2021-09-14_12h03_45

Custom Antibody Services

Custom Rabbit mAb Development

Accelerate the development of therapeutic and diagnostic lead candidates with RabwizTM—our platform for custom rabbit monoclonal antibody production. By combining the high precision of the rabbit immune response with the unique selection conditions only available by phage display, RabwizTM delivers antibodies with higher affinity and specificity, especially for difficult targets.

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Overview

Why Abwiz?

Founded in 2012, we at Abwiz Bio, have decades of collective experience making hundreds of rabbit immune libraries to develop high affinity and functional rabbit mAbs for difficult targets including GPCRs and ion channels or rabbit mAbs possessing extremely fine specificity targeting idiotypes or small molecules.

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

  • Our patented cloning method WizAmp uses a single primer to uniformly amplify antibody genes from immune cells, efficiently covering the maximum diversity including from antibody-producing plasma cells. By covering higher diversity, we can obtain clones against minor and rare but important epitopes
  • Our proprietary Fab-phage display vector is engineered to express fully rabbit Fabs, including Ck1 clones which contain extra cysteines in framework region 3. These Ck1 clones make up 70-90% of rabbit serum antibodies, but could not historically be obtained by phage display due to poor expression in E. coli. We solved this issue by engineering our plasmid vector, enabling us to access the entire rabbit immune repertoire.

By paring these innovative technologies, we can obtain antibodies with affinities typically attained only through immunization, but with the ability to obtain rare and functional clones by performing unique selection strategies only accessed through phage display.

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Other companies use sub-optimal systems that cannot access the full immune repertoire. 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 epitopes that are more immunogenic and may not show favorable specificity and function, as important clones are often against less immunogenic and/or minor epitopes. 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 requires 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.

Some companies use alternate phage display methods paired with rabbit immunization, but at Abwiz Bio, we are the only laboratory that has solved the historical problem with rabbit Fab expression in E. coli. Other phage display methods must use alternatives such as scFv display or chimeric rabbit Fv-human CH1/Ck constructs. These platforms the miss 70-90% of the rabbit immune response composed of Ck1 clones possessing extra cysteines. By limiting the diversity of their libraries, these platforms miss rare and important mAbs.

High affinity, highly specific rabbit antibodies can be created in less than 6 months from the date of rabbit immunization. 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.

Why rabbits?

Rabbit recombinant monoclonal antibodies represent a novel source of untapped potential. The rabbit immune repertoire uniquely possesses higher diversity within antigen binding regions (CDRs) compared to humans or mice. Notably, light chain CDR diversity in rabbits is nearly as high as heavy chain CDR diversity. This enables rabbit mAbs to possess exquisitely high specificity and affinity. Lead candidates from rabbit immune libraries typically possess pM KD affinity, compared to nM KD for lead candidates from mice. Additionally, due to their evolutionary distance, 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.

 

For therapeutic lead candidates, rabbit antibodies are relatively straightforward to humanize. Though rabbit CDR diversity is extremely high, germline usage is relatively limited, enabling a similar humanization strategy among varied lead candidates. Humanized therapeutic mAbs from rabbit sources are well established for clinical use (LINK). Don’t miss out on top quality antibodies developed using Abwiz Bio’s therapeutic monoclonal antibody discovery platform.

Why phage display?

While monoclonal antibodies against immunodominant epitopes can be easily obtained by nearly any antibody discovery platform, functional antibodies often recognize minor epitopes. These rare antibodies can only be accessed by capturing the full diversity of the immune response. B-cell and hybridoma methods can only access a subset of the true immune diversity, while our patented library cloning method amplifies all antibody genes uniformly to capture even rare clones.

 

Phage display allows access to extremely large libraries (up to 2E10) compared to alternative display formats, such as yeast display. Additionally, phage display allows for the use of a variety of selection methods to isolate rare, functional clones. This includes selecting highly-specific mAbs by competition ELISA (see anti-idiotype or small molecule antibody development case studies below) or cross-reactive clones by alternating selection on recombinant protein and cell expressed antigens. 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 to block immunodominant epitopes to obtain neutralizing clones (Example by Abwiz scientists).

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 WizAmp gene cloning protocol
•Clone into phagemid vector engineered for optimized expression of rabbit Fab in bacteria
QC report confirming high quality libraries 2 weeks
Selection •Phage panning
•Fab screening
•Sequencing/H3 grouping
•Fab ELISA/flow cytometry data
•Full VH/VL sequence data
2 weeks
IgG production •IgG cloning
•Transfection in HEK293
•IgG expression/purification
•IgG ELISA/flow cytometry
•IgG validation data
•~1mg antibody
•Affinity measurement (optional)
3 weeks
  From rabbit immunization to validated IgG   ~4-5 months
 

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.

Case Studies

We specialize in traditionally difficult targets. If you desire high affinity, highly specific, and functional rabbit monoclonal antibodies, then our platform is right for you. These type of mAbs can often only be obtained when rabbit immune libraries are paired with the unique selection conditions available only with phage display, and only at Abwiz have we solved the rabbit Fab expression issue in E. coli. We have often gone head-to-head with traditional hybridoma or B cell rabbit monoclonal antibody discovery platforms and have had more success obtaining antibodies with the desired function. With over 400 rabbit immune libraries made against hundreds of targets, below are only some examples of which our platform is capable.

Anti-idiotype antibodies for companion diagnostics

Anti-idiotype antibodies recognize the idiotype of a target antibody, typically a therapeutic drug antibody. They bind within the complementarity determining regions (CDRs) of the target antibody and can serve as useful tools during therapeutic antibody drug development as companion diagnostics. Anti-idiotype mAbs can be used in pharmacokinetic assays to measure either the amount of free drug antibody alone or the amount of antibody-drug complex, or they can be used as reference controls in immunogenicity assays to determine the presence of anti-drug antibodies elicited by patients.

The high specificity of rabbit mAbs makes them excellent candidates for anti-idiotype antibodies, and when coupled with our phage panning methods, our Rabwiz system has successfully generated multiple types of anti-idiotype antibodies. We have successfully created both (1) Type 1 anti-idiotype mAbs specific to a therapeutic drug antibody and (2) Type 2 anti-idiotype mAbs that bind the drug antibody/antigen complex only and not the drug antibody or the antigen alone.

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GPCR and ion channel antibodies

G protein-coupled receptors (GPCRs) and ion channels play major roles in signal transduction and human pathophysiology. Over 30% of all FDA-approved drugs target a GPCR, and ion channels are promising targets in many diseases. Yet these proteins are extremely difficult to target with antibodies, as they are primarily composed of transmembrane domains with small extracellular loops. Targeting the functional site of these receptors can be difficult.

Rabbit antibodies are an excellent source of therapeutic lead candidates for targeting GPCRs and ion channels. At Abwiz Bio, we have successfully generated multiple recombinant rabbit monoclonal antibodies against the GPCRs cannabinoid receptor 1, cannabinoid receptor 2, and protease-activated receptor 1 and against the ion channel Kv1.3, among others. All mAbs recognize the native, cell-displayed receptor.

Our unique immunization and selection strategies use a combination of formats including peptides, recombinant extracellular loop-Fc fusion proteins, transfected or native cells, and RNA. The high specificity of rabbit mAbs and the unique selection conditions of phage display enable us to obtain these otherwise elusive, functional antibodies.

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Highly specific small molecule antibodies

Anti-hapten antibodies can recognize small molecules with exquisite specificity. These antibodies are extremely difficult to create, as they require discrimination against highly similar antigens, which sometimes differ by as little as a single functional group. Yet these antibodies serve as important tools in diagnostic assays or for basic research.

Rabbit monoclonal antibodies are ideal sources of anti-hapten antibodies due to their extremely high affinity and specificity. However, target-specific antibodies can only be obtained when using a competitive selection strategy to eliminate cross-reactive clones from an immune library, as is possible with phage display.

In one example rabbit monoclonal antibody development project, we successfully obtained a rabbit mAb specific to an oxidized nucleoside that showed no reactivity to other highly similar small molecules. These extremely rare mAbs can only be selected by using directed evolutionary methods and would be missed when using screening-based methods such as hybridoma or B cell cloning. Only our Rabwiz system can efficiently display the full rabbit immune repertoire, including Ck1 clones containing extra cysteines.

(small molecule image here)

VHH alpaca antibodies

VHH antibodies, or nanobodies, lack a light chain and are naturally found in camels, alpacas, and sharks. Their small size, high stability, quick clearance from blood, and deep tissue penetration can make them ideal diagnostics or therapeutics for certain diseases. To compensate for the lack of light chain, VHH antibodies have extra cysteines that aid in stabilization. Our proprietary phagemid vector, to our knowledge the only phagemid engineered to efficiently display rabbit Ck1 Fabs that contain extra cysteines, is also ideally suited to display cysteine-rich VHH libraries.

Alpaca VHH immune libraries panned using our vector have resulted in clones possessing extremely high diversity, including CDR H3 sequences ranging from 8 to 27 amino acids in length. In one instance, the top candidates were further characterized as possessing 1 pM KD for a highly flexible loop of complement factor H. By pairing immune VHH libraries with our robust phagemid vector, we can obtain excellent nanobodies for use as diagnostics or therapeutic lead candidates.

(VHH image here)

Antibodies against post-translational modifications

Site-specific antibodies that recognize post-translational modifications (PTMs) are useful tools for the study of important biochemical pathways. With the protein target differing by only a single functional group such as a methyl or phosphoryl group, antibodies recognizing these types of modifications must be extremely sensitive and specific, and thus rabbit monoclonal antibodies are often a common choice.

While obtaining PTM-specific mAbs that work in ELISA against modified over unmodified peptides is relatively straightforward, it can be much more difficult to create PTM-specific mAbs that also recognize native protein in Western blot or cells. As our extensive (catalog of phospho-specific mAbs shows, we are experts in obtaining these highly specific rabbit monoclonal antibodies.

With our phage display selections, we have pioneered a method that uses cell panning on native antigen in combination with peptide panning to elicit anti-PTM mAbs that recognize native antigen and show high specificity in flow cytometry, Western blot, and immunohistochemistry (IHC). These types of highly specific monoclonal antibodies can only be obtained by combining rabbit immunization with the unique selection conditions compatible with phage display, as we have pioneered with our Rabwiz system.

(PTM image here)

Anti-cancer antibodies from cell immunization

Obtaining antibodies against native cancer proteins can be difficult, especially when the cancer proteins are poorly defined or unknown. One approach is to immunize with whole primary cancer cells to extract a recombinant monoclonal antibody specific to a particular cell-displayed protein associated with the cancer. However, given the complex makeup of the cell, antibodies obtained by hybridoma or B cell screening methods often recognize dominant, non-functional proteins.

Using our Rabwiz system, in one example we immunized with a cancer cell line and obtained cell line-specific rabbit mAbs by selection on (1) cancer cells while removing binders against non-cancerous cells or on (2) a cancer cell specific protein antigen. Because our platform is entirely recombinant, DNA libraries can be archived and stored for repeated selection to obtain new candidates once more is understood about a specific target. This is in direct contrast to hybridoma or B cell screening methods, which are a one-shot solution that cannot be repeatedly screened.

(cancer image here)

 

FAQs

Rabbit antibodies are an exceptional source of lead candidates for therapeutic applications and as tools for diagnostic assays for many reasons:

  • Higher diversity within antigen binding regions (light chain CDR diversity as high as heavy chain CDR diversity)
  • 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 rabbit mAbs is straightforward, as they predominantly use a single VH and VK germline 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.

Recently, single B cell cloning and deep sequencing approaches are used for rabbit monoclonal antibody development. However, these methods lack directed evolution/selection steps required to obtain rare, functional clones—instead, each mAb must be tested individually, often recovering only clones recognizing immunodominant, less important epitopes.

In contrast, Abwiz Bio's proprietary recombinant rabbit monoclonal antibody development platform, RabWizTM, combines the power of the rabbit immune response with the large sizes (>1010) and unique selection conditions enabled by 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.

Only when expressed using our proprietrary phagemid vector. 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 GPCRs, ion channels, small molecules, and anti-idiotype antibodies.

The level of antibody diversity that can be obtained depends on the organ used for library construction. 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 against 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 PCR amplification of antibody genes using two separate gene-specific primers. This causes biased and non-specific amplification, resulting in poor coverage of the immune repertoire.

AbWiz Bio has invented a unique method called WizAmpTM (US 9,890,414) that engineers cDNA specifically for antibody genes and uses a single non-gene-specific primer via single primer amplification. This method ensures wide, uniform coverage of the antibody repertoire, including rare clones possessing 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 creating 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 (a service often offered by other companies), the light and heavy chains are most likely randomly paired. However, the light and heavy chain pairing of clones isolated from an immune library is not random at all. After constructing and selecting from hundreds of rabbit immune libraries, we have observed that clones that belong to the same heavy chain clusters based on HCDR3 sequences are always paired with the same light chain clusters based on LCDR3, indicating that native pairing is retained.

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