IgE/IgG targeting of cancer antigens ErbB-1 & ErbB-2 in dog

Most of the antibodies applied for passive immunotherapy in cancer patients belong to the IgG class, ignoring the potential capacity of other immunoglobulin isotypes for anti-cancer therapy, such as IgE. This study is intended to create the necessary tools and methods needed for examining the anti-tumor efficacy of IgG versus IgE antibodies side by side, in vitro and in vivo by a comparative oncology strategy. The major aims of the study thus are: a) Characterization of canine ErbB-1 and ErbB-2. Our preliminary experiments identified ErbB-1 and -2 as being highly conserved among human and canine species (dog, Canis domesticus). However, the prevalence and biological relevance of ErbB-1 and ErbB-2 expression in canine breast cancer is today not known. For the design of targeted therapies, we aim to complete this information by histological and gene expression profiling. Further, we aim to analyse the cellular and signalling effects of antibody-mediated targeting of the canine versus human ErbB-1 and ErbB-2 homologues. b) Construction of caninized anti ErbB-1 cetuximab and ErbB-2 trastuzumab antibodies. ErbB-1 and -2 are of great relevance in human targeted cancer therapy, but are not exploited for cancer therapies in dogs. For targeted therapy in dogs, "caninization" of trastuzumab and cetuximab antibody will be needed. We aim thus to generate canCetuximab and canTrastuzumab IgG. As the biological relevance of targeting the canine homologues has not been documented so far, we plan to stringently test their in vitro and in vivo effects in comparison with the outstanding anti-cancer properties of the original antibodies. c) Investigation of IgE anti-cancer immunity in the canine system. Numerous studies indicate a great potency of IgE-targeted anti-cancer therapy. For proof of concept studies, therefore also canCetuximab and canTrastuzumab of the IgE species will be generated. We plan head-to-head comparison with the canine and human IgG isotype by in vitro and in vivo tumor transplant studies to provide the basis for clinical applications in dog patients. Taken together, this project will contribute to the understanding of ErbB biology in mammalians other than the human species, and simultaneously develop and preclinically evaluate novel immunotherapeutics for dog and human patients suffering from cancer.

Nearly every second dog develops cancer from the age of ten years onward. As in humans, cancers in dogs have complex causes. The interaction of the environment, food, and genetic disposition are the most well known factors. Today nearly all methods of human medicine are basically available for dogs with cancer, but this was not true of cancer immunotherapy so far. So-called cancer immunotherapy - which is the treatment of tumors by the use of antibodies - has been established and used very successfully in human medicine for about 20 years. Since cancer cells bear very specific antigens on the surface, the corresponding antibodies bind to these molecules and thus inhibit tumor growth. Cetuximab is a prototype of these antibodies and directed against epidermal growth factor receptor or EGFR, a receptor frequently found on human tumor cells. The mechanism that becomes effective is a destructive signal sent by the antibody to the inside of the cancer cell and initiates its death. In a second mechanism, the immune system of the patient also destroys the "marked" tumor in a more efficient way. Results We discovered that EGFR is nearly 100 percent identical with the EGF receptor in dogs. In human medicine EGFR is frequently used as the target of cancer immunotherapy because many cancer cells bear this receptor on their surface. The so-called anti-EGFR antibody binds to cancer cells and thus triggers the destruction of the cells. Due to the high similarity of the receptor in humans and dogs, we anticipated that this type of therapy should work well in dogs too. The binding site of the antibody to EGFR in man and dogs differs only in respect of four amino acids. To ensure best possible binding of the antibody to cancer cells in dogs, the human antibody had to be trimmed to "dog" in the laboratory. In human medicine this process is known as the "humanization" of an antibody, hence corresponding to caninization for dogs. The antibody originally produced in the mouse has to be adjusted to the species for which it is used. We replaced the corresponding elements in the humanized antibody with elements from the dog. In experiments on dog cancer cells in the laboratory it was found that the newly developed antibodies did, in fact, bind to canine cancer cells with great specificity. It is thus expected that greater tolerability of the caninized antibody, can225, will be achieved in treatment and diagnostics of canine cancer. The study was published in Molecular Cancer Therapeutics, a journal of the American Association of Cancer Research. This may indicate that our attempt, to acquire information from dog- for human cancer patients, has been appreciated by the (human) AACR. Can225 in the meanwhile has been further developed to a "theranostic" (therapy and diagnostics), in collaboration with the radio-pharmacy in The General Hospital Vienna, AKH. First results with mice are presently evaluated. In a parallel project line can225 has been modified from the usual IgG immunoglobulin class to an IgE variant. IgE is known from allergies, but it has a very high potential to kill cancer cells. We are presently purifying or new can225-IgE and in a collaboration with Kings College, London, we apply a method that allows to enrich sufficient quantities for lab- and clinical studies. Only a head-to-head comparison of can225-IgG and IgE will be able to provide the answer which of the classes will be more successful. Taken together, this project contributed to the world first canine immunoglobulin for dogs suffering from cancer. Thereby we hope having paved the way for improvements in cancer therapy and diagnosis not only for dogs, but also for human patients.