Developing Knockout Rats for Antibody Discovery

Feb 01 2010

PALO ALTO, California—From a scientific point of view, there is little if any reason anyone needs a new platform technology for producing therapeutic antibodies, concedes Roland Buelow, the founder and CEO of Open Monoclonal Technology Inc. (OMT). More than 20 years have passed since the first monoclonal antibody was approved as a drug; since then dozens have come to market worldwide and more are advancing through clinical trials for all manner of disorders. The products themselves, as well as intellectual property related to making them, have earned fortunes for key patent holders and drug developers alike.

Buelow believes that new antibody discovery platforms represent an excellent business opportunity, in part because companies that have waited to pursue drugs in this class can no longer access some of the key technology platforms available earlier on. At the end of 2005, Amgen Inc. acquired Abgenix (now known as Amgen Fremont Inc.) and its XenoMouse platform for generating high-affinity, fully human antibodies in mice. [200510208] Until then, Abgenix had made the technology available to numerous companies. Similarly, Medarex Inc. granted many firms license to use its UltiMAb system before and after going public in 1987. But that channel closed in July 2009 when Bristol-Myers Squibb Co. agreed to acquire Medarex for approximately $2 billion in cash.

Regeneron Pharmaceuticals Inc. still offers license to its VelocImmune antibody production system, Buelow notes, “but their mouse is extremely expensive, and presumably the company is very busy now.” In November 2009 Regeneron for the second time expanded the antibody -development deal it signed with Sanofi-Aventis in late 2007. [200720829] For at least the next five years, Regeneron will receive $160 million annually from this partner, now a 19% stakeholder, to develop and commercialize antibodies.  OMT’s platform will be much like Regeneron’s, with a few exceptions, Buelow contends: OMT’s method relies on transgenic rats rather than mice and he describes the platform as “virgin” or “unencumbered,” meaning that it is available to raise human antibodies against any target. Although early developers of monoclonal antibody technologies sometimes licensed off development rights piecemeal, OMT plans to give open access to select partners and offer exclusive licenses for specific monoclonal antibodies.

“I don’t think anyone has generated an animal-based antibody platform as fast as we have,” he notes. Whereas early companies like Abgenix and Medarex spent seven to 10 years fine-tuning their platforms, Buelow says he has put the main pieces in place in just two. OMT intends to develop transgenic rats with six key genetic traits and so far the start-up has animals expressing five. Buelow insists the sixth and final trait needed to produce human antibodies in rats will soon materialize. Eventually, the final OMT rat expressing a human antibody repertoire will be accomplished through traditional breeding of genetically mutated animals.

OMT’s method for creating rats with inactivated immunoglobulin loci, the genes encoding antibodies, earned a publication in the journal Science in July 2009, spotlighting the creation of the first genetic ” knockout ” rat. Buelow thought the scientific feat of making genetically engineered rats expressing human antibodies would be possible, because he had himself previously made transgenic rabbits through his company Therapeutic Human Polyclonals Inc. (THP), which Roche acquired in April 2007 for $56.5 million cash. [200710052] Buelow founded OMT following THP’s acquisition, and immediately brought aboard Marianne Brüggemann, head of the Antibody Development Laboratory at the Babraham Institute in Cambridge, UK. Brüggemann, along with Michael S. Neuberger, now joint head of the Division of Protein and Nucleic Acid Chemistry at the Medical Research Council in Cambridge, UK, 20 years ago created and patented the first mouse expressing human antibodies. Until 2009 when the patent expired, anyone working with animals expressing human antibodies had to pay a licensing fee, as the patent claim extended to animals of any kind, not just mice.

Both Brüggemann and Neuberger now sit on OMT’s Scientific Advisory Board, and both consult for other companies including Crescendo Biologics Ltd., a start-up company that aims to develop therapeutic antibody fragments. Even Buelow was surprised at how quickly he was able to actualize his idea for creating a transgenic rat capable of producing human antibodies. “Generation of knockout rats was a breakthrough for us, and we accomplished it way faster than anticipated,” he says. Buelow says he came up with the idea to use restriction enzymes known as zinc finger nucleases (ZFN) to mutate the rat’s own immunoglobulin genes. The technique had been used to knockout genes in certain cell lines, and in the fruit fly Drosophila, but never before in a mammalian embryo.  “The concern was that when you put a ZFN into an embryo either it wouldn’t cut because the genetic loci would be inaccessible, or it would cut too late and result in a mutant animal that would not transmit the mutation to its offspring. The ZFN could have also proved too active and caused an abortion of the embryo,” Buelow explains. As it turned out, none of these concerns bore out, and he says, “The method is very effective at knocking out the genes we want to knock out.” Buelow says he is thankful to Sangamo BioSciences Inc. for agreeing to make zinc fingers to his specifications, so they would cut out just what he wanted. Now that OMT has proved the concept, Sigma-Aldrich Corp. has taken a license from Sangamo to make and sell ZFNs as research reagents.

The knockout rats were created by injecting messenger RNA encoding ZFNs into the nuclei of rat embryos that were then transplanted into a mother rat. In the embryo, the ZFNs cut the embryo’s genomic DNA. In some embryos the cut in the DNA was repaired and resulted in a mutation inactivating the target gene. To create rats capable of expressing human antibodies, rat embryos were injected with DNA encoding different components of human antibodies. In some embryos, the injected DNA was incorporated into the rat genome and the newborn transgenic animals expressed human immunoglobulins. Breeding of knockout rats with transgenic rats will result in rats that express human antibodies instead of rat antibodies. “We are breeding a lot of animals to combine genetic traits in one animal,” Buelow declares, explaining that when the procedure works as intended, young rats inheriting genes from both parents “are fully immunocompetent. They look normal and make human antibodies.”

Actually, as with other technologies for producing “human” antibodies in mice, only the binding portion of the antibody (which looks like the V atop the stem of the letter Y) has a fully human idiotype. The constant region (the stem of the Y) is still formed as it is in true rodent antibodies, so drug manufacturers replace this portion with a human constant region, and then express the antibody in cell lines. OMT will do the same.  Buelow notes that sometimes, if a mouse antigen looks almost identical or very similar to a human antigen, it is difficult to immunize the mouse and get a good immune response. This is because the mouse has difficulty mounting an immune response against one of its own proteins, because they are perceived as “self.” In such cases, the antibodies are weak in terms of their binding ability and therefore of no therapeutic use. To minimize such difficulties in OMT rats , the company is working with “out-bred”  rats versus the usual in-bred laboratory rats , where every creature is genetically identical to its brothers and sisters. Buelow says he expects the greater diversity among immunized rats will yield a greater variety of antibodies and increase the potential for identifying tight-binding, clinically effective monoclonals.

Antibody variety is a subject to which Buelow has given much thought through the years, and which he is eager to pursue clinically. He points out that when he started his previous company Therapeutic Human Polyclonals, he did so in hopes of developing mixtures of antibodies, or “polyclonals” in transgenic rabbits. While young venture capitalists “looked at us like we were crazy,” Buelow says some older VCs knew that serum from animals injected with antigens had been used to treat certain diseases 100 years ago, and had even been tested against antibiotics in the 1940s. Even today, animal antibodies are still used as anti-venom, because venoms contain more than one toxin and polyclonal preparations are needed to counteract them. Back in 2000, Buelow recalls, “nobody wanted to hear about polyclonals,” and THP eventually abandoned those efforts and sold its rabbit technology to Roche as a monoclonal production platform.

Now, a decade later, Buelow believes the time could be right for companies like OMT to develop monoclonal antibody cocktails as therapeutics. By now, he says, many people recognize that combinations of antibodies specific for several different targets are more potent than a single monoclonal specific for one target. One example is the combination of monoclonals against CD-19 and/or CD-22 receptors on cancer cells with rituximab (Rituxan), an antibody specific for CD-20 receptor target. The cocktail works better than Rituxan alone. “The community has realized that if that’s what you need to do, the FDA will come around,” he notes, adding that the regulatory agency has already allowed Crucell NV’s mixture of two monoclonal antibodies to be tested in humans, post-exposure to rabies. The agency also permits sale of specialized intravenous immunoglobulin (IVIG) preparations made by immunizing human blood donors.
Beyond the growing stacks of clinical evidence, commercial factors are now also well aligned to support development of therapeutic monoclonal mixtures, Buelow declares—at least for OMT. “If you had to pay licensing fees and royalties on each antibody , mixtures would not be feasible. But we want to take advantage of the fact that we own the platform, so we intend to develop monoclonal cocktails.”

In the near term, however, OMT has more basic challenges to face. For one thing, the start-up has yet to produce a transgenic rat with all six of the genetic traits required to produce human antibodies. Buelow is confident that it’s just a matter of time, and a short matter at that, till this task is accomplished. Once it is, OMT will begin pursuing simple, initial partnerships focused on single targets. Just as Medarex, Abgenix and others did years ago when their technologies were new, OMT needs to demonstrate that its genetically engineered rats do, in fact, make antibodies that perform as well as anticipated.

OMT presently has just two employees, and about 20 other individuals working as contractors in the UK, US, France, Germany, Hungary and Switzerland. When the platform is completed, OMT will ramp up staffing.

Essex Woodlands Healthcare Ventures provided OMT’s Series A funding in 2008; Buelow declines to disclose the amount invested but says the money should last the company until 2011.

By Deborah Erickson
START UP, February 2010
Open Monoclonal Technology Inc.
Phone: (650) 224-6835
Web Site:
Contact: Roland Buelow, PhD, CEO
Industry Segment: Biotechnology
Business: Monoclonal antibody platform in rats
Founded: April 2007
Founder: Roland Buelow
Employees: 2
Financing to Date: Undisclosed Series A
Investors: Essex Woodlands Health Ventures
Board of Directors: Roland Buelow; Richard Murdock; Ron Eastman (Essex Woodlands Health Ventures); Thomas D. Kiley
Scientific Advisory Board: Ignacio Anegon, MD (INSERM); Marianne Brüggemann, PhD (Babraham Institute); C. Geoffrey Davis, PhD (Angelica Therapeutics Inc.); Michael S. Neuberger, PhD, FRS (Medical Research Council); Andrew M. Scharenberg, MD (University of Washington, Seattle)