Is immunotherapy the key to wiping out canine cancer?

An intact and functional immune system is essential to protect an organism against invading pathogens and infectious disease. However, the immune system also plays a pivotal role in identifying and eliminating transformed cells that, if left unchecked, would progress to cancer. Clinical evidence of the immune system’s ability to control cancer comes from a number of observations in both the veterinary and medical settings. Consider the following examples: Cats receiving chronic immunosuppression following renal transplant have a higher incidence of lymphoma compared with the general feline population1,2 Spontaneous regression of transmissible venereal tumor is associated with an increased proportion of tumor-infiltrating cytotoxic CD8+ T cells3 Canine osteosarcoma patients that develop bacterial infections after limb-sparing surgery experience significantly prolonged survival times.4 The presence of tumor-specific, cytotoxic T cells within tumors, such as ovarian carcinoma, confers a favorable prognosis,5 whereas infiltration with regulatory or suppressor T cells confers a worse prognosis6 These observations, coupled with two decades of experimentation in murine cancer models, indicate that finding ways to initiate, augment, and broaden a patient’s antitumor immune response holds promise for the treatment and possible prevention of cancer. Indeed, this is the aim of cancer immunotherapy, and recent advances in this field have delivered unprecedented clinical results, including the induction and maintenance of durable remissions in patients with relapsed, refractory hematological and solid tumors, such as malignant melanoma. Together, this raises hope immunotherapy treatment regimens will one day lead to a cure. Figure 1: Cartoon of a chimeric antigen receptor (CAR) T cell. The CAR is introduced into the T cell using a retroviral vector. When the CAR engages its cognate antigen through its scFv, the T cell becomes activated. Activation leads to CAR-T cell proliferation, cytokine production and cytotoxicity against the target tumor cell. The numbers of CAR-T cells increase exponentially providing a living army of serial killers that serve to eliminate target tumor cells. Chimeric antigen receptor T cells The term “cancer immunotherapy” applies to any approach aiming to promote effective antitumor immunity. One of the most promising proposed treatments to date has been the use of adoptive cell therapy (ACT) using chimeric antigen receptor (CAR) T cells. CAR-T cells are autologous T cells that are genetically engineered to express a CAR that specifically recognizes a tumor-associated antigen expressed on the cancer cell’s surface. The CAR consists of an antibody fragment (single-chain variable fragment [scFv]) responsible for antigen recognition that is linked to a transmembrane domain and the CD3ζ intracellular signaling domain of T cells, either alone or more commonly in combination with T cell co-stimulatory domains, such as CD28 (Figure 1). CARs endow T cells with the ability to recognize target antigens on the tumor cell’s surface, circumventing their usual requirement for antigen presentation in the context of major histocompatibility complex (MHC) molecules. This is relevant because tumors often down-regulate their antigen-presenting MHC molecules, becoming “invisible” to the patient’s T cells, which enables them to grow unchecked. Engagement of the CAR with its target antigen leads to robust activation of the CAR-T cell without the need for additional co-stimulatory signals usually supplied by other immune cells, such as dendritic cells or macrophages. Activation leads to proliferation, increasing the numbers of CAR-T cells generated. Given their ability to function as serial killers, even bulky disease can be eliminated following a single CAR-T infusion.7 Figure 2: Schematic of CAR-T cell generation and administration. Patients undergo a single leukapheresis procedure to obtain peripheral blood mononuclear cells from which T cells can be isolated. T cells are activated, transduced with a retroviral vector containing the CAR construct and expanded ex vivo to generate the CAR-T cell product. Prior to adoptive transfer of the CAR-T cells, the patient undergoes a lymphodepleting regime of high dose cyclophosphamide to promote CAR-T cell engraftment and prevent formation of antibodies against the CAR-T cells. An overview of CAR-T cell generation and administration is provided in Figure 2. Patient T cells are isolated from a leukapheresis product, activated in vitro, and infected with retroviral vectors that integrate the CAR construct into the cell’s DNA. The CAR-T cell product is then expanded ex vivo to increase numbers for administration. The fold expansion that occurs in vitro during this time is predictive of clinical response in human patients with chronic lymphocytic leukemia (CLL), suggesting that successful CAR-T cell therapy is directly related to the “fitness” of the T cell product.8 Unfortunately, intrinsic T cell dysfunction and an inability of patient T cells to proliferate means that as many as 10 percent of patients with acute lymphocytic leukemia (ALL) presenting for CAR-T cell therapy cannot be treated.9 Prior to CAR-T administration, patients undergo a lymphodepleting chemotherapy regimen aimed at generating space within the bone marrow for CAR-T cell engraftment, increasing the production of homeostatic cytokines that promote CAR-T expansion and preventing anti-CAR immune responses that reduce CAR-T cell persistence. Following administration, CAR-T cells engage with target-positive malignant cells, leading to their activation, proliferation, cytokine production, and destruction of tumor cells. An exponential increase in CAR-T cell numbers occurs within days of administration, providing more tumor-specific T cells to eliminate the neoplastic cells. Furthermore, by using a specific T cell subset (e.g. central memory T cells or stem cell memory T cells) to generate CAR-T cells, long-term CAR-T persistence is more likely to occur. CAR-T persistence also correlates with improved clinical response.8 CAR-T cell responses in hematological malignancies CD19 is a molecule expressed on the surface of healthy and malignant B cells, and has emerged as an important target for CAR-T cell therapy. The adoptive transfer of autologous CD19 targeting CAR-T cells (CART-19) into appropriately preconditioned, lymphopenic patients with relapsed, refractory, B-acute lymphoblastic leukemia (B-ALL) or B-chronic lymphocytic leukemia (B-CLL) has produced dramatic clinical results, with patients experiencing durable remissions that have persisted for years.9 Impressive responses have also been observed with CART-19 in human patients with B cell lymphoma, although the response rate is lower than that achieved in leukemia. CART-19 is approved by the U.S. Food and Drug Administration (FDA) for the treatment of relapsed, refractory B-ALL in children and young adults, and for aggressive, refractory non-Hodgkin’s lymphoma. CAR-T cells targeting the B cell maturation antigen (BCMA) have also shown therapeutic potential in multiple myeloma.10 Challenges of CAR-T cell therapy Although highly effective in hematological malignancies, CAR-T cells have shown limited effect in solid tumors. This is in part due to the immunosuppressive tumor microenvironment (TME) in solid tumors that inhibits CAR-T cell function and survival. Tumor stromal elements, including regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages—together with the immunosuppressive cytokines (e.g. TGF-β and interleukin 10 [IL-10]) they secrete—present a formidable barrier for CAR-T cell function. Checkpoint molecules, including indolamine dioxygenase (IDO),2,3 and PD-L1, also inhibit CAR-T cell proliferation and survival in the TME. These inhibitory factors must be addressed for CAR-T cells to achieve meaningful clinical responses in solid tumors. Therefore, combination strategies (e.g. CAR-T cells plus checkpoint inhibitors or additional modifications of CAR-T cells that render them resistant to these immunosuppressive factors) are now being explored. Severe side effects—including cytokine release syndrome (CRS), neurotoxicity, and on-target, off-tumor cytotoxicity—have been reported following CAR-T cell therapy in some human patients.11 Those with high tumor burden experience more severe CRS symptoms, which include fever and headache and can progress to hypotension, disseminated intravascular coagulation (DIC), and respiratory failure if left untreated. CRS is primarily associated with the release of pro-inflammatory cytokines, including IL-6. Early intervention using the IL-6R antagonist, tocilizumab, has significantly improved the outcome of patients with CRS. A poorly understood neurotoxicity that manifests as headaches, confusion, cerebral edema, seizures, and in some cases death can occur with CAR-T cells that target either CD19 or BCMA. Mild symptoms can resolve with symptomatic management and supportive care alone. Finally, on-target, off-tumor effects occur when CAR-T cells attack healthy cells that also express the tumor-associated target antigen. The severity of effects depends on which healthy cells are targeted. For CART-19, all B cells are eliminated, leaving the patient with B cell aplasia and hypogammaglobulinemia, a condition that can be managed using intravenous gamma globulin. However, CAR-T cells directed against the human epidermal growth factor receptor 2 (HER2/neu) were shown to directly target pulmonary epithelial cells that express low levels of HER2/neu, and this resulted in acute respiratory distress and death of the human patient being treated on an early clinical trial.12 Approximately one third of CAR-T cell patients relapse, with one third of these relapses being associated with CD19-negative disease.9 Evidence from human patients that develop CD19- B cell leukemia after CART-19 therapy suggest a subset of CD19- malignant clones may exist prior to CART-19 treatment. CAR-T-mediated elimination of the CD19+ cells then enables CD19- disease to emerge.13 In other instances, CD19- disease may arise de novo from tumor cells that have undergone epitope loss following CART-19 treatment. Targeting two separate antigens expressed on B cell (i.e. CD19 and CD20) using CAR-T cells may circumvent this escape mechanism in the future. Canine CAR-T cells and their clinical use Given the remarkable success of CAR-T cell therapy in treating aggressive hematological malignancies in human patients, attention has turned to CAR-T cell use in canine patients with similar aggressive malignancies to improve outcomes. Despite intensive cytotoxic chemotherapy regimens, the prognosis for long-term survival for dogs with B cell lymphoma remains guarded. Could CAR-T cells induce durable remissions in dogs with B cell lymphoma? Could CAR-T cells be employed in the future to treat solid malignancies, such as osteosarcoma, hemangiosarcoma, and glioblastoma, and provide better outcomes than our current standard of care? In the past five years, several groups have demonstrated the ability to genetically redirect canine T cells using CARs and have demonstrated their potent ability to kill antigen-positive target cells in vitro in cell-based killing assays. In the first report, Gottschalk’s group used a murine scFv (FRP5) that cross-reacts with human and canine HER2/neu to generate HER2/neu-specific canine CAR-T cells, and showed they were able to kill human fibroblast cells engineered to express human or canine HER2 in an antigen-dependent manner.14 More recently, Panjwani et al. demonstrated that primary canine T cells could be genetically redirected using a CAR specific for CD20, a molecule expressed on the surface of B cells.15,16 When cultured with a canine B cell lymphoma cell line that expresses CD20, the canine CAR-T cells produced high levels of IFN-g and efficiently killed lymphoma cells. Furthermore, when these CART-20 cells were used therapeutically in dogs with B cell lymphoma, the CART-20 cells were shown to expand in vivo, and in one instance, CART-20 treatment led to the emergence of a CD20- B cell lymphoma. Analysis of this patient’s tumor following CART-20 treatment revealed the target epitope had been spliced out, paralleling mechanisms of resistance to CART-19 that have been reported in human patients. Canine T cells have also been redirected against the IL13Rα2 subunit, expressed on canine glioma and osteosarcoma cells.17 Canine IL-13Rα2-specific CAR-T cells killed IL-13Rα2+ glioma cells in vitro and were highly effective at eliminating IL-13Rα2+ brain tumors derived from the canine glioma cell line (J3T) in mouse models of glioma.17 Taken together, these data demonstrate unequivocally that canine T cells can be genetically redirected against clinically relevant, tumor-associated antigens and can kill target-positive tumor cells in the experimental setting. While the generation of functional canine CAR-T cells is an important milestone, durable clinical responses to CAR-T cell therapy have yet to be achieved in dogs with B cell lymphoma.16 Possible explanations for this include the highly aggressive nature of canine B cell lymphoma, the choice of target antigen (CD20 compared with CD19), the use of a less aggressive preconditioning regimen compared with human patients, and the presence of immunosuppressive cells and molecules within the tumor microenvironment that may effectively inhibit CAR-T function.18 As each element is explored and addressed, one may expect successful manufacturing and clinical protocols will emerge that will lead to comparable positive results in canine B cell lymphoma patients as seen in human patients. Indeed, the promise of a therapy that could induce durable clinical remissions in this canine patient population without the need for multiple cycles of cytotoxic chemotherapy using a single autologous CAR-T cell infusion is extremely exciting and might soon be within our reach. Nicola Mason BVetMed, PhD, DACVIM, is an associate professor of medicine in the Department of Clinical Sciences and Advanced Medicine and holds the Paul A. James and Charles A. Gilmore Endowed Chair Professorship at the University of Pennsylvania’s School of Veterinary Medicine. She received her veterinary degree from the Royal Veterinary College, London and her immunology PhD from the University of Pennsylvania. Dr. Mason performed her postdoctoral fellowship in the lab of Carl June at the Abramson Cancer Center at the School of Medicine at UPenn. Her translational research group focuses on developing safe and effective immunotherapies for use in dogs with cancer and autoimmunity, and research interests include adoptive cell therapy using genetically modified T cells in canine patients with B cell malignancies, glioblastoma, and osteosarcoma, and in dogs with autoimmune skin disease. Through a comparative approach, Mason’s work aims to accelerate the clinical translation of effective immunotherapies, improving the lives of both dogs and humans. REFERENCES Wormser, C., Mariano, A., Holmes, E.S., Aronson, L.R. & Volk, S.W. Post-transplant malignant neoplasia associated with cyclosporine-based immunotherapy: prevalence, risk factors and survival in feline renal transplant recipients. Vet Comp Oncol 14, e126-e134 (2016). Trail, P.A. & Yang, T.J. Canine transmissible venereal sarcoma: quantitation of T-lymphocyte subpopulations during progressive growth and spontaneous tumor regression. J Natl Cancer Inst 74, 461-467 (1985). Barber, M.R. & Yang, T.J. Tumor infiltrating lymphocytes: CD8+ lymphocytes in canine transmissible venereal sarcomas at different stages of tumor growth. Anticancer Res 19, 1137-1142 (1999). Lascelles, B.D., et al. Improved survival associated with postoperative wound infection in dogs treated with limb-salvage surgery for osteosarcoma. Ann Surg Oncol 12, 1073-1083 (2005). Zhang, L., et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med 348, 203-213 (2003). Curiel, T.J., et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 10, 942-949 (2004). Porter, D.L., Levine, B.L., Kalos, M., Bagg, A. & June, C.H. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med 365, 725-733 (2011). Fraietta, J.A., et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med 24, 563-571 (2018). Majzner, R.G. & Mackall, C.L. Clinical lessons learned from the first leg of the CAR T cell journey. Nat Med 25, 1341-1355 (2019). Brudno, J.N., et al. T Cells Genetically Modified to Express an Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor Cause Remissions of Poor-Prognosis Relapsed Multiple Myeloma. J Clin Oncol 36, 2267-2280 (2018). Bedoya, F., Frigault, M.J. & Maus, M.V. The Flipside of the Power of Engineered T Cells: Observed and Potential Toxicities of Genetically Modified T Cells as Therapy. Mol Ther 25, 314-320 (2017). Morgan, R.A., et al. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther 18, 843-851 (2010). Fischer, J., et al. CD19 Isoforms Enabling Resistance to CART-19 Immunotherapy Are Expressed in B-ALL Patients at Initial Diagnosis. J Immunother 40, 187-195 (2017). Mata, M., 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 37, 407-415 (2014). Panjwani, M.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 24, 1602-1614 (2016). M. Kazim Panjwani, M.J.A., Martha A. MaloneyHuss, Kumudhini P. Haran, Ailian Xiong, Minnal Gupta, Irina Kulikovsaya, Simon F. Lacey & Nicola J. Mason. Establishing a model system for evaluating CAR T cell therapy using dogs with spontaneous diffuse large B cell lymphoma. Oncoimmunology (2019). Yin, Y., et al. Checkpoint Blockade Reverses Anergy in IL-13Ralpha2 Humanized scFv-Based CAR T Cells to Treat Murine and Canine Gliomas. Mol Ther Oncolytics 11, 20-38 (2018). Pinheiro, D., et al. Dissecting the regulatory microenvironment of a large animal model of non-Hodgkin lymphoma: evidence of a negative prognostic impact of FOXP3+ T cells in canine B cell lymphoma. PLoS One 9, e105027 (2014).

Cookie	Duration	Description
cookielawinfo-checkbox-analytics	11 months	This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Analytics".
cookielawinfo-checkbox-functional	11 months	The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Functional".
cookielawinfo-checkbox-necessary	11 months	This cookie is set by GDPR Cookie Consent plugin. The cookies is used to store the user consent for the cookies in the category "Necessary".
cookielawinfo-checkbox-others	11 months	This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Other.
cookielawinfo-checkbox-performance	11 months	This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Performance".
JSESSIONID	session	The JSESSIONID cookie is used by New Relic to store a session identifier so that New Relic can monitor session counts for an application.
viewed_cookie_policy	11 months	The cookie is set by the GDPR Cookie Consent plugin and is used to store whether or not user has consented to the use of cookies. It does not store any personal data.

Cookie	Duration	Description
vuid	2 years	Vimeo installs this cookie to collect tracking information by setting a unique ID to embed videos to the website.
_ga	2 years	The _ga cookie, installed by Google Analytics, calculates visitor, session and campaign data and also keeps track of site usage for the site's analytics report. The cookie stores information anonymously and assigns a randomly generated number to recognize unique visitors.
_gat	1 minute	This cookie is installed by Google Universal Analytics to restrain request rate and thus limit the collection of data on high traffic sites.
_gat_UA-	1 minute	A variation of the _gat cookie set by Google Analytics and Google Tag Manager to allow website owners to track visitor behaviour and measure site performance. The pattern element in the name contains the unique identity number of the account or website it relates to.
_gid	1 day	Installed by Google Analytics, _gid cookie stores information on how visitors use a website, while also creating an analytics report of the website's performance. Some of the data that are collected include the number of visitors, their source, and the pages they visit anonymously.
__gads	1 year 24 days	The __gads cookie, set by Google, is stored under DoubleClick domain and tracks the number of times users see an advert, measures the success of the campaign and calculates its revenue. This cookie can only be read from the domain they are set on and will not track any data while browsing through other sites.

Cookie	Duration	Description
ADV_u_id	3 months 8 days	Unique customer identifier used to track unique ad views and interactions with some ads.
anj	3 months	AppNexus sets the anj cookie that contains data stating whether a cookie ID is synced with partners.
fr	3 months	Facebook sets this cookie to show relevant advertisements to users by tracking user behaviour across the web, on sites that have Facebook pixel or Facebook social plugin.
IDE	1 year 24 days	Google DoubleClick IDE cookies are used to store information about how the user uses the website to present them with relevant ads and according to the user profile.
loc	never	AddThis sets this geolocation cookie to help understand the location of users who share the information.
OAGEO	session	OpenX sets this cookie to avoid the repeated display of the same ad.
OAID	1 year	Cookie set to record whether the user has opted out of the collection of information by the AdsWizz Service Cookies.
test_cookie	15 minutes	The test_cookie is set by doubleclick.net and is used to determine if the user's browser supports cookies.
uuid2	3 months	The uuid2 cookie is set by AppNexus and records information that helps in differentiating between devices and browsers. This information is used to pick out ads delivered by the platform and assess the ad performance and its attribute payment.
_fbp	3 months	This cookie is set by Facebook to display advertisements when either on Facebook or on a digital platform powered by Facebook advertising, after visiting the website.

Is immunotherapy the key to wiping out canine cancer?

How adoptive cell therapy with autologous CAR-T cells may offer durable clinical remissions

Membership Required