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Adoptive cell transfer: new perspective treatment in veterinary oncology


Cancer immunotherapy is recently considered the most promising treatment for human patients with advanced tumors and could be effectively combined with conventional therapies such as chemotherapy or radiotherapy. Patients with hematological malignancies and melanoma have benefited greatly from immunotherapies such as, adoptive cell transfer therapy, experiencing durable remissions and prolonged survival. In the face of increasing enthusiasm for immunotherapy, particularly for the administration of tumor-specific T lymphocytes, the question arises whether this method could be employed to improve treatment outcomes for canine patients. It is warranted to determine whether veterinary clinical trials could support comparative oncology research and thus facilitate the development of new cell-based therapies for humans. Herein, we discuss adoptive transfer of T lymphocytes and lymphokine-activated cells for application in veterinary oncology, in the context of human medicine achievements. Furthermore, we discuss potential benefits of using domestic dog as a model for immunotherapy and its advantages for translational medicine. We also focus on an emerging genome-editing technology as a useful tool to improve a T cells’ phenotype.


Cancer is a complex disease caused by the impairment in a cells’ physiology leading to uncontrolled proliferation and inhibition of apoptosis [1]. Disease progression results from a complicated interplay between genetic alterations of transformed cells and cancer immunoediting by the hosts immune defense mechanisms [2]. It has been indicated in multiple human and canine studies that the dysfunction of immune system, enabling tumor growth and metastasis, is associated with tumor immune escape. This process is mainly manifested by downregulated expression of major histocompatibility complex (MHC) class I and tumor specific antigens, as well as, by production of anti-inflammatory cytokines such as IL-10 and TGF-β by malignant cells [3, 4]. Local immunosuppression is further supported by active recruitment of myeloid-derived suppressor cells (MDSC) into tumor microenvironment and activation of suppressive T regulatory cells (Tregs). This unfavorable niche alters the fate of immune cells and contributes to the functional inhibition of effector T and NK cells (Natural Killer cells), resulting in immunologic tolerance [5]. Unresponsiveness of T cells is caused by chronic stimulation and the expression of co-inhibitory receptors such as Programmed cell death protein 1 (PD-1) and cytotoxic T cell antigen 4 (CTLA-4), which leads to T cell exhaustion [6]. Moreover, cancer cells can induce deactivation of circulating monocytes and polarization of macrophages to M2-like phenotype, which not only foster existing tumor but also facilitate spread of transformed cells [7, 8]. Promotion of cancer progression is also linked with production of pro-angiogenic and pro-metastatic factors by tumor-associated macrophages (TAMs) and MDSCs [8,9,10]. Given the complex and dynamic crosstalk within the tumor microenvironment, the development of an effective anticancer immunotherapy has been a challenging endeavor.

The first report of ACT therapy date back to mid-1960’s, when allogeneic T lymphocytes have been transferred into rats to treat primary fibrosarcoma [11]. The goal of the study was to harness cytotoxic CD8+ T cells (CTLs), capable of mediating direct target cell lysis, to fight against cancer. These landmark experiments paved the way for the development of cellular immunotherapy. Further advances have resulted in the discovery of cancer-associated antigens and the improvement of genetic engineering.

Currently, ACT therapy has demonstrated great promise in eliciting curative responses against hematological malignancies and melanoma in human patients. Veterinary oncology is highly translatable for human medicine and results obtained in the canine patients can facilitate the design of the next-generation clinical trials to treat advanced solid tumors in humans.

Search strategy

This review is based on a search in PubMed ( using the terms “adoptive cell transfer” OR “adoptive cell transfer in dogs” AND “tumor infiltrating lymphocytes” OR “TILs” AND “TCR engineered T cells” AND “CAR T cells” OR “canine CAR T cells” AND “canine T-LAK” AND “genome editing” OR “genome editing therapy”. Only papers written in English were included in the review. The vast majority of the literature cited, is less than 15 years old. Exceptions are the papers that describe for the first time the crucial method or discovered phenomenon in the field of immunotherapy (i.e. first studies that paved the way for immunotherapy as a historical link).

All original research related to the canine immunotherapy (more specifically canine adoptive cell transfer and T-LAK therapy) were incorporated. Studies related to adoptive cell immunotherapy and genome editing, were evaluated and the most relevant to the review were selected. Our systematic review comprises the current knowledge on adoptive cell transfer therapy in canine oncology, in the context of human medicine achievements.

Advantages of using a dog model for comparative oncology

The domestic dog (Canis lupus familiaris) is an attractive and useful model for comparative medicine for the evaluation and development of novel therapeutic strategies and ensuing immunological assessments [12,13,14,15,16]. Unlike transplantable xenograft rodent models, canine tumors share with human tumors similar epidemiology, genetic, biology, treatment responses, prognosis factors and clinical outcomes. Cancer occurs in dogs naturally and spontaneously and the progression of disease (e.g. metastatic cascade) is similar to humans. Finally, dogs share the same life environment and thus cancerogenic and risk factors with people [17]. Similar to people, the incidence of cancer in dogs increases with age, with a frequency of 45% in animals older than 10 years [18]. Therefore, the domestic dog is a recommended model for multiple human diseases including cancers such as non-Hodgkin lymphoma, osteosarcoma, leukemia, melanoma and lung, head and neck, prostate, mammary, and bladder carcinomas [12, 19, 20].

Cancer research has been revolutionized through the advances in genome sequencing and assembly. Knowledge about the genetic basis and molecular mechanisms of cancer progression greatly accelerated the development of novel therapies. In 2005, the canine genome was sequenced, leading to more advanced studies in the field of veterinary sciences [21]. It was an important step for comparative studies that improved the understanding of mammalian evolution as well as enhanced our knowledge of tumorigenesis, cancer growth and metastasis, and tumor immunology in animals.

The innate and adaptive immune system in dogs is comparable to humans. Additionally, the main immune cells subsets identified in dogs, as well as their surface markers, exhibit similarities to human [16]. Although many similarities have been found our understanding of canine immune system functioning is still highly affected by lack of specific reagent to fully define all immune subsets. Furthermore, factors regulating immune response and subsequent signaling pathways responsible for induction of anticancer immunity are yet to be determined. Nevertheless, domestic dog model offer possibilities of clinical studies that will benefit and inform design of human clinical trials. Advantages of using canine animal models are summarized in Fig. 1.

Fig. 1
figure 1

The domestic dog can serve as an attractive model in comparative oncology. Dogs and humans share the same environment and possess multiple similarities in genetics, physiology as well as tumorigenesis and cancer progression. For this reason, canine models are of great importance to cancer and immunological studies and can contribute to improvement of human immunotherapy

Adoptive T cell transfer therapy

Among the four different categories of immunotherapies including: checkpoint inhibitors, monoclonal antibodies, therapeutic vaccines and cellular therapies, it is adoptive T cells transfer (ACT) therapy that has recently shown clinical outcomes of record efficiency in human hematological malignances and melanoma [22, 23].

ACT treatment involves the infusion of tumor-specific T lymphocytes into the circulatory system of cancer patients. The immune cells can be derived from one of two sources: (1) natural host T cells identified in the tumor mass—the autologous tumor infiltrating lymphocytes (TILs), (2) autologous T cells from patients’ peripheral blood that have been genetically engineered ex vivo to express specific antitumor T cell receptors (TCRs) or chimeric antigen receptors (CARs) [24, 25]. The cells obtained from patients are activated and extensively expanded to large numbers (1 × 1011) ex vivo in the presence of high dose IL-2 (a T cell growth factor) before re-infusion. Recently, additional genome editing has been proposed to enhance the function of engineered T lymphocytes.

In veterinary medicine, due to the similarities between canine and human antitumor immunity, a parallel approach can be applied (Fig. 2). Herein, we will compare ACT therapy in human versus veterinary settings.

Fig. 2
figure 2

Tumor microenvironment consists of malignant cells, stroma and different populations of immune cells. Complex crosstalk between them shapes the final outcome of neoplastic disease. Anticancer response is driven mainly by cytotoxic CD8+ T cells and NK cells, which release IFN-γ and granzymes, thus are involved in direct lysis of the tumor cells. Th1 subpopulation of CD4+ T cells, M1 macrophages and activated dendritic cells (DCs) support anticancer immunity by antigen presentation and cytokine production (IL-12, IFN-γ). CD8+ and CD4+ T cells recognize tumor antigens in the context of MHC class I and II respectively, followed by costimulatory signaling via CD28 molecule, necessary for their full activation, proliferation and function. Tumor progression, in turn, is associated with the presence of the Th2 and T regulatory CD4+ lymphocytes, M2 macrophages and MDSC. These cells secrete immunosuppressive factors such as IL-4, IL-10, or TGF-β and exhibit high activity of arginase, respectively. Unresponsiveness of cytotoxic CD8+ T cells is caused by decreased expression of MHC I on the cancer cells surface and activation of coinhibitory receptors such as PD-1. Adapted from Servier Medical Art

Tumor infiltrating T lymphocytes

The tumor microenvironment consists of transformed cancer cells and non-transformed stromal and inflammatory cells including fibroblasts, endothelial cells, T lymphocytes, dendritic cells, MDSCs and macrophages. Dynamic crosstalk between all of them via released cytokines and growth factors, and direct cell–cell interactions is responsible for either promotion or inhibition of tumor growth and metastasis (Fig. 3).

Fig. 3
figure 3

Adoptive cell immunotherapy possibilities for cancer in dogs include transfer of TILs, TRC-modified and CAR-engineered T lymphocytes. Tumor reactive T cells can be derived from tumor mass or peripheral blood lymphocytes can be genetically engineered to recognize tumor-specific antigens. Obtained T cells are expanded ex vivo and then administrated back to the tumor-bearing patient. Recently, genome editing technologies are using to confer additional modifications to T cells such as disruption of endogenous TCR and MHC. Introduction of ACT treatment into veterinary medicine can greatly facilitate the design of the new clinical trials for humans. Adapted from Servier Medical Art

Among the immune cells that penetrate a tumor, the tumor infiltrating lymphocytes (TILs), are capable of mediating cancer cells eradication. Increased T cell infiltration is a good prognostic marker for multiple solid tumors including, breast cancer (reviewed: [26, 27]) head and neck squamous cell carcinoma [28], melanoma [29, 30], muscle-invasive bladder cancer [31], seminoma [32], pancreatic adenocarcinoma [33], cervical [34], ovarian [35], gastric [36] and colorectal [37] tumors.

Most of the TILs in the tumor environment, due to the immunoediting process, experience chronic activation and exhaustion leading to the attenuation of proliferation and effector functions via activation of checkpoint co-inhibitory receptors such as PD-1 or CTLA-4 [6]. In some solid tumors, TILs are present in too low frequency to mediate a potent immune response [38]. Thus, TILs are retrieving from tumor mass, expanded ex vivo and re-infused back to the cancer patients. Tumor-specificity of TILs is selected based on IFN-γ production while co-cultured with cancer cells. Rosenberg et al. [39] were the first to demonstrate the regression of metastatic melanoma in patients that received autologous TILs along with high doses of IL-2 treatment.

However, after initial success with TIL therapy, further investigation revealed that transferred T cells do not persist long-term. In fact, infused T cells diminished over time and were undetectable in the blood of some cancer patients 2 weeks post-transfer, which resulted in a lack of objective responses [40]. The cytotoxic T cells used for transfer were comprised of terminally differentiated effector cells, without self-renewal capacity. Moreover, for CTLs to induce a potent antitumor response they depend on the activity of effector CD4+ T cells and the availability of homeostatic cytokines [41,42,43]. Thus, upon transfer into an immunocompetent recipient, CTLs were not able to survive and mount an effective response.

Preconditioning of cancer patients significantly improves the therapeutic efficiency of TIL therapy. This involves the systemic destruction of the recipient immune system (lymphodepletion) before ACT-based treatment. It was shown that chemotherapy consisting of high-dose of cyclophosphamide and fludarabine or total body irradiation of non-myeloablative (2 Gy) dose caused lymphodepletion, which significantly enhanced ACT efficiency [44, 45]. Such a regimen, prior to infusion, is necessary to destroy host immune cells and create ‘space’ for expansion of adoptively transferred T lymphocytes. The mechanism relies also on the elimination of immunosuppressive Tregs and MDSCs and by augmenting the innate immune system. Lymphodepletion excludes immune cells termed “cytokine sinks” that compete with infused T cells for homeostatic cytokines, thereby increasing access to those cytokines, particularly IL-15 and IL-7, which promote T lymphocyte proliferation in vivo [46].

Autologous TIL treatment was currently demonstrated to induce durable, complete, and in some cases curative metastatic melanoma regression with objective tumor response observed in around 50% of patients [47, 48]. Nevertheless, adaptation of this therapy for other types of solid tumor is limited due to low availability of TILs from tumor mass [49, 50]. To date, melanoma remains most immunogenic type of tumor and even patients in advanced stage IV of melanoma can benefit from autologous TIL transfer. However, still short-term persistence of infused T cells that are fully differentiated and exhausted constitutes an obstacle [51, 52]. Thus, extensive studies are ongoing to improve the protocols of ex vivo expansion and preparation of TILs (i.e. using pharmacological modifications). As of September 2018 there are 158 open clinical studies regarding administration of TILs to human cancer patients ( Advantages and disadvantages of using TILs are summarized in Table 1.

Table 1 The major advantages and disadvantages of various methods of generating T cells for the purpose of adoptive transfer

TILs in veterinary oncology

Tumor infiltrating lymphocytes in canine cancers are not well characterized, therefore there is still much to be gleaned. An increase in T lymphocyte infiltration was correlated with spontaneous, rapid regression of transmissible venereal sarcoma [53], oral papilloma [54] and cutaneous histiocytoma [55].

Lymphocyte infiltration has been studied in canine mammary gland tumors as it is an attractive biological model for the investigation of human breast cancer immunology [56]. Initial studies showed an increase in infiltrating T cells in the tumors that gave metastasis, suggesting that TILs, especially CD4+ T cells promote tumor expansion [57, 58]. In contrast, the lab of Carvalho [59] demonstrated that malignant mammary tumors had a reduced number of intratumoral T cells compared to benign tumors. However, the proportion was reversed when CD3+ T cells were counted in the peripheral parts of the tumor or in the adjacent non-malignant mammary gland, indicating that T cells from that region might be engaged in tumor progression. In addition, an increase of TILs was associated with increased canine mammary tumor malignancy [60]. Unfortunately, the published data lacks in-depth T cell subset analysis, specifically the identification of Tregs, which are known to exhibit immunosuppressive properties thereby supporting cancer development [60].

Recent data confirms that a high frequency of T cells found within malignant tumors is due to a high percentage of Tregs [61]. Similarly, Mucha et al. [62] demonstrate an increased population of FoxP3+ (regulatory T cell-associated transcription factor) cells in canine malignant and metastatic mammary cancers compared to benign tumors. Likewise, Tominaga et al. [63] found, high Treg frequency in the peripheral blood and even higher in the tumor mass of dogs with oral melanoma.

Although, no studies have utilized TILs for ACT therapy in dogs with solid tumor, there is ongoing research which exploits T cells from canine patients with lymphoma [64]. In this case, however, the use of the term “TILs” can be speculative since in the hematological malignancies T cells do not truly infiltrate the tumor. T cells used in the following study were memory T cells which exhibit anticancer activity. These studies revealed that the transfer of autologous T lymphocytes from dogs with non-Hodgkin lymphoma, previously treated with chemotherapy, improved clinical outcomes. T cells isolated from the blood of lymphoma-bearing dogs were co-cultured with γ-irradiated K562 cells genetically modified to function as artificial antigen presenting cells (aAPC), in the presence of rhIL-21 and rhIL-2. Recently, this approach was also used to expand canine CAR T cells [65]. For the purpose of ACT, the majority of the cells were CD3+/CD8+ (88%). Importantly, 70% of the cells were memory T cells (CD3+, CCR7+) characterized by high production of IFN-γ. Dogs treated with chemotherapy and ACT therapy achieved complete remission lasting at least 338 days post T cell transfer. Overall percentage of CD8+ T cells was higher in the blood for up to 42 days of canine patients that received ACT therapy compared to their non-treated counterparts. The increase of CD8+ T cells was correlated with increased expression of thymidine kinase, enhanced granzyme B production and decreased neutrophil to lymphocyte ratio. The study demonstrated the successful application and clinical outcomes of tumor immunotherapy for canine patients. Of note, this was the first study to show the power of this effective therapy in in vivo study providing a solid foundation for further studies in the field of veterinary oncology.

Utilizing T cell receptor transduction for ACT therapy

Antitumor activity of T cells is based on the ability of a T cells to recognize antigen via its endogenous T cell receptor (TCR). TCR is a transmembrane protein complex, consisting of α- and β-chains (more rarely δ- and γ-chains). Although, it was demonstrated that tumor-specific T cells can be successfully isolated from both tumor microenvironment and the blood for the purpose of ACT [66, 67], the limitations of this approach still hinder its wide application in medicine. The major drawback is related to accessibility of TILs in certain tumor types [50]. To overcome this limitation, the use of TCR-engineered T cells has been extensively studied.

The first studies, performed with mouse models, demonstrated that transfer of TCR α and β genes, targeted to tumor antigens, into T cells confers tumor antigen-specificity [68, 69]. These pioneering studies led to significant progress in the field of TCR-engineering for T cell therapy. To date, TCR-engineered T cells have been used against renal cell carcinoma antigen (RCC) [70], Wilms tumor antigen-1 (WT1) for leukemia cells [71], melanoma antigen recognized by T cells 1 (MART1) for patients suffering from melanoma [72] or cancer–testis antigen (NY-ESO-1) to treat myeloma [73]. Each of the studies showed the benefit with objective responses and potent antitumor activity.

The advantage of TCR-engineered T cells is that lymphocytes can be easily obtained from the peripheral blood and then transformed into tumor-reactive cells. Nevertheless, the major limitation of this method arises from off-target TCR heterodimer formation (Table 1). With the addition of a second α and β TCR chain, it is probable for the expression of four different TCR receptors due to interactions of endogenous and introduced α and β TCR chains [74]. Studies by Sommermeyer laboratory [75] have indicated that TCR with strong antigen affinity may replace TCR with weak antigen affinity, resulting in T cells with altered or novel antigen specificity. Furthermore, simultaneous expression of two distinct TCR was associated with an induction of autoimmunity [76,77,78].

Numerous clinical trials reveal potential toxicity of TCR-redirected T cell therapy, arising from on-target but off-tumor effects that in extreme cases lead to lethal outcomes [79]. For this reason, the crucial step in development of effective and safe treatment is selection of tumor-specific TCR.

TCR-engineered T cells have not been used in veterinary oncology primarily due to the fact that antigen recognition is based on MHC-restricted epitopes. Recently, however, Barth et al. [80] successfully identified a binding motif of canine MHC I allele DLA-88*50101. Interestingly, this binding motif exhibited high similarity to human leukocyte antigen HLA-A*02:01. Therefore, the authors stressed that canine models would be suitable for testing TCR-transduced T cell-based immunotherapies. Importantly, the canine genome has been sequenced which will help in the development of novel targets that can be implemented in both human and veterinary medicine.

Chimeric antigen receptor (CAR) engineered T cells

The main restriction in using TCR-engineered T cells results from heterodimer formation resulting in undefined antigen specificity. Importantly, T cells can recognize antigen via its TCR only when it is processed into a peptide and presented in the context of an MHC molecule. However, malignant cells often downregulate expression of MHC molecules in order to avoid immune response [81]. Due to these limitations, researchers have developed a novel solution. The chimeric antigen receptor (CAR) fuses a single chain variable fragment (scFv) of an antibody that recognizes antigen with the intracellular signaling domains of the CD3ζ. The initial studies, conducted by Gross et al. [82], proved that T cells can successfully express chimeric receptors that recognize specific antigens and trigger T cell response. To date, three generations of CARs can be distinguished, based on the signaling endodomains [83]. First generation CARs consist solely of an antigen binding domain and the CD3ζ signaling domain necessary for T cell activation, while the 2nd generation incorporated a co-stimulatory signaling such as CD28 or inducible T cell costimulator (ICOS) [84]. Third generation of CARs combine additional co-stimulatory molecules to enhance the potency [85]. It was demonstrated that each generation of CARs exhibited distinctive properties with regard to the T cells’ antitumor activity, proliferation, cytotoxic properties, and persistence [86]. Studies by Hombach and Abken [87] have revealed significant differences, mainly related to proliferation and cytokine secretion, between lymphocytes expressing CARs recognizing the same epitope but with various costimulatory endodomains. Also, Bridgeman et al. [88] pointed out that results may differ significantly from each other even if the same chimeric receptors were used. Consequently, the design of artificial chimeric receptors requires further refinements and definition of the most optimal CAR structure.

There is great potential for adoptive therapy with CAR-engineered T cells as a treatment for some types of the cancer such as neuroblastoma and leukemia [89, 90], yet in other cancer types (e.g. ovarian cancer and metastatic renal cell carcinoma) the therapy have demonstrated poor responses [91, 92]. Additionally, these studies have documented on-target/off-tumor toxicities after CAR-T cell treatments. Nonetheless, it was proposed that on-target toxic effects of CAR-T cell therapy can be abolished by administration of agents that block specific epitopes, such as monoclonal antibodies [93].

Many aspects of CAR-T cell therapy require further investigation however, the therapy induce a powerful antitumor response. The latest trends in CAR-T cell research are designing the next-generation CAR-T cells that will be engineered with suicide genes or dual-antigen receptors. It is believed that it will be easier to control the function of the next generation CAR-T cells and thereby minimizing their side effects [94].

CAR-T cells in veterinary oncology

ACT therapy in dogs is not as advanced as in humans and therefore the literature on the subject is still limited. To date, just two groups have attempted to use CAR technology to treat canine osteosarcoma (OS) and B cell lymphoma. These tumors are considered as valuable model of human cancers as they share similar tumor-associated antigens such as HER2 (osteosarcoma associated antigen) and CD20 (marker of transformed B cells).

The ACT study to treat osteosarcoma was performed with T cells obtained from peripheral blood of healthy dogs [65]. The T lymphocytes were activated using irradiated K562 aAPCs, genetically modified to express human CD80, CD83, CD86, 41BBL. Additionally, cells were stimulated with phytohemagglutinin (PHA) and rhIL-21. Interestingly, other methods of activation, such as administration of Concanavalin A (ConA) or anti-human CD3 (OKT-3 clone) antibodies coated-plates were insufficient to effectively stimulate canine T cell proliferation for ACT therapy purpose [65, 95]. Activated T cells were subsequently transfected with a 2nd generation α-canine HER2 CAR (containing CD3ζ and CD28 domains). The authors demonstrated that HER2-CAR-T cells in co-culture with several HER2 positive canine OS cell lines secreted more IFN-γ and exhibited superior ability to eliminate HER2+ OS cells in vitro. The results were not observed in HER2 OS-phenotype cell lines or non-transfected T cells. Despite these promising results, this approach has not yet been evaluated in vivo.

Building on these encouraging in vitro data, Panjwani et al. [95] performed in vivo studies on dogs suffering from B cell lymphoma. Autologous canine T cells were transiently transfected with α-canine CD20 CAR using electroporation. In the presence of canine lymphoma cells the CD20-CAR-T cells secreted significantly more of IFN-γ, than non-modified T cells or CD19-CAR-T cells (used as non-specific CAR transfection control). Additionally, antigen-specific CAR-T cells actively lysed the target cancer cells. Dogs with B cell lymphoma that relapsed after chemotherapy (l-asparaginase, vincristine, cyclophosphamide, doxorubicin or prednisone) were given three doses of 700,000 CD20-CAR-T cells per kg. Serious adverse side effects were not observed. Each injection resulted in an enlargement of the target lymph node, decreased frequency of cancer CD79a+/CD20+ cells and increase of non-transformed CD5+ B lymphocytes in the lymph nodes. In addition, increased serum levels of IL-6 and IFN-γ were measured after the first dose of CD20-CAR-T cells [95]. Unfortunately, due to the transient transfection of these CAR-T cells, durable remission was not attained.

Lymphokine-activated killer (LAK) cells for adoptive cell therapy

In contrast to adoptive T cell transfer therapy which exploits tumor-specific T cells, passive immunotherapy involves administration of autologous lymphocytes, without cancer specificity. In human medicine, this type of immunotherapy is referred to as lymphokine-activated killer (LAK) cell therapy and was among one of the first cell transfers [96] discarded in a favor of newer technologies (ACT therapy using TCR- or CAR-engineered T cells). However, LAK cells transfer was preferentially investigated in veterinary medicine, due to the low cost of cell generation. LAK cells are derived from PBMC stimulated with α-CD3 antibody-coated plates and soluble hrIL-2. Initially, LAK cell therapy was tested in healthy beagles [97]. Sequential administration of LAK cells increased host immune cell proliferation and IFN-γ levels in the serum without inducing severe side effects. These results suggested that LAK cell therapy is safe and could promote an immune response. Several papers described LAK cells’ generation and anticancer activity against canine thyroid carcinoma and melanoma cell lines in vitro [97,98,99]. Recently, LAK cell therapy, in combination with surgery, was evaluated in vivo in 15 tumor-bearing dogs [100]. Patients received five rounds of LAK cell transfer with 2–4 weeks intervals. Single transfer resulted in an increase of CD8+ T cells’ frequency and a decreased ratio of CD4+ to CD8+ T cells. Although the immunoenhancing effect of LAK cell transfer was confirmed, this type of immunotherapy alone has not mediated potent anticancer immune response or complete tumor eradication. Therefore, LAK cell transfer is not suitable as a monotherapy, but its application is promising as an adjuvant treatment.

A summary of different immunotherapeutic approaches in veterinary medicine have been compiled in Table 2.

Table 2 Adoptive cell transfer immunotherapies in veterinary medicine

Exploiting genome-editing technology to improve T cell phenotype

Genetic engineering technology is used for modifying of both TCR- and CAR-engineered T cells. Novel approaches focused on modulation of chemokine receptor expression and cytokine secretion of T lymphocytes or resistance to immunosuppressive tumor microenvironment by effector cells.

To date, three methods of genome editing have been implemented in the clinic: (1) zinc finger nucleases (ZFN), (2) transcription activator-like effector nuclease (TALEN) and the most recent (3) clustered regularly interspaced short palindromic repeats (CRISPR) associated protein system (CRISPR/Cas9) (Fig. 4) [101]. ZFN and TALEN are chimeric nucleases that contain a DNA-binding domain (engineered to recognize specific sequences) and a DNA cleavage enzyme (usually FokI) [102,103,104]. CRISPR/Cas9 system is based on RNA-guided DNA sequence recognition followed by cleavage by Cas9 endonuclease [105]. Each of these methods introduces nuclease-induced DNA double strand breaks into the sequence of interest. Importantly, these technologies not only improve gene silencing, but also allows for other genetic manipulations such as gene correction, deletion or insertion (Fig. 4) [106]. Thus, they appear to be a promising and useful tool for biomedical research.

Fig. 4
figure 4

Genome editing systems are used to introduce double-strand breaks into DNA, which allow for gene correction, deletion or addition ZFNs, TALENs and CRISPR/Cas9 are versatile nuclease-based platforms of genome editing technology. ZFNs and TALENs consist of DNA-binding domain, which is engineered to recognize specific sequences and nuclease domain—FokI—responsible for DNA cleavage. CRISPR/Cas9 is based on RNA-guided DNA recognition complex which interacts with Cas9 nuclease catalyzing site-specific breaks in DNA. Genome editing technologies can be used for the immunotherapy purposes to enhance T cells properties

The first in vivo success of this technology was achieved using ZFN to treat haemophilia in a murine model-correcting this genetic disorder and restorating normal blood coagulation [107]. Genome-editing technologies were also employed for treating of hereditary tyrosinemia (HTI), Duchenne muscular dystrophy (DMD) and hepatitis B virus (HBV) infection [108,109,110].

Recently, T cells modified using TALEN-based technology were transferred to leukemia patients [111]. Allogeneic healthy donor T cells were genetically engineered to prevent graft-versus-host disease (GVHD) upon adoptive cell transfer. Infused T cells did not cause an immune response in the recipient. This technique has been used just to prolong a patients’ survival time until suitable donor is found. In followup studies, CD19 CAR-T cells with disrupted TCR and CD52 coding genes were used to effectively treat B cell malignancies in infant [112]. Authors speculated that due to the lack of TCR expression, heterologous CAR-T cells can potentially be utilized for every patient with B cell leukemia [112, 113]. These study confirmed that gene editing technology may help to solve problems associated with engineering T cells. Similarly, ZFN-mediated disruption of the endogenous TCR genes in WT1 antigen-specific T cells prevented GVHD upon transfer into mice. This procedure prevents TCR heterodimmer formation, which is one of the major drawbacks of TCR-engineered T cell transfer therapy [114]. Recent advances in genetic manipulations can allowed to reduce adverse effects of ACT therapy and make it more widely applicable for the treatment for a range of malignancies. However, there are still several limitations of using genome editing technologies, which comprise: low efficiency of the method in some cell types, construct delivery problems, single-instead of double-stranded breaks, and off-target effects of nucleases and genetically modified cells (Table 1) [106, 115].

Genome editing has not been fully exploited in canine patients. Nevertheless, CRISPR/Cas9 method has been used to generate beagles with a myostatin gene knockout [116]. The study proved the feasibility of canine genome editing technique and has paved the way to generate other knockout dogs, potentially serving as models of multiple disease in the biomedical sciences.


In the past few years, we have witnessed amazing successes in harnessing immune system to fight against cancer. The American Society of Clinical Oncology selected cancer immunotherapy as the 2016 “Advance of the Year” for its unprecedented effectiveness in eradicating various types of tumors. In the future, more efforts should be made to utilize translational veterinary medicine to benefit human health. The domestic dog model offer multiple advantages to move cancer immunotherapies forward. Most importantly, these treatments may be also the silver lining in veterinary oncology giving hope for canine patients.



artificial antigen presenting cells


adoptive cell transfer


antibody-dependent cellular cytotoxicity


chimeric antigen receptor


chemokine (C-C motif) ligand 20


chemokine receptor 5


C-C chemokine receptor type 7


cluster of differentiation


concanavalin A


clustered regularly interspaced short palindromic repeats (CRISPR) associated protein system with Cas9 endonuclease


cytotoxic T cell antigen 4


cytotoxic T lymphocytes, CD8+ positive cells


dendritic cells


dog leukocyte antigen


Duchenne muscular dystrophy


graft-versus-host disease


hepatitis B virus


human epidermal growth factor receptor 2


human leukocyte antigen


hereditary tyrosinemia I


inducible T cell costimulator






lymphokine-activated killer cells


melanoma antigen recognized by T cells 1


myeloid-derived suppressor cells


major histocompatibility complex

NK cells:

natural killer cells

NKT cells:

natural killer T cells


cancer–testis antigen


canine osteosarcoma


programmed cell death protein 1




renal cell carcinoma antigen


recombinant human interleukin


retinoic acid-related orphan receptor


single chain variable fragment


transcription activator-like effector nuclease


tumor-associated macrophages


T cell receptor


tumor growth factor β

Th cells:

helper CD4+ T cells


tumor infiltrating lymphocytes


tumor necrosis factor α


T regulatory cells


Wilms tumor antigen-1


zinc finger nucleases


  1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70.

    CAS  Article  PubMed  Google Scholar 

  2. Mittal D, Gubin MM, Schreiber RD, Smyth MJ. New insights into cancer immunoediting and its three component phases–elimination, equilibrium and escape. Curr Opin Immunol. 2014;27:16–25.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. Kim R, Emi M, Tanabe K. Cancer immunoediting from immune surveillance to immune escape. Immunology. 2007;121:1–14.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell. 2010;140:883–99.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. Horna P, Sotomayor EM. Cellular and molecular mechanisms of tumor-induced T-cell tolerance. Curr Cancer Drug Targets. 2007;7:41–53.

    CAS  PubMed  Article  Google Scholar 

  6. Intlekofer AM, Thompson CB. At the bench: preclinical rationale for CTLA-4 and PD-1 blockade as cancer immunotherapy. J Leukoc Biol. 2013;94:25–39.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. Mytar B, Wołoszyn M, Szatanek R, Baj-Krzyworzeka M, Siedlar M, Ruggiero I, et al. Tumor cell-induced deactivation of human monocytes. J Leukoc Biol. 2003;74:1094–101.

    CAS  PubMed  Article  Google Scholar 

  8. Chanmee T, Ontong P, Konno K, Itano N. Tumor-associated macrophages as major players in the tumor microenvironment. Cancers. 2014;6:1670–90.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. Król M, Pawłowski KM, Majchrzak K, Dolka I, Abramowicz A, Szyszko K, et al. Density of tumor-associated macrophages (TAMs) and expression of their growth factor receptor MCSF-R and CD14 in canine mammary adenocarcinomas of various grade of malignancy and metastasis. Pol J Vet Sci. 2011;14:3–10.

    PubMed  Article  CAS  Google Scholar 

  10. Mucha J, Majchrzak K, Taciak B, Hellmén E, Król M. MDSCs mediate angiogenesis and predispose canine mammary tumor cells for metastasis via IL-28/IL-28RA (IFN-λ) signaling. PLoS ONE. 2014;9:e103249.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  11. Delorme E. Treatment of primary fibrosarcoma in the rat with immune lymphocytes. Lancet. 1964;284:117–20.

    Article  Google Scholar 

  12. Paoloni M, Khanna C. Translation of new cancer treatments from pet dogs to humans. Nat Rev Cancer. 2008;8:147–56.

    CAS  PubMed  Article  Google Scholar 

  13. Schiffman JD, Breen M. Comparative oncology: what dogs and other species can teach us about humans with cancer. Philos Trans R Soc B Biol Sci. 2015;370:20140231.

    Article  Google Scholar 

  14. Mata M, Gottschalk S. Man’s best friend: utilizing naturally occurring tumors in dogs to improve chimeric antigen receptor T-cell therapy for human cancers. Mol Ther. 2016;24:1511–2.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. Stroud C, Dmitriev I, Kashentseva E, Bryan JN, Curiel DT, Rindt H, et al. A one health overview, facilitating advances in comparative medicine and translational research. Clin Transl Med. 2016;5. Accessed 30 Jun 2017.

  16. Park JS, Withers SS, Modiano JF, Kent MS, Chen M, Luna JI, et al. Canine cancer immunotherapy studies: linking mouse and human. J Immunother Cancer. 2016; 4. Accessed 29 Jun 2017.

  17. Khanna C, Lindblad-Toh K, Vail D, London C, Bergman P, Barber L, et al. The dog as a cancer model. Nat Biotechnol. 2006;24:1065–6.

    CAS  PubMed  Article  Google Scholar 

  18. Bronson RT. Variation in age at death of dogs of different sexes and breeds. Am J Vet Res. 1982;43:2057–9.

    CAS  PubMed  Google Scholar 

  19. Anderson KL, Modiano JF. Progress in adaptive immunotherapy for cancer in companion animals: success on the path to a cure. Vet Sci. 2015;2:363–87.

    PubMed  PubMed Central  Article  Google Scholar 

  20. Nishiya A, Massoco C, Felizzola C, Perlmann E, Batschinski K, Tedardi M, et al. Comparative aspects of canine melanoma. Vet Sci. 2016;3:7.

    PubMed Central  Article  Google Scholar 

  21. Lindblad-Toh K, Wade CM, Mikkelsen TS, Karlsson EK, Jaffe DB, Kamal M, et al. Genome sequence, comparative analysis and haplotype structure of the domestic dog. Nature. 2005;438:803–19.

    CAS  PubMed  Article  Google Scholar 

  22. Muranski P, Restifo NP. Adoptive immunotherapy of cancer using CD4(+) T cells. Curr Opin Immunol. 2009;21:200–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Rosenberg SA, Restifo NP. Adoptive cell transfer as personalized immunotherapy for human cancer. Science. 2015;348:62–8.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  24. Rosenberg SA, Restifo NP, Yang JC, Morgan RA, Dudley ME. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer. 2008;8:299–308.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. Raval RR, Sharabi AB, Walker AJ, Drake CG, Sharma P. Tumor immunology and cancer immunotherapy: summary of the 2013 SITC primer. J Immunother Cancer. 2014;2:14.

    PubMed  PubMed Central  Article  Google Scholar 

  26. Mahmoud SMA, Paish EC, Powe DG, Macmillan RD, Grainge MJ, Lee AHS, et al. Tumor-infiltrating CD8+ lymphocytes predict clinical outcome in breast cancer. J Clin Oncol Off J Am Soc Clin Oncol. 2011;29:1949–55.

    Article  Google Scholar 

  27. Wang K, Xu J, Zhang T, Xue D. Tumor-infiltrating lymphocytes in breast cancer predict the response to chemotherapy and survival outcome: a meta-analysis. Oncotarget. 2014. Accessed 29 Jun 2017.

  28. Nguyen N, Bellile E, Thomas D, McHugh J, Rozek L, Virani S, et al. Tumor infiltrating lymphocytes and survival in patients with head and neck squamous cell carcinoma: tumor infiltrating lymphocytes. Head Neck. 2016;38:1074–84.

    PubMed  PubMed Central  Article  Google Scholar 

  29. Piras F, Colombari R, Minerba L, Murtas D, Floris C, Maxia C, et al. The predictive value of CD8, CD4, CD68, and human leukocyte antigen-D-related cells in the prognosis of cutaneous malignant melanoma with vertical growth phase. Cancer. 2005;104:1246–54.

    CAS  PubMed  Article  Google Scholar 

  30. van Houdt IS, Sluijter BJR, Moesbergen LM, Vos WM, de Gruijl TD, Molenkamp BG, et al. Favorable outcome in clinically stage II melanoma patients is associated with the presence of activated tumor infiltrating T-lymphocytes and preserved MHC class I antigen expression. Int J Cancer. 2008;123:609–15.

    PubMed  Article  CAS  Google Scholar 

  31. Sjödahl G, Lövgren K, Lauss M, Chebil G, Patschan O, Gudjonsson S, et al. Infiltration of CD3+ and CD68+ cells in bladder cancer is subtype specific and affects the outcome of patients with muscle-invasive tumors. Urol Oncol. 2014;32:791–7.

    PubMed  Article  Google Scholar 

  32. Hadrup SR, Braendstrup O, Jacobsen GK, Mortensen S, Pedersen LØ, Seremet T, et al. Tumor infiltrating lymphocytes in seminoma lesions comprise clonally expanded cytotoxic T cells. Int J Cancer. 2006;119:831–8.

    CAS  PubMed  Article  Google Scholar 

  33. Fukunaga A, Miyamoto M, Cho Y, Murakami S, Kawarada Y, Oshikiri T, et al. CD8+ tumor-infiltrating lymphocytes together with CD4+ tumor-infiltrating lymphocytes and dendritic cells improve the prognosis of patients with pancreatic adenocarcinoma. Pancreas. 2004;28:e26–31.

    PubMed  Article  Google Scholar 

  34. Piersma SJ, Jordanova ES, van Poelgeest MIE, Kwappenberg KMC, van der Hulst JM, Drijfhout JW, et al. High number of intraepithelial CD8+ tumor-infiltrating lymphocytes is associated with the absence of lymph node metastases in patients with large early-stage cervical cancer. Cancer Res. 2007;67:354–61.

    CAS  PubMed  Article  Google Scholar 

  35. Li J, Wang J, Chen R, Bai Y, Lu X. The prognostic value of tumor-infiltrating T lymphocytes in ovarian cancer. Oncotarget. 2017. Accessed 29 Jun 2017.

  36. Lee HE, Chae SW, Lee YJ, Kim MA, Lee HS, Lee BL, et al. Prognostic implications of type and density of tumour-infiltrating lymphocytes in gastric cancer. Br J Cancer. 2008;99:1704–11.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. Mei Z, Liu Y, Liu C, Cui A, Liang Z, Wang G, et al. Tumour-infiltrating inflammation and prognosis in colorectal cancer: systematic review and meta-analysis. Br J Cancer. 2014;110:1595–605.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Hershkovitz L, Schachter J, Treves AJ, Besser MJ. Focus on adoptive T cell transfer trials in melanoma. Clin Dev Immunol. 2010;2010:1–11.

    Article  CAS  Google Scholar 

  39. Rosenberg SA, Packard BS, Aebersold PM, Solomon D, Topalian SL, Toy ST, et al. Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. N Engl J Med. 1988;319:1676–80.

    CAS  PubMed  Article  Google Scholar 

  40. Dudley ME, Wunderlich J, Nishimura MI, Yu D, Yang JC, Topalian SL, et al. Adoptive transfer of cloned melanoma-reactive T lymphocytes for the treatment of patients with metastatic melanoma. J Immunother. 2001;24:363–73.

    CAS  PubMed  Article  Google Scholar 

  41. Dudley ME. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science. 2002;298:850–4.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Walter EA, Greenberg PD, Gilbert MJ, Finch RJ, Watanabe KS, Thomas ED, et al. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N Engl J Med. 1995;333:1038–44.

    CAS  PubMed  Article  Google Scholar 

  43. Matloubian M, Concepcion RJ, Ahmed R. CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection. J Virol. 1994;68:8056–63.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Dudley ME, Wunderlich JR, Yang JC, Sherry RM, Topalian SL, Restifo NP, et al. adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J Clin Oncol. 2005;23:2346–57.

    CAS  PubMed  Article  Google Scholar 

  45. Dudley ME, Yang JC, Sherry R, Hughes MS, Royal R, Kammula U, et al. Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens. J Clin Oncol. 2008;26:5233–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. Gattinoni L, Finkelstein SE, Klebanoff CA, Antony PA, Palmer DC, Spiess PJ, et al. Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells. J Exp Med. 2005;202:907–12.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. Besser MJ, Shapira-Frommer R, Treves AJ, Zippel D, Itzhaki O, Hershkovitz L, et al. Clinical responses in a phase II study using adoptive transfer of short-term cultured tumor infiltration lymphocytes in metastatic melanoma patients. Clin Cancer Res Off J Am Assoc Cancer Res. 2010;16:2646–55.

    CAS  Article  Google Scholar 

  48. Pilon-Thomas S, Kuhn L, Ellwanger S, Janssen W, Royster E, Marzban S, et al. Efficacy of adoptive cell transfer of tumor-infiltrating lymphocytes after lymphopenia induction for metastatic melanoma. J Immunother. 2012;35:615–20.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. Clay TM, Custer MC, Sachs J, Hwu P, Rosenberg SA, Nishimura MI. Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity. J Immunol. 1999;163:507–13.

    CAS  PubMed  Google Scholar 

  50. Kunert A, Straetemans T, Govers C, Lamers C, Mathijssen R, Sleijfer S, et al. TCR-engineered T cells meet new challenges to treat solid tumors: choice of antigen, T cell fitness, and sensitization of tumor milieu. Front Immunol. 2013;4:363.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  51. Robbins PF, Dudley ME, Wunderlich J, El-Gamil M, Li YF, Zhou J, et al. Cutting edge: persistence of transferred lymphocyte clonotypes correlates with cancer regression in patients receiving cell transfer therapy. J Immunol. 2004;173:7125–30.

    CAS  PubMed  Article  Google Scholar 

  52. Zhou J, Shen X, Huang J, Hodes RJ, Rosenberg SA, Robbins PF. Telomere length of transferred lymphocytes correlates with in vivo persistence and tumor regression in melanoma patients receiving cell transfer therapy. J Immunol. 2005;175:7046–52.

    CAS  PubMed  Article  Google Scholar 

  53. Knowles G, O’Neil BW, Campo MS. Phenotypical characterization of lymphocytes infiltrating regressing papillomas. J Virol. 1996;70:8451–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Nicholls PK, Moore PF, Anderson DM, Moore RA, Parry NR, Gough GW, et al. Regression of canine oral papillomas is associated with infiltration of CD4+ and CD8+ lymphocytes. Virology. 2001;283:31–9.

    CAS  PubMed  Article  Google Scholar 

  55. Pires I, Queiroga FL, Alves A, Silva F, Lopes C. Decrease of E-cadherin expression in canine cutaneous histiocytoma appears to be related to its spontaneous regression. Anticancer Res. 2009;29:2713–7.

    PubMed  Google Scholar 

  56. Carvalho MI, Pires I, Prada J, Queiroga FL. A role for T-lymphocytes in human breast cancer and in canine mammary tumors. Biomed Res Int. 2014;2014:130894.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  57. Kim J-H, Yu C-H, Yhee J-Y, Im K-S, Sur J-H. Lymphocyte infiltration, expression of interleukin (IL)-1, IL-6 and expression of mutated breast cancer susceptibility gene-1 correlate with malignancy of canine mammary tumours. J Comp Pathol. 2010;142:177–86.

    CAS  PubMed  Article  Google Scholar 

  58. Estrela-Lima A, Araujo MS, Costa-Neto JM, Teixeira-Carvalho A, Barrouin-Melo SM, Cardoso SV, et al. Immunophenotypic features of tumor infiltrating lymphocytes from mammary carcinomas in female dogs associated with prognostic factors and survival rates. BMC Cancer. 2010;10. Accessed 29 Jun 2017.

  59. Carvalho MI, Pires I, Prada J, Queiroga FL. T-lymphocytic infiltrate in canine mammary tumours: clinic and prognostic implications. Vivo Athens Greece. 2011;25:963–9.

    Google Scholar 

  60. Saeki K, Endo Y, Uchida K, Nishimura R, Sasaki N, Nakagawa T. Significance of tumor-infiltrating immune cells in spontaneous canine mammary gland tumor: 140 cases. J Vet Med Sci. 2012;74:227–30.

    PubMed  Article  Google Scholar 

  61. Kim ST, Jeong H, Woo OH, Seo JH, Kim A, Lee ES, et al. Tumor-infiltrating lymphocytes, tumor characteristics, and recurrence in patients with early breast cancer. Am J Clin Oncol. 2013;36:224–31.

    CAS  PubMed  Article  Google Scholar 

  62. Mucha J, Rybicka A, Dolka I, Szymańska J, Manuali E, Parzeniecka-Jaworska M, et al. Immunosuppression in dogs during mammary cancer development. Vet Pathol. 2016;53:1147–53.

    CAS  PubMed  Article  Google Scholar 

  63. Tominaga M, Horiuchi Y, Ichikawa M, Yamashita M, Okano K, Jikumaru Y, et al. Flow cytometric analysis of peripheral blood and tumor-infiltrating regulatory T cells in dogs with oral malignant melanoma. J Vet Diagn Investig. 2010;22:438–41.

    Article  Google Scholar 

  64. 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.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  65. 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;37:407–15.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  66. Yee C, Thompson JA, Byrd D, Riddell SR, Roche P, Celis E, et al. Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells. Proc Natl Acad Sci USA. 2002;99:16168–73.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  67. Butler MO, Friedlander P, Milstein MI, Mooney MM, Metzler G, Murray AP, et al. Establishment of antitumor memory in humans using in vitro-educated CD8+ T cells. Sci Transl Med. 2011;3:80ra34.

    PubMed  Article  CAS  Google Scholar 

  68. Gabert J, Langlet C, Zamoyska R, Parnes JR, Schmitt-Verhulst AM, Malissen B. Reconstitution of MHC class I specificity by transfer of the T cell receptor and Lyt-2 genes. Cell. 1987;50:545–54.

    CAS  PubMed  Article  Google Scholar 

  69. DembiĆ Z, Haas W, Weiss S, McCubrey J, Kiefer H, von Boehmer H, et al. Transfer of specificity by murine α and β T-cell receptor genes. Nature. 1986;320:232–8.

    PubMed  Article  Google Scholar 

  70. Engels B, Noessner E, Frankenberger B, Blankenstein T, Schendel DJ, Uckert W. Redirecting human T lymphocytes toward renal cell carcinoma specificity by retroviral transfer of T cell receptor genes. Hum Gene Ther. 2005;16:799–810.

    CAS  PubMed  Article  Google Scholar 

  71. Xue S-A, Gao L, Hart D, Gillmore R, Qasim W, Thrasher A, et al. Elimination of human leukemia cells in NOD/SCID mice by WT1-TCR gene-transduced human T cells. Blood. 2005;106:3062–7.

    CAS  PubMed  Article  Google Scholar 

  72. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science. 2006;314:126–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  73. Rapoport AP, Stadtmauer EA, Binder-Scholl GK, Goloubeva O, Vogl DT, Lacey SF, et al. NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat Med. 2015;21:914–21.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  74. Thomas S, Stauss HJ, Morris EC. Molecular immunology lessons from therapeutic T-cell receptor gene transfer. Immunology. 2010;129:170–7.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  75. Sommermeyer D, Neudorfer J, Weinhold M, Leisegang M, Engels B, Noessner E, et al. Designer T cells by T cell receptor replacement. Eur J Immunol. 2006;36:3052–9.

    CAS  PubMed  Article  Google Scholar 

  76. Sarukhan A, Garcia C, Lanoue A, von Boehmer H. Allelic inclusion of T cell receptor alpha genes poses an autoimmune hazard due to low-level expression of autospecific receptors. Immunity. 1998;8:563–70.

    CAS  PubMed  Article  Google Scholar 

  77. Gladow M, Uckert W, Blankenstein T. Dual T cell receptor T cells with two defined specificities mediate tumor suppression via both receptors. Eur J Immunol. 2004;34:1882–91.

    CAS  PubMed  Article  Google Scholar 

  78. Bendle GM, Linnemann C, Hooijkaas AI, Bies L, de Witte MA, Jorritsma A, et al. Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy. Nat Med. 2010;16:565–70 (1p following 570).

    CAS  PubMed  Article  Google Scholar 

  79. Linette GP, Stadtmauer EA, Maus MV, Rapoport AP, Levine BL, Emery L, et al. Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma. Blood. 2013;122:863–71.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  80. Barth SM, Schreitmüller CM, Proehl F, Oehl K, Lumpp LM, Kowalewski DJ, et al. Characterization of the Canine MHC Class I DLA-88*50101 Peptide binding motif as a prerequisite for canine T cell immunotherapy. PLoS ONE. 2016;11:e0167017.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  81. Garrido F, Ruiz-Cabello F, Cabrera T, Pérez-Villar JJ, López-Botet M, Duggan-Keen M, et al. Implications for immunosurveillance of altered HLA class I phenotypes in human tumours. Immunol Today. 1997;18:89–95.

    CAS  PubMed  Article  Google Scholar 

  82. Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci USA. 1989;86:10024–8.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  83. Jena B, Dotti G, Cooper LJN. Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor. Blood. 2010;116:1035–44.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  84. Gilham DE, Debets R, Pule M, Hawkins RE, Abken H. CAR–T cells and solid tumors: tuning T cells to challenge an inveterate foe. Trends Mol Med. 2012;18:377–84.

    CAS  PubMed  Article  Google Scholar 

  85. Berry LJ, Moeller M, Darcy PK. Adoptive immunotherapy for cancer: the next generation of gene-engineered immune cells. Tissue Antigens. 2009;74:277–89.

    CAS  PubMed  Article  Google Scholar 

  86. Dai H, Wang Y, Lu X, Han W. Chimeric antigen receptors modified t-cells for cancer therapy. J Natl Cancer Inst. 2016.

    Article  PubMed  PubMed Central  Google Scholar 

  87. Hombach AA, Abken H. Costimulation by chimeric antigen receptors revisited the T cell antitumor response benefits from combined CD28-OX40 signalling. Int J Cancer. 2011;129:2935–44.

    CAS  PubMed  Article  Google Scholar 

  88. Bridgeman JS, Hawkins RE, Hombach AA, Abken H, Gilham DE. Building better chimeric antigen receptors for adoptive T cell therapy. Curr Gene Ther. 2010;10:77–90.

    CAS  PubMed  Article  Google Scholar 

  89. Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 2013;368:1509–18.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  90. Louis CU, Savoldo B, Dotti G, Pule M, Yvon E, Myers GD, et al. Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma. Blood. 2011;118:6050–6.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  91. Kershaw MH, Westwood JA, Parker LL, Wang G, Eshhar Z, Mavroukakis SA, et al. A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer. Clin Cancer Res. 2006;12:6106–15.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  92. Lamers CHJ, Sleijfer S, Vulto AG, Kruit WHJ, Kliffen M, Debets R, et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. J Clin Oncol. 2006;24:e20–2.

    PubMed  Article  Google Scholar 

  93. Lamers CH, Sleijfer S, van Steenbergen S, van Elzakker P, van Krimpen B, Groot C, et al. Treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: clinical evaluation and management of on-target toxicity. Mol Ther J Am Soc Gene Ther. 2013;21:904–12.

    CAS  Article  Google Scholar 

  94. Zhang E, Xu H. A new insight in chimeric antigen receptor-engineered T cells for cancer immunotherapy. J Hematol Oncol. 2017;10:1.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  95. Panjwani MK, Smith JB, Schutsky K, Gnanandarajah J, O’Connor CM, Powell DJ, 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;24:1602–14.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  96. Rosenberg SA, Lotze MT. Cancer immunotherapy using interleukin-2 and interleukin-2-activated lymphocytes. Annu Rev Immunol. 1986;4:681–709.

    CAS  PubMed  Article  Google Scholar 

  97. Hoshino Y, Takagi S, Osaki T, Okumura M, Fujinaga T. Phenotypic analysis and effects of sequential administration of activated canine lymphocytes on healthy beagles. J Vet Med Sci. 2008;70:581–8.

    CAS  PubMed  Article  Google Scholar 

  98. Helfand SC, Soergel SA, Modiano JF, Hank JA, Sondel PM. Induction of lymphokine-activated killer (LAK) activity in canine lymphocytes with low dose human recombinant interleukin-2 in vitro. Cancer Biother. 1994;9:237–44.

    CAS  PubMed  Article  Google Scholar 

  99. Itoh H, Kakuta T, Kudo T, Sakonju I, Hohdatsu T, Ebina T, et al. Bulk cultures of canine peripheral blood lymphocytes with solid phase anti-CD3 antibody and recombinant interleukin-2 for use in immunotherapy. J Vet Med Sci. 2003;65:329–33.

    CAS  PubMed  Article  Google Scholar 

  100. Mie K, Shimada T, Akiyoshi H, Hayashi A, Ohashi F. Change in peripheral blood lymphocyte count in dogs following adoptive immunotherapy using lymphokine-activated T killer cells combined with palliative tumor resection. Vet Immunol Immunopathol. 2016;177:58–63.

    CAS  PubMed  Article  Google Scholar 

  101. Cox DBT, Platt RJ, Zhang F. Therapeutic genome editing: prospects and challenges. Nat Med. 2015;21:121–31.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  102. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. Genome editing with engineered zinc finger nucleases. Nat Rev Genet. 2010;11:636–46.

    CAS  PubMed  Article  Google Scholar 

  103. Nemudryi AA, Valetdinova KR, Medvedev SP, Zakian SM. TALEN and CRISPR/Cas genome editing systems: tools of discovery. Acta Nat. 2014;6:19–40.

    CAS  Google Scholar 

  104. Joung JK, Sander JD. TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol. 2013;14:49–55.

    CAS  PubMed  Article  Google Scholar 

  105. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157:1262–78.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  106. Gaj T, Gersbach CA, Barbas CF. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 2013;31:397–405.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  107. Li H, Haurigot V, Doyon Y, Li T, Wong SY, Bhagwat AS, et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature. 2011;475:217–21.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  108. Yin H, Xue W, Chen S, Bogorad RL, Benedetti E, Grompe M, et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol. 2014;32:551–3.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  109. Lin S-R, Yang H-C, Kuo Y-T, Liu C-J, Yang T-Y, Sung K-C, et al. The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo. Mol Ther Nucleic Acids. 2014;3:e186.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  110. Ousterout DG, Perez-Pinera P, Thakore PI, Kabadi AM, Brown MT, Qin X, et al. Reading frame correction by targeted genome editing restores dystrophin expression in cells from duchenne muscular dystrophy patients. Mol Ther. 2013;21:1718–26.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  111. Reardon S. Leukaemia success heralds wave of gene-editing therapies. Nature. 2015;527:146–7.

    CAS  PubMed  Article  Google Scholar 

  112. Qasim W, Zhan H, Samarasinghe S, Adams S, Amrolia P, Stafford S, et al. Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Sci Transl Med. 2017;9:eaaj2013.

    PubMed  Article  Google Scholar 

  113. Torikai H, Reik A, Liu P-Q, Zhou Y, Zhang L, Maiti S, et al. A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR. Blood. 2012;119:5697–705.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  114. Provasi E, Genovese P, Lombardo A, Magnani Z, Liu P-Q, Reik A, et al. Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer. Nat Med. 2012;18:807–15.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  115. Peng R, Lin G, Li J. Potential pitfalls of CRISPR/Cas9-mediated genome editing. FEBS J. 2016;283:1218–31.

    CAS  Article  PubMed  Google Scholar 

  116. Zou Q, Wang X, Liu Y, Ouyang Z, Long H, Wei S, et al. Generation of gene-target dogs using CRISPR/Cas9 system. J Mol Cell Biol. 2015;7:580–3.

    PubMed  Article  Google Scholar 

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Authors’ contributions

JKB provided the design of the paper, reviewed the literature and wrote the major part of the manuscript, prepared figures and tables. RP reviewed the literature, wrote the TILs and Lymphokine-activated killer cells paragraphs, formatted references list, and completed the tables, significantly contributed to the final outcome of the draft. MHN provided the human immunotherapy expertise, critically reviewed and contributed to the final draft of the manuscript. KM helped in conception and design of the paper, critically reviewed the manuscript, provided veterinary oncology expertise, and significantly contributes to the final draft of the manuscript. All authors read and approved the final manuscript.

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The authors declare that they have no competing interests.

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This work was supported, in part, by NCN UMO-2016/21/N/NZ6/02137 grant (to JKB) and programme First TEAM/2017-3/25 of Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund (to KM).

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Correspondence to Kinga Majchrzak.

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Bujak, J.K., Pingwara, R., Nelson, M.H. et al. Adoptive cell transfer: new perspective treatment in veterinary oncology. Acta Vet Scand 60, 60 (2018).

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  • Adoptive cell transfer
  • Canine oncology
  • Gene editing
  • Immunotherapy
  • T lymphocytes