Preclinical murine models of chimeric antigen receptor (CAR) T cell therapy are widely applied, but are greatly limited by their inability to model the complex human tumor microenvironment and adequately predict safety and efficacy in patients. We therefore sought to develop a system that would enable us to evaluate CAR T cell therapies in dogs with spontaneous cancers. We developed an expansion methodology that yields large numbers of canine T cells from normal or lymphoma-diseased dogs. mRNA electroporation was utilized to express a first-generation canine CD20-specific CAR in expanded T cells. The canine CD20 (cCD20) CAR expression was efficient and transient, and electroporated T cells exhibited antigen-specific interferon-gamma (IFN-γ) secretion and lysed cCD20+ targets. In a first-in-canine study, autologous cCD20-ζ CAR T cells were administered to a dog with relapsed B cell lymphoma. Treatment was well tolerated and led to a modest, but transient, antitumor activity, suggesting that stable CAR expression will be necessary for durable clinical remissions. Our study establishes the methodologies necessary to evaluate CAR T cell therapy in dogs with spontaneous malignancies and lays the foundation for use of outbred canine cancer patients to evaluate the safety and efficacy of next-generation CAR therapies and their optimization prior to translation into humans.

We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells’ efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor in vitro. Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested in vitro by co-culture with canine tumor cells and in vivo in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells.

Mason (2020) Establishing a model system for evaluating CAR T cell therapy using dogs with spontaneous diffuse large B cell lymphoma, OncoImmunology, 9:1, 1676615,

NKG2C + CD8 + T cells, found in 40% of HCMV-seropositive individuals, have lost BCL11B expression and adopt an innate-like identity.

No abstract

Human CMV (HCMV) is a ubiquitous pathogen that indelibly shapes the NK cell repertoire. Using transcriptomic, epigenomic, and proteomic approaches to evaluate peripheral blood NK cells from healthy human volunteers, we find that prior HCMV infection promotes NK cells with a T celllike gene profile, including the canonical markers CD3e, CD5, and CD8b, as well as the T cell lineagecommitment transcription factor Bcl11b. Although Bcl11b expression is upregulated during NK maturation from CD56 bright to CD56 dim , we find a Bcl11b-mediated signature at the protein level for FceRIg, PLZF, IL-2Rb, CD3g, CD3d, and CD3e in later-stage, HCMV-induced NK cells. BCL11B is targeted by Notch signaling in T cell development, and culture of NK cells with Notch ligand increases cytoplasmic CD3e expression. The Bcl11b-mediated gain of CD3e, physically associated with CD16 signaling molecules Lck and CD247 in NK cells is correlated with increased Ab-dependent effector function, including against HCMV-infected cells, identifying a potential mechanism for their prevalence in HCMV-infected individuals and their prospective clinical use in Ab-based therapies.

Blood samples from the patients have been analyzed for CAR T cells (PCR) and immunosuppressive cells such as T regulatory cells (Treg), myeloid-derived suppressor cells (MDSCs) and M2 macrophages (CD163+). The CAR gene could be detected at the highest levels 1w post infusion and then the copy number varied over time. The levels were not correlated to ongoing response in terms of CR or PD but patients with higher levels commonly had a response to treatment and/or an ongoing cytokine release syndrome (CRS). Mild CRS or signs of neurological toxicity were noted in several patients but only 3 CRS and 1 neurological toxicity required hospitalization. The level of suppressive cells at enrollment did not correlate to response but a decline of suppressive cells over time was more often noted in responding patients. Proteomic analysis (ProSeek platform) has been done on patient plasma and is under evaluation to define response biomarkers. In summary, 14 patients have been treated with increasing doses of CAR T cells in Sweden. The conditioning has been relatively mild as compared to previous published studies and no lethal toxicity occurred. Six of the 14 treated patients had an initial CR, two of them and four other patients are still alive.

Chimeric antigen receptor (CAR) therapy has demonstrated great promise in treating human leukemias, but preclinical murine models are limited in their ability to predict safety and efficacy in humans. Given the rapid and on-going advances in CAR T cell technology in the laboratory, it now becomes necessary to identify and develop an outbred, large animal spontaneous cancer model in which the safety of novel targets and therapeutic effectiveness of re-directed T cells can be evaluated and optimized. Canines naturally develop spontaneous B cell lymphoma, and we have previously shown the feasibility of evaluating CAR T cell therapy in dogs using CD20-targeting RNA CAR T cells. To parallel human CAR T cell approaches, we have now optimized primary canine T cell transduction with lentivirus. Once peak T cell activation and artificial antigen presenting cell elimination timeframes were established, transduction yielded up to 26% CAR+ canine T cells. Canine T cells transduced with a second generation CD20-targeting CAR (CD20-28-ζ) demonstrated CAR-mediated, antigen-specific proliferation and efficient cytolysis of a CD20+ canine B cell lymphoma line in vitro. Three relapsed canine B cell lymphoma patients were treated with autologous CD20-28-ζ CAR T cells. Despite the administration of low numbers of CAR T cells, transient anti-tumor effects were observed in the first 2 patients. Following administration of ~700,000 CAR T cells/kg intravenously to the third patient, CAR T cells were found to persist and expand in the periphery and malignant nodes and disease progression was halted. This work establishes the feasibility of using the dogs with spontaneous cancer as pre-clinical models for advancing human CAR T cell therapy.