It is critical we achieve a better understanding of the canine immune system, meaning what “makes it tick,” or fail, in the case of cancers, such as osteosarcoma. "Running behind humans” is the title of a manuscript I drafted about a year ago that attempted to summarize what is known in the field of immunotherapy for dogs. Since then, this topic has been at the center of other studies, and was a focus of the midyear conference of the Veterinary Cancer Society.2 However, it is fair to say the distance between the two “running partners” still remains the same, if not wider. More now than ever before, immunotherapy dominates headlines when it comes to cancer treatments in humans, exciting oncologists of the possibility of finally having an alternative to conventional chemotherapy and radiation. The question remains: Shouldn’t veterinarians expect immunotherapy to follow a similar trend by increasingly becoming an option for canines with cancer, too? This brief review is not an exhaustive discussion on immunotherapy for canine lymphoma and osteosarcoma. (There are excellent articles offering in-depth analysis of both.3,4) Rather, it points to the challenges dogs and their owners face regarding access to novel immunotherapies similar to human patients. Stumbling blocks There are many reasons why development of immunotherapies in dogs has been slow. Although cancer, and in particular lymphoma and osteosarcoma, is frequent in dogs, just one percent of owners have pet insurance; those who don’t are willing to spend only a limited amount of money to treat their dog, especially when initial treatment results are not as dramatic as in humans. Let’s keep in mind that treatment with monoclonal antibodies (mAbs) can cost several thousands of dollars per cycle, while newer cellular immunotherapies, such as with chimeric antigen receptor- (CAR-) engineered T-lymphocytes, at least in humans, can easily reach the $350,000 mark. Given these drawbacks, the main thrust for developing immunotherapy for dogs is in the area of “tumor vaccines.” A review of the American Veterinary Medical Association’s (AVMA’s) Animal Health Studies Database of ongoing immunotherapy studies for cancer shows the vast majority refer to cancer vaccines,5 which are often generated from extracted tumor tissue. Alternatively, antigens from human tumors that rely on cross-reactivity (e.g. Oncept)6 are another option. Some veterinarians offer intratumor or intradermal injection of immune-stimulatory cytokines, such as IL-12, often by electrogene therapy.7 Do any of these vaccines really work or do they mostly satisfy the owner’s desire that some form of immunotherapy is offered to their dog? Many studies have documented the fact the immune system of patients with cancer is deeply dysfunctional. For example, cancer cells produce exosomes, which are tiny vesicles measuring 100 to 200 nm. These circulate in the blood, carrying and releasing molecules (e.g. TGF-beta) that are known to paralyze immune cells.8 Moreover, the tumor’s microenvironment is cluttered with cells and cytokines that create an inhibitory “landscape” for immune cells. Tumor cells also change over time, escaping the cytotoxic effect of immune cells. They may initially express a defined neoantigen but over time express several antigens, thereby escaping any therapy directed against the initial neoantigen. Such antigen escape is well-known in humans, a result of treatment with the monoclonal CD20 antibody or CD19 chimeric antigen receptor (CAR) T cells.9 Considering these challenges, it is critical we achieve a better understanding of the canine immune system, meaning what “makes it tick,” or fail, in the case of cancer. Unfortunately, we are working with an incomplete understanding of how cells in the canine immune system and their mediators work, as well as inadequate comprehension of suppressive factors of the tumor microenvironment. Compared to mice, an insufficient number of reagents and assay systems have been developed for canines that would allow identification of relevant immune cells, including natural killer (NK) cells, T-regulatory cells, and myeloid-derived suppressor cells in the blood or the tumor microenvironment. In fact, NK cells are considered the main effector cells in (human) mAb treatment. Unfortunately, NK cells in dogs are poorly defined.10 What treatment options are available? Many immunotherapy studies in dogs lack convincing outcome data when they compare a new treatment with historical controls that often were done in different breeds and age ranges, and include other confounding factors. Single-case reports can be convincing when they are properly documented and can be clearly linked to the effect of the immunotherapy agent. Realizing the limitations of enrolling larger numbers of dogs in a more randomized fashion, even a Simon 2 stage design for a controlled clinical trial, could have some convincing power, but those are rarely found in the veterinary literature. Overall, survival is a poor end point, as dogs will get other treatments once the experimental therapy has failed, while disease-free survival requires proper documentation, often including more advanced and frequent imaging. With these more general comments in mind, what are the immunotherapy options for dogs with lymphoma and osteosarcoma, two of the most common canine cancers? Lymphoma Most frequently, initial disease control in dogs with lymphoma is achieved with chemotherapy. In contrast to human lymphoma, treatment with canine-specific mAbs against the CD20 B-cell antigen and the CD52 T cell lymphoma antigen lack persuasive results.11,12 Human mAbs do not cross-react with canine lymphoma cells. This lack of convincing efficacy of mAbs in canine lymphoma is likely related to how these antibodies kill their target, meaning their Fc “tail” binds to corresponding Fc-receptors on NK cells and macrophages, which then release cytotoxic molecules (e.g. perforin, granzymes, and caspases) that kill tumor cells. This is how human mAbs, such as Rituxan and Herceptin, eliminate cancer cells. Unfortunately, canine immunology has not been able to optimize the effect of mAbs, largely due to the fact the roles of NK cells and macrophages are poorly characterized in dogs. There is some “buzz” Elanco’s anti-CD20 mAb may have some efficacy in treating canine B-cell lymphoma. Recently, mAbs inhibiting/blocking checkpoint molecules (i.e. PD-1 and CTLA-4) on immune cells (especially T cells) have shown in humans to “take the brakes off” and allow immune cells to attack malignant cells that express the “do not eat me” ligands of the PD-L1 family.13 In humans, studies involving mAbs against the checkpoint receptor PD-1 have shown encouraging results in some lymphoma types, although not as dramatic as in melanoma, lung cancer, and bladder cancer.14 This divergent effect of checkpoint inhibitors among various cancers is believed to be due to the rate of somatic mutations of different cancers. The study by Coy et al15 showed only approximately 25 percent of canine lymphocytes express PD-1. This relatively moderate expression of PD-1 could explain why the only clinical study that looked at a canine PD-1 mAb in dogs with oral melanoma showed a (partial) response in one out of seven patients.16 However, larger trials are underway (Merck Animal Health) that should better define the role of these antibodies in the treatment of canine cancers. In human medicine, the infusion of a patient’s own (autologous) CAR-engineered lymphocytes has shown positive results in patients with relapsed lymphoblastic leukemia and lymphoma.17 The lymphocytes are obtained from the patient by leukapheresis, and are then genetically manipulated in the lab to express a CAR that will recognize the CD19 lymphoma antigen. These cells subsequently undergo expansion in culture for several days to arrive at bigger numbers. The main thrust for developing immunotherapy for canine cancers is in the area of “tumor vaccines.” Not every patient qualifies for this treatment, and side effects—which are mostly related to a release of cytokines from the patient’s body cells in addition to some neurological side effects—are not uncommon. As mentioned previously, the treatment for human patients costs approximately $350,000. Due to the logistics, the cost, and incomplete knowledge regarding appropriate tumor antigens in dogs, this treatment has not been developed for dogs with lymphoma with the exception of one study.18 Researchers treated one dog with CAR-T cells directed against the CD20 lymphoma antigen, resulting in only a temporary partial tumor response. Scientists at the University of Pennsylvania are engineering human T cells as a source for CAR-T cells for dogs by using clustered, regularly interspaced short palindromic repeats (CRISPR) technology to eliminate human leukocyte antigens (HLA) and the T cell receptor. Unfortunately, there is a problem with this approach: the lack of HLAs can trigger NK cells from the dog that recognize “non-self” to attack the T cells. Autologous lymphocyte therapies for lymphoma, which are less complex than CAR engineering, are currently being explored by academia and veterinary immunotherapy companies.19 One such therapy is offered by Aurelius Biotherapeutics, which collects 20 ml of blood from dogs with lymphoma to develop the therapy. That said, no details are available as to how the lymphocytes are activated and expanded—more conclusive outcome data are awaited. Osteosarcoma Osteosarcoma is about 75 times more common in dogs than in humans. Standard therapy generally includes amputation and radiation. Due to the high potential for microscopic metastases early on, tumor progression is often rapid and chemotherapy has limited efficacy. No monoclonal antibodies against tumor antigens or checkpoint inhibitors are available. About 40 percent of canine osteosarcoma cells express the Her-2 (Herceptin) antigen, which is common in some human tumors, especially breast cancer. Despite 90 percent homology, the human mAb (Herceptin) is ineffective in canine Her-2 positive cancers. However, when researchers at MD Anderson Cancer Center engineered dog lymphocytes to express either a human or a canine Her-2 CAR, they showed that both CARs were able to recognize and kill canine osteosarcoma cells. This suggests CAR-mediated recognition and killing mechanisms are different from those of mAbs.20 ELIAS Animal Health is developing an autologous lymphocyte infusion approach. Prior to lymphocyte collection from the dog, the animal receives a vaccine that is prepared from the excised tumor material. The vaccine is then given intradermally several weeks before lymphocyte collection. Preparation of the vaccine is proprietary information. The collected lymphocytes are expanded in the presence of human IL-2 and then infused into the patient, usually a couple of weeks later. First data are promising in that, besides having no significant side effects, the treatment seems to prolong survival. Based on the positive data, ELIAS is seeking USDA approval, which would make it the first approved cellular therapy for dogs. A novel immunotherapy developed at the UPenn and currently tested at various veterinary centers in the U.S. takes a different approach. It combines the “Coley toxin” observation from more than 100 years ago with modern cell engineering technology. In the early 1890s, William Coley reported accidental acquisition or intentional inoculation of patients with the bacterium responsible for Streptococcus infections could result in regression or delayed recurrence of various cancers. Based on that observation, Coley developed a vaccine consisting of two killed bacteria, Streptococcus pyogenes and Serratia marcescens, which was named “Coley’s toxins.” It was shown to have some benefit in the treatment of a variety of tumor types, including bone sarcomas. The UPenn group engineered the Listeria bacterium to express a chimeric human Her-2 protein, at the same time removing harmful genes from the bacterium.21 The rationale is the dog’s immune system attacks the bacteria, “discovers” the Her-2 antigen, and gets cross-primed for the osteosarcoma antigens. Encouraging results from a pilot study in 18 dogs have prompted a multicenter randomized study to hopefully show efficacy. Conclusion The most successful immune therapies in humans have occurred in the field of CAR therapy and with monoclonal antibodies against checkpoint molecules expressed on immune cells. However, CAR therapies are logistically challenging to prepare and administer, and checkpoint inhibitors have so far been inconclusive due to insufficiently defined checkpoint mAbs. The cost of therapy also is a significant drawback. Moreover, it is unlikely that single agent immunotherapy will control cancer in dogs, although a combination approach based on scientifically sound observation could be the answer. Such therapies could include rather inexpensive components, such as low-dose metronomic chemotherapy to induce immunogenic cell death, local low-dose irradiation to stimulate an anti-inflammatory tissue response combined with vaccination, and possibly infusion of immune cells. References 1 Klingemann H, Immunotherapy for dogs—Running behind humans. Front Immunol 2018; 9: 133. doi: 10.3389/ mmu.2018.00133 2 http://vetcancersociety.org/midyear-conference/ 3 Regan D, Dow S. Manipulation of innate immunotherapy for cancer therapy in dogs. Vet Sci (2015) 2:423–39. doi:10.3390/vetsci2040423 4 Wycislo KL and Fan TM. The immunotherapy of canine osteosarcoma: A historical and systematic review. J Vet Int Med 2015; 29:759-69 5 Enter ‘tumor’ in the search field at https://ebusiness.avma.org/aahsd/study_search.aspx 6 Ottnod JM, Smedley RC, Walshaw R, Hauptman JG, Kiupel M, Obradovich JE. A retrospective analysis of the efficacy of Oncept vaccine for the adjunct treatment of canine oral malignant melanoma. Vet Comp Oncol 2013; 11:219–29. doi:10.1111/vco.12057 7 Pavlin D, Cemazar M, Cor A, Sersa G, Pogacnik A, Tozon N. Electrogenetherapy with interleukin-12 in canine mast cell tumors. Radiol Oncol (2011) 45(1):31–9. doi:10.2478/v10019-010-0041-9 8 Szczepanski MJ, Szajnik M, Welsh A, Whiteside TL, Boyiadzis M. Blast-derived microvesicles in sera from patients with acute myeloid leukemia suppress natural killer cell function via membrane-associated transforming growth factor-b1. Haematologica 2011; 96:1302-09. doi:10.3324/haematol.2010.039743 9 Jackson, HJ, Brentjens RJ. Overcoming antigen escape with CAR-T cell therapy. Cancer Discov 2015; 12: 1238-40. doi: 10.1158/2159-8290 10 Michael HT, Ito D, McCullar V, Zhang B, Miller JS, Modiano JF. Isolation and characterization of canine natural killer cells. Vet Immunol Immunopathol (2013) 155(3):211–7. doi:10.1016/j.vetimm.2013.06.013 11 Rue SM, Eckelman BP, Efe JA, Bloink K, Deveraux QL, Lowery D, et al. Identification of a candidate therapeutic antibody for treatment of canine B-cell lymphoma. Vet Immunol Immunopathol (2015) 164(3–4):148–59. doi:10.1016/j.vetimm.2015.02.004 12 Rodriguez C, Hansen G. Bioavailability and safety of caninized anti-CD52 monoclonal antibody in dogs with T cell lymphoma. Proceedings: 34th Annual Veterinary Cancer Society Conference. St Louis, MO: (2014) 13 Ribas A & Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science 359; 2018: 1350-55. DOI: 10./1126/science.aar4060 14 Merryman RW, Armand P, Wright KT, Rodig SJ. Checkpoint blockade in Hodgkin and non-Hodgkin lymphoma. Blood Adv 2017; 1: 2643-54. DOI: 10.1182/bloodadvances.2017012534 15 Coy J, Caldwell A, Chow L, Guth A, Dow S. PD-1 expression by canine T cells and functional effect of PD-1 blockade. Vet Comp Oncol 2017; 15: 1487-1502 16 Nemoto, Y Shosu K, Okuda M, Noguchi S, Mizuno T. Development and characterization of monoclonal antibodies against canine PD-1 and PD-L1. Vet Immunol Immunopathol 2018; 198: 19-25 17 June CH, O’Connor RS, Kawalekar OU,Ghassemi S, Milone MC. CAR-T cell immunotherapy for human cancer. Science 359; 6382: 1361-65 DOI: 10.1126/science.aar6711 18 Panjwani MK, Smith JB, Schutsky K, et al. Feasibility and safety of RNA transfected CD20 specific chimeric antigen receptor T cells in dogs with spontaneous B cell lymphoma. Mol Ther (2016) 24:1602. doi:10.1038/mt. 2016.146 19 O’Connor CM, Sheppard S, Hartline CA, Huls H, Johnson M, Palla SL, et al. Adoptive T cell therapy improves treatment of canine non-Hodgkin lymphoma post chemotherapy. Sci Rep (2012) 2:249. doi:10.1038/ srep00249 20 Mata M, Vera JF, Gerken C, Rooney CM, Miller T, Pfent C, et al. Toward immunotherapy with redirected T cells in a large animal model: ex vivo activation, expansion, and genetic modification of canine T cells. J Immunother (2014) 37(8):407–15. doi:10.1097/CJI.0000000000000052 21 Mason N, Gnanandarajah JS, Engiles JB et al. Immunotherapy with a HER2-targeting Listeria induces HER2-specific immunity and demonstrates potential therapeutic effects in a phase I trial in canine osteosarcoma. Clin Canc Res (2016); 22 (17): 4380-90 doi: 10.1158/1078-0432.CCR-16-0088 Hans Klingemann, MD, PhD, has served in hematology/oncology academic leadership positions in the U.S. and Canada. He discovered NK-92 cells, which are now in clinical trials worldwide. Dr. Klingemann is currently vice president of research and development at NantKwest, a company that develops a portfolio of immunotherapy approaches for cancer. He maintains an academic appointment at Tufts University in Boston and can be reached at hans.klingemann@tufts.edu.
