBMC or GALT.

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.

In general, library construction is performed by the PCR amplification of antibody genes utilizing 2 gene-specific primers. This causes biased amplification and non-specific amplification resulting in poor coverage of antibody repertoire in the immunized animals.

AbWiz Bio invented a unique method called WizAmpTM (US 9,890,414) that engineers cDNA specifically for antibody genes and utilizes only 1 non-gene-specific primer (single primer amplification). This method ensures the widest coverage of antibody repertoire including the rare clones that have the desired function and specificity.

Yes! Abwiz Bio has developed a validated in-house rabbit antibody humanization platform (See our humanized COVID neutralizing therapeutic antibody), and our recombinant monoclonal antibody development platform is ideal for engineering humanized variants after the initial lead identification. Learn more about our Antibody Humanization and Optimization services.

If the antibodies are isolated from a naïve library the answer is they are most likely randomly paired but, if the library is an immune library, the light and heavy chain pairing of the isolated clones is not random at all. The clones that belong to the same heavy chain clusters based on HCDR3 sequences are paired with the same light chain clusters based on LCDR3.

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