The Cloning, Optimization, and Validation of Caninized-Phage Monoclonal Antibody Library For Regulation of Cancer Responses in Canus lupus

Cancer has emerged as a major cause of morbidity and mortality in the dog, with an estimated incidence of 1 in 4. Despite advances in conventional chemotherapy and radiation, complete cures for most cancer types remains elusive, the challenge being to develop highly targeted therapies that kill cancer cells but leave normal cells healthy. The same seemingly insurmountable problem holds true for the treatment of human cancer and a big advance in oncology in the past 20 years has been the discovery and application of monoclonal antibodies as novel therapeutic tools to improve cancer treatments in patients. In fact, one of the biggest areas of R and D in the pharmaceutical industry is now in the development of monoclonal antibodies.

What are monoclonal antibodies (MAb's)? These are proteins made by our bodies that form a normal part of a healthy immune system and is the way nature helps to rid our bodies of foreign viruses, pathogenic bacteria, and also minimize disease development. Indeed, one reason why disease develops when we age or why we are more sensitive to viruses when we are older is essentially due to a "broken" immune system. However, scientists have discovered spectacular ways to harness the power of the immune system, by first cloning MAb's and then turning the MAb's on to treat particular diseases. The MAb's then act essentially like "magic bullets" as they can specifically hone in on rogue cells and cure disease. However, a major obstacle in this strategy of using MABs as magic bullets is that the immune system can recognize as foreign any particle that looks in any way different from normal proteins in the body; even a MAb given to patients with disease can paradoxically look foreign and be eliminated from the body before the MAb has a chance to act and elicit a cure.

To overcome this obstacle, scientists in turn have pioneered a way to "tailor" the MAb so that it appears invisible to the body's immune system and can circulate and target disease cells. This tailoring of a MAb to treat humans is called "human-izing" the MAb and this approach provides further hope that complex diseases can be treated in the future. Despite these advances, the treatment of dog cancer, though in principle could be carried out like that in humans, has not been attempted in any significant way using MAb's. The main reason is that "dog-ized" MAb's would have to be used that are not recognized as invaders by the dog's immune system and dog-ized MAb's have not been developed that would greatly facilitate this process. In our research programme, we have developed novel methods to capture and clone the large majority of MAb's that can be made by the dog's immune system. Our application aims to optimize our dog-ized MAb library and validate it by isolating potential dog-ized "magic bullets" that can be used to inhibit specific receptor proteins that are implicated in causing dog cancers. The successful validation of these dog-ized MAb's will lead to clinical trials that would hopefully improve the health and longevity of dogs with cancer, but also produce a road map on how to develop dog-ized MAb's to treat other age related diseases in the dog.

Breeds of the domestic dog Canus lupus suffer from a number of age-related sporadic cancers, including lymphoma and osteosarcoma. Treatments for canine cancer are rather limited. The use of caninized (i.e. "dog-ized") monoclonal antibodies that are tolerated by the canine (Canus lupus) immune system could be used to begin to diagnose and treat canine diseases like cancer. In this application we aim to:
(1) optimize the cloning of separate heavy and light chain framework and variable regions from normal and cancerous spleen tissue to produce a high titer single-domain and single-chain (scFV) phage-antibody library with very high diversity;
(2) begin the selection of caninized single-domain and scFV antibodies in vitro from our canine-phage antibody library towards two specific receptor domains implicated in canine lymphoma, including the clinically validated CD20 receptor and a novel receptor (IFITM5) that was identified from in-house expression screen of canine lymphoma;
(3) optimize the canine single-domain and scFV antibody leads for developing high, medium, and low affinity scFV canine antibodies towards target canine antigen, and
(4) develop and use canine lymphoma cell lines for the in vitro validation of the high, medium, and low affinity scFV canine antibodies in order to link antibody affinity and specificity to biological outcome.
Together, these approaches aim to optimize tools that are specifically used to target canine antigens important for canine cancer, validate our canine-phage antibody library, and open the door for future in vivo animal trials to improve the treatment of canine cancer. This approach also forms a foundation for a general strategy to improve the diagnosis and treatment of a number of age-related canine diseases such as cancer, arthritis, and susceptibility to virus-infections using species-specific monoclonal antibodies.

Developing an inter-disciplinary programme to improve the health and welfare of canines using MAB therapeutics is a fundamental long term goal of this application. The impacts are listed below:
1. Academic impacts. This research can have an impact on UK/European government and policy makers by supporting UK and pan-European funding in a systems biology approach to understand and improve canine health. Various disciplines will be involved including basic science and technology platforms, immune system function and evolution in animals, novel chemical probes and whole body animal imaging, bioinformatics, clinical-veterinary proteomics and transcriptomics, veterinary and animal clinical trials to improve animal health and welfare.
2. Society impacts. Cancer in dogs is a major concern in terms of health and welfare of this species. It is estimated that 1 in 3 dogs will develop cancer in their lifetime. The UK dog population is estimated at around 7 million dogs, and thus cancer represents a major cause of morbidity and mortality. The development of the key reagents and tools in this programme offers the improved diagnosis and treatment of many canine cancer types that reflect the breed-specific canine cancers including, lymphoma, melanoma, breast cancer, and other common cancer types. The impact is higher considering the numbers of dogs that also develop cancer in western societies outwith the UK.
3. Comparative oncology. The field of "comparative oncology" maintains that insights gained into how to treat and cure cancer in one species might form novel approaches for improved treatment of cancer in other animal species. For example, approaches used to treat human cancer can have a translational impact on animal cancer; this is specifically highlighted here where the establishment of human-ized MAB for treating human cancer is used as logic to develop dog-ized antibodies to treat canine cancers. Thus, identifying novel pro-oncogenic targets validated in canine lymphoma cancer (like IFITM5 in this application) could have an impact in novel pathway areas in other animal cancers and in humans.
4. Commercial and pharmaceutical impacts. As Immunosolv have been our collaborators in developing the canine antibody library, we are now in the position to begin to discuss commercialization of our invention describing the generation of the canine-phage antibody library, possibly to develop a spinout company. We also anticipate some of the recipients of this technology would be investors such as Pfizer animal health (who already have an established collaboration with David Argyle to identify novel targets in canine lymphoma) and Canfel therapeutics (canfeltherapeutics.com), as well as veterinary clinicians involved in canine clinical trials.
5. Impacts for the wider international community.
a.Impact on European funding policy. Based on our phage-antibody library developments, we have already been in consultation with veterinarians and scientists in France through INCa (Institut national du cancer (INCa)). Based on our meetings, INCa have established a call to fund up to £2M Euros (for French recipients only) to exploit the canine phage antibody library to improve canine health (cancer).
b.Impact on European Framework networks. Meetings have already taken place with INCa in Paris to plan a European wide canine research consortium to solicit funding begin to use our canine-phage antibody library to diagnose and treat canine disease. A key impact will be on canine clinical trials in Europe and the USA.
c.Impact on comparative oncology. Meetings have already taken place with INCa in Paris and NCRI in London to plan a special session at the 2011 NCRI cancer meeting to highlight to the international community the fact that we are developing a technology platform that can be used to bring together veterinary and human clinical oncologists to drive innovation in their respective fields.