In the recent years, immunotherapy has been emerged as a major breakthrough in cancer treatment. Recently, two renowned scientists got nobel prize for their incredible work in the field of immunotherapy. James Allison discovered CTLA-4 protien and Honjo discovered PD-1 protein, known to be the checkpoint molecule. Inhibition of these T cell checkpoint proteins has achieved remarkable success against some of the advanced tumors in humans, including melanoma, renal cell carcinoma and nonsmall cell lung cancer
(Topalian et al., 2012; Wu et al., 2012; Ott et al., 2013; Quezada and Peggs, 2013;
Perez-Gracia et al., 2014). Amongst all, the most successful human immunotherapies to date include using monoclonal antibodies against T-cell checkpoint molecules, namely PD-1 and CTLA-1. Blocking these pathways unleashes the cytotoxic power of T lymphocytes and may also activate other immune responses such as antigen presentation and cytokine release (
Pardoll, 2012). Tumor-specific T cell was second major breakthrough in tumor immunotherapy. Impressive tumor responses observed following adoptive transfer of tumor-specific T cells have stimulated enormous exhilaration in the field
(Aldrich et al., 2010; McCormack et al., 2013; Riches and Gribben, 2013;
O’Connor and Wilson-Robles, 2014;
Raval et al., 2014). The third leading advancement in tumor immunotherapy is engineered monoclonal antibodies, which directly target the cells for destruction
(Maleki et al., 2013; Miller et al., 2013; Sliwkowski and Mellman, 2013;
Zigler et al., 2013; Zhou et al., 2014). Most of the recent immunotherapies are now being practiced in human medicine but still having limitations in veterinary medicine probably due to high finances involved. However, over the past many years the economics of some recent techniques has reduced therefore immunotherapy will find its application in veterinary medicine.
Recently available immunotherapies in veterinary medicine
Activated macrophage
Macrophages and monocytes when activated shows selective cytotoxicity against tumor cells either directly, or indirectly by producing cytokines, such as tumor necrosis factor- α (TNF- α). However, when not activated they may stimulate tumor progression and can promote tumor metastases
(Ruffell et al., 2012). The NLR agonist MTP (administered as a liposomal formulation known as L-MTP-PE) is one of the most thoroughly evaluated macrophage targeted immune therapeutics in veterinary medicine, with demonstrated anti-metastatic activity in canine osteosarcoma
(Kurzman et al., 1995). According to
Zhang and Tizard (1996) macrophages in tumors can also be activated by directinjection of fungal (
e.g., acemannan) or mycobacterial cell wall (
e.g., immunocidin) extracts directly into tumors.
NK cells
Activated natural killer cells are considered the first line of defence against tumor cells. They are capable of lysing cancerous cells without prior sensitization through direct destruction by inducing cytotoxicity or via release of cytokine IFN-α antitumor activity. Human NK cells are identified by the expression of CD56 receptors while they lack CD3 and the T-cell receptor (TCR)
(Cooper et al., 2001). On the other hand, canine NK cells are incompetent in expressing the typical NK cell surface markers. They do not express the characteristic T-lymphocyte markers CD3 and express CD5dim
(Huang et al., 2008; Michael et al., 2013 ). Placenta and CD34-expressing progenitor cells from cord blood are the chief sources of NK cells for humans while in dogs, only the blood lymphocytes have been utilized as NK cell source. NK cells plays a very important role in Antibody dependent cellular cytotoxicity (ADCC), a mechanism where NK cells act as effector cell of the immune system and thereby account for antitumor effect of mAb. Activation of NK cells can also be achieved via recombinant cytokines, like IFN-α and IFN-γ and these two synergize to expand and activate NK cells in lung and other tissues to exert an enhanced antitumor response. Interleukin-12 (IL-12) also activates NK cells and triggers the release of IFN-γ. A multitude of clinical research evaluating the efficacy of IL-12 gene therapy have been performed in different veterinary species
(Heinzerling et al., 2001; Siddiqui et al., 2007; Chuang et al., 2009; Reed et al., 2010; Pavlin et al., 2011). Chuang et al., (2009) reported that IL-12 electrogene therapy resulted in a significant delay in tumor growth and eventually complete tumor remission in canine transmissible venereal tumor (TVT). In another study,
Pavlin et al., (2011) opined that IL-12 electrogene therapy resulted in a median reduction in tumor volume of 50% in dogs with mast cell tumors.
Tumor vaccine - oncept
In humans, approach of tumor vaccine against cancerous cells has shown limited effect, on the contrary in case of dogs, these tumor vaccine found to be quite effective against canine melanoma. Oncept, the canine melanoma vaccine is a tumor-specific vaccine being utilised in veterinary medicine. Being the first USDA approved vaccine, it is commercially available for use in dogs and targets the human tyrosinase protein, a molecule that is very similar though not identical to the canine tyrosinase protein. Oncept consists of a plasmid DNA encoding human tyrosinase (h-Tyr) and is administered via intradermal immunization using a needle-free injection system. Although Immune responses to the Oncept vaccine have not been extensively evaluated in tumor-bearing dogs, yet antibody responses have been detected in dogs with advanced melanoma
(Liao et al., 2006) and hTyr-specific T cell responses have been demonstrated in healthy laboratory Beagle dogs
(Goubier et al., 2008). It is approved for the treatment of stage II and III melanoma in dogs. Reports suggest that administration of the vaccine results in enhanced survivability
(Bergman et al., 2003; Liao et al., 2006). This vaccine was greeted with huge exuberance, but researchers are evaluating true therapeutic benefits of the vaccine, beyond the peace of mind for dog owners in knowing that some intervention is being provided for their pet
(ottnod et al., 2013).
CAR engineered T cells
Acknowledged as a chimeric antigen receptor technology in which T cells are transfected with genes encoding chimeric antibody receptors specific for a tumor antigen, hence resulting in homing of the T cells directly to the desired target cell
(Casucci et al., 2012; Cruz et al., 2013; Riches and Gribben, 2013;
Cheadle et al., 2014; Cieri et al., 2014). CAR T-cells targeting the CD19 receptor on human leukemia and lymphoma cells have resulted in some favourable outcomes, and recently FDA also approved the first CAR T-cell treatment for patients with CD19-positive acute lymphoblastic leukemia (ALL). Humongous success has been achieved using the CAR approach targeting T cells to CD19+ B cells in human patients with relapsed chronic lymphocytic leukemia
(Cruz et al., 2013; Riches and Gribben, 2013). The CAR approach has recently been evaluated for future use in dogs with osteosarcoma
(Mata et al., 2014). Canine lymphocytes transfected with a human HER-2 CAR revealed that the human CAR can target and kill HER-2-positive canine osteosarcoma cell lines
(Mata et al., 2014). CAR T-cell treatment can also cause significant toxicities such as cytokine release syndrome (CRS), encephalopathy or bone marrow suppression in about one third of treated patients
(Dotti et al., 2014).
Monoclonal antibodies (mAbs)
US Department of Agriculture has provided the affirmation for the use of monoclonal antibodies for CD20-positive B cell (Blontress®) or CD52 positive T cell lymphoma (Tactress®) and are commercially available in the USA and Canada (
Regan and Dow, 2015). Antibody-dependent cellular cytotoxicity (ADCC), exclusively mediated via the Fc receptor (FcR) on natural killer (NK) cells defines the major mechanism of action for most human mAbs
(Lehrnbecher et al., 1999). Treatment outcomes with mAbs are far more superior in human patients if their NK cells express a high-affinity FcR
(Musolino et al., 2008; Gavin et al., 2017). Pertaining to the restricted information about the FcR distribution on canine immune cells, especially on NK cells, optimizing mAb efficacy for dogs becomes difficult
(Bergeron et al., 2014). This may explain the suboptimal response to canine CD20 mAb as compared to those seen in humans. Additional canine CD20 mAbs are under development and preliminary studies have shown depletion of peripheral B lymphocytes in beagle dogs
(Rue et al., 2015). Two large, well controlled studies concomitance with cytotoxic chemotherapy has been performed and efficacy of a caninized mAb against the T-cell antigen CD52 (Tactress®) has been tested for the treatment of canineT-cell lymphoma. Unfortunately, the mAb could not improve progression free survival in this more aggressive form of lymphoma (
Rodriguez and Hansen, 2014). Bevacizumab, a humanized mAb inhibits angiogenesis by blocking the vascular endothelial growth factor (VEGF). It is used to treat a variety of cancers in humans.
Scharf et al., (2013) conducted a study to assess the effect of bevacizumab on angiogenesis and growth of canine osteosarcoma cells xenografted in athymic mice. Bevacizumab has shown some efficacy in murine models of canine mesenchymal neoplasms. The results of the above study suggested that some mAbs can have cross-species reactivity, which may vary with the type of epitope recognized by the mAb. Lately, monoclonal antibodies against checkpoint inhibitors has proven to be a huge success in humans and considered the most recent immunotherapy in human medicine.
Checkpoint inhibitory mechanism
Checkpoint molecule are the proteins having a significant role in regulation of our immune system. These two proteins are CTLA-4 and PD-1 working as a brake for the immune system. CTLA-4 (cytotoxic T-lymphocyte antigen) is a homolog of CD28 and are expressed over the T cell surface following the T-cell activation, thereby resulting in the inhibition of T-cell mediated responses. Since these CTLA-4 are stored in the vesicles at microtubule organizing centre (MTOC) therefore they need to be translocated for their expression over the surface of T-cell. However, the surface expression of CTLA-4 depends on the signal strength of T-cell receptors leading to a much more expression in case the signal is strong and way lesser if it’s weak. Once expressed, CTLA-4 competes with CD28 for binding to same ligand
i.e. B7 and eventually this binding with B7 decides whether the immune system will get stimulated or it gets inhibited. In case the TCR signal is weak, CD28:B7 binding increases providing a positive activating signal, eventually leading to the production of cytokines and resulting in the proliferation and growth of T-cells. while if the TCR signal is strong, CTLA-4:B7 binding is more which produce a negative signal resulting in diminished cytokine secretion, reduced proliferation and survival. Accordingly for the successful immunotherapy, inhibition of CTLA-4 molecule is a prerequisite activating the immune system against tumor cells. Signaling through CTLA-4 (and other checkpoint molecules in the family including PD-1, TIM3 and Lag3) delivers negative signals to T-cells, thereby effectively blocking the stimulatory signals delivered through co-stimulatory molecules such as CD28 (Allison
et al.,1998). When CTLA-4 expression is eliminated genetically or by antibody blockade, mice may succumb to spontaneous autoimmune disease
(Allison et al., 1998; Shin et al., 2012; Wu et al., 2012; Kawano et al., 2013; Son et al., 2014). CTLA-4 blockade in human melanoma patients demonstrated significant antitumor activity in melanoma patients. Owing to this accomplishment, the first cancer immunotherapeutic drug got approval (Yervoy; Ipilimumab)
(Lebbe et al., 2014).
Another major checkpoint molecule is PD-1 (Programmed cell death protein 1) present on the T-cell surface and regulates the immune response by binding with two ligands,
i.e. PD-L1 and PD-L2. When T-cell comes in contact with tumor cell or antigen presenting cell (APC) it leads to the expression of PDL1/L2 on the surface of APC. PDL1/L2 binds with PD1 and eliciting an inhibitory pathway of T-cell leading to lesser proliferation, decreased cytokine production and reduced survivability of T-cell. However, if the PD1 protein is inhibited
via monoclonal antibodies, Immunity will not get hampered leads to T-cell activation which fight against cancer cells. Substantial results from the recent PD-1 blocking monoclonal antibody trials have further energized the tumor immunotherapy field and newly discovered PD-1 targeted mAb (Keytruda; Pembrolizumab) has recently been approved for humans with melanoma, renal cell carcinoma and non-small cell lung cancer (
Riley, 2013;
Robert et al., 2013; Poole, 2014). The mAbs against the checkpoint molecules
viz; CTLA-4 and PD-1 have yielded remarkable responses in humans, especially for melanoma lung, kidney, and bladder cancer (
Wang and Wu, 2017). Canine lymphocytes also express the PD-1 protein molecule
(Coy et al., 2017). Studies using canine tumor biopsy samples and a human mAb having cross reactivity with canine PD-L1, have confirmed the expression of PD-L1 on a number of canine tumors
(Scharf et al., 2013). However, a recent clinical trial with a “caninized” mAb against canine PD-L1 showed a limited response in dogs with advanced melanoma
(Mackawa et al., 2014).
Cancer immunotherapy: A challenge in veterinary medicine
Immunotherapy in veterinary medicine is lagging behind and there are several reasons falls for it, finance involved being most crucial. Biotechnology enterprises are well informed that there is limited scope of expensive immunotherapies in Veterinary field and are therefore reluctant to provide adequate funds for clinical trials and sometimes the expenses of these immunotherapies makes them unaffordable. Moreover, the cellular biology of tumor in canines is still unexplored to much extent and even the immune system of dogs has not been concisely evaluated, thereby making immunotherapy a big challenge in the field of veterinary medicine. Though the progress has been escalated in the last few years in terms of clinical trials, financial boost and changing perception, yet we are far away from immunotherapy being the mainstream approach for cancer therapeutics.
Major challenges in canine cancer immunotherapy (According to Klingemann 2018)
√ Costs for clinical trials.
√ Profit margin for pharmaceutical companies limited.
√ Randomized trials are challenging due to-
• Breed variability.
• Heterogeneous nature of tumors.
√ Outcome measurements require close follow up-
• Immunotherapy can cause initial tumor enlargement -leading to early discontinuation.
• At progression, owners often prefer euthanasia.
• Owners may consider treatment interventions palliative, often resulting in reduced drug dosing.
√ Limited knowledge about canine immune system.
