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Monoclonal antibodies (mAbs) provide effective therapies for previously intractable diseases. Approximately 60 mAbs have been approved by the FDA during the past 20 years, and during the past several years 5–6 new antibodies have been approved annually.
The therapeutic antibody space is growing rapidly with several hundred mAbs currently under development. Historically, antibody discovery for therapeutics relied on derivation of mAbs using phage display technology and making hybridomas from B cells of human or murine origin. Both approaches have theoretical and practical limitations, although the in vivo approach has yielded most of the approved therapeutic mAbs.
Immunized mice have provided B cells for mAb discovery since Milstein and Kohler were awarded a Nobel Prize for developing mAb technology in 1975. Beginning in the 1990s, strains of transgenic mice (e.g., Xenomouse) in which the murine immunoglobulin genes are replaced with their human counterparts have been available. The advantage of these mice is their production of fully human sequence antibodies; this attribute obviates the need to “humanize” mAbs derived from wild-type mice to reduce their immunogenicity.
Since 2010, the OmniRat® has provided another source of fully human sequence mAbs for therapeutic antibody discovery. Because mice and rats are different species of rodents, they produce different antibodies. The realization that divergent species produce antibodies that cannot be obtained from mice or humans was recently expanded to include genetically engineered chickens that are licensed by Ligand Pharmaceuticals under the OmniChicken® trademark. Like their murine counterparts, these transgenic chickens produce fully human sequence mAbs.
OmniChicken, OmniRat, and OmniMouse® make up Ligand’s OmniAb®️ platform, along with OmniFlic® and OmniClic™, which are engineered rats and chickens, respectively, that produce human sequence antibodies with a common light chain for bispecific applications. Ligand is uniquely positioned to offer five platforms from three species for discovery of therapeutic mAbs under one OmniAb license. There are currently 10 OmniAb derived drugs in clinical trials.
The OmniChicken platform
“Chickens diverged from humans about 300 million years ago; rodents and humans diverged only about 100 million years ago,” explains Marie-Cecile Van De Lavoir, DVM, PhD, vice president of operations at Ligand Pharmaceuticals. “Due to genetic drift as the species diverged, chickens will recognize protein sequences in human antigens that are shared between mice and humans (but differ from chickens) as well as those that differ between chickens and humans. Even in highly conserved proteins there will often be sufficient differences between chicken and human orthologs to promote a robust immune response”.
According to Van De Lavoir, “the chicken host leverages evolutionary distance to enhance the epitope coverage of antibody panels that we generate. As we are fond of saying, antibody discovery in OmniChicken is ‘powered by evolution.’”
“We often see OmniChicken providing more epitope coverage than rodents for any given human target,” notes William Harriman, PhD, vice president of antibody discovery services at Ligand Pharmaceuticals. “One example that comes to mind is BDNF (brain-derived neurogenic factor); the protein sequence is 99% conserved between mice and humans. Prior attempts to raise antibodies against human BDNF in rodents failed. Since chicken BDNF is only 91% conserved with the human ortholog, we were able to raise titers in OmniChicken and generate antibodies.”
Partners access OmniChicken for their antibody campaigns directly through Ligand. Working in conjunction with their partners, the team at Ligand develops an immunization strategy for each antigen and immunizes animals held in their avian
vivarium. “We believe this is a much more cost-effective solution for our partners than building a chicken vivarium from scratch, getting licensed and certified, and training staff in the art of chicken husbandry,” says Harriman.
Post immunization, partners can utilize Ligand’s antibody discovery services, or they (or their CRO’s) can receive tissues or B cells from immunized OmniChickens and mine those repertoires using their own processes, including phage/yeast display, single B-cell cloning, and/or NGS. “Some partners have already built up internal capabilities that they want to use in conjunction with OmniChicken,” adds Harriman.
Complementary OmniRat and OmniMouse platforms
In addition to the OmniChicken platform, Ligand also offers OmniRat and OmniMouse platforms for mAb discovery, as previously noted. The rodent platforms are ideal for when the target is not highly conserved and the partner desires to control all aspects of the discovery process from immunization to antibody recovery. The rodents can be shipped directly to a partner facility or to an approved CRO. All three of the models have been engineered to improve the ease of generating antibodies for therapeutic applications.
“We have the intellectual property rights to all three species, offering convenient, one-stop shopping for our partners. Other platforms are available from other companies, but Ligand is the only one with freedom to operate in all three species,” says Harriman.
All OmniAb platforms are designed to express human immunoglobulin genes. This prevents development of an anti-drug immune response in patients who are treated with antibody-based therapies. Often, the portion of the antibody that generates the anti-drug
immune response is a portion of mouse immunoglobulin. With careful planning, Ligand has developed mouse, rat, and chicken platforms in which this issue is addressed by the replacement of endogenous sequence with their human counterparts.
Relationship with partners
When working with Ligand, the client relationship is developed as a partnership to meet the individual needs of each collaboration. “Often clients turn to Ligand for help with their more problematic targets,” points out Harriman.
“It is an interactive process that Ligand uses in assisting our partners. We aid our collaborators in tailoring the approach to antibody discovery by offering our expertise during the entire process. The Ligand team can aid in optimizing the immunization process, the initial selection of candidate antibodies for validation, and the initial optimization of the antibodies,” says Van De Lavoir. “It is very much an interactive process that begins with defining the goals of the campaign. Our experts work with our partners to modify immunization and screening schemes as the immune response is monitored and the panels of mAbs emerge.
“Typically, there are only one or two iterations of the research plan before panels of antibodies that meet the design goals are provided to our partners. The program ends when our partners have the mAbs they need to advance into in vivo studies.”
Ligand offers a wide range of services to pharma companies both large and small. “A partner might come to us for a chicken program after conducting conventional campaigns in rodents,” explains Harriman. In these cases, our job is to expand the breadth of the mAb panel using the OmniChicken platform.
Cross-reactivity: Another advantage of the OmniChicken platform
In relative terms, humans and mice are more closely related than chickens are to either humans or mice. The consequence of the phylogenetic relatedness of mice and humans is shared sequences in some portions of antigens of therapeutic interest. In these cases, chickens will frequently generate mAbs that recognize a shared epitope on the target antigen. These pan-mammalian antibodies are important to Ligand’s partners because they can be used to assess the therapeutic potential of candidate mAbs in mouse models of disease. Previously, surrogate mAbs would have been used to gain an indirect assessment of the therapeutic candidate and therefore, drug developers appreciate the ability to by-pass this step in the drug discovery process.
“In one case, our partner had a rodent model of their disease of interest. To validate the antibody in the model animal, a cross-reactive protein that recognized both the human and the rodent versions was imperative,” says Harriman. “Using the OmniChicken technology, we were able to help them find a cross-reactive antibody.”
“Bottom line: essentially every time we generate panels of mAbs against a human target, at least some members of the panel will also recognize the murine homolog,” adds Van De Lavoir.
How are antibodies isolated from OmniChicken?
Chickens are not amenable to hybridoma generation. Hence, over 10 years ago, Ligand developed a proprietary technology to screen single B cells for antibodies of interest. This unique method, developed by Harriman and Shelley Mettler-Izquierdo, PhD, who serves as the director of antibody discovery services at Ligand, was designated as the Gel Encapsulated Microenvironment (GEM) assay.
The GEM assay is uniquely positioned as the only single B cell screening technology that simultaneously assesses specificity and, in some cases, biological activity of therapeutic candidates within the population of B cells harvested following immunization.
Furthermore, the candidates are identified before sequencing to obviate the time, effort, and expense of working up mAbs that do not meet the goals of the campaign. In addition, the GEM assay is designed to identify rare mAbs in the population whereas competing technologies are often directed at the most frequent mAb generated following immunization.
“Once the chickens are challenged with the target of interest, we look at serum titers and do an initial screening of the polyclonal response by ELISA or FACS. Based on serum titers, we select an OmniChicken producing interesting antibodies. Next, we remove the chicken’s spleen, which yields two to three billion spleen cells. About 100 million of these cells are used in the initial analysis; the remainder are frozen down in 20 to 30 vials of 100 million cells each,” explains Harriman. “The remaining frozen cells can later be mined for additional target-specific antibodies with slightly different characteristics, if needed.”
To evaluate the cells for desired characteristics, spleen cells are isolated into droplets using a proprietary oil emulsification technique. Each droplet in the emulsion is about 150 microns across with (on average) one cell per droplet. Harriman points out that “in each droplet there are colored beads coated with the antigen of interest, allowing selection of droplets based on the color profile on the beads. This allows us to move straight to a primary B-cell repertoire.”
Beads of different colors coated with different proteins bound to them can be used to further screen for B cells producing mAbs that recognize specific forms of the antigen.
“We can also incorporate whole cells expressing receptors of interest into the GEMs during the emulsion step. By incorporating cells expressing the antigen in situ and coating beads with different isoforms, the GEM assay allows us to isolate highly specific mAbs. We use a big, open well about the size of a cell phone to screen for and pick clones of interest in the droplets,” explains Harriman.
“The droplets containing clones expressing the desired color profile are recovered to isolate the top one or two hundred candidate mAbs from 50–100 million spleen cells contained in the GEM preparation. To date, the power of the GEM assay to concentrate the pool of candidates by 5–6 orders of magnitude in a single experiment is unmatched in the field. The pool of candidate mAbs is then sequenced and expressed in small amounts to verify that they meet the design goals of the campaign.
“Although the OmniChicken and GEM assay is our ‘in-house’ workhorse, we are always conscious of our partner’s unique circumstances. If they choose to use OmniRat and/or OmniMouse and conduct a campaign using their own processes, we are happy to accommodate them. We structure our collaborations to meet their needs and capabilities.”
As described above, there are many advantages to employing OmniChicken for antibody discovery.
“Ligand has tapped into a previously underutilized antibody repertoire to create efficacious mAbs,” summarizes Van De Lavoir. “There are now many technologies built around both in vitro and in vivo systems for therapeutic antibody discovery. At Ligand, we encourage our collaborators to think outside the box and consider the attributes of the most recent advances at Ligand. We believe that by offering the OmniRat, OmniMouse, and OmniChicken platforms we can better serve our partner’s needs and move programs from concept to reality more quickly.”