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.

Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here, we isolated a high affinity (HA) folate receptor beta (FRβ)-specific scFv (2.48nM KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T-cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ+ AML in vitro and in vivo compared to a low affinity (LA) FRβ CAR (54.3nM KD). Using the HA-FRβ IgG, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34+ hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T-cells lysed mature CD14+ monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T-cells retained effective anti-tumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T-cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.

Acute stress impairs the retrieval of hippocampus-dependent memory, and this effect is mimicked by exogenous administration of stress-responsive glucocorticoid hormones. It has been proposed that glucocorticoids affect memory by promoting the release and/or blocking the reuptake of norepinephrine (NE), a stress-responsive neurotransmitter. It has also been proposed that this enhanced NE signaling impairs memory retrieval by stimulating β1-adrenergic receptors and elevating levels of cAMP. In contrast, other evidence indicates that NE, β1 and cAMP signaling are transiently required for the retrieval of hippocampus-dependent memory. To resolve this discrepancy, wild-type rats and mice with and without gene-targeted mutations were stressed or treated with glucocorticoids and/or adrenergic receptor drugs prior to testing memory for inhibitory avoidance or fear conditioning. Here we report that glucocorticoids do not require NE to impair retrieval. However, stress- and glucocorticoid-induced impairments of retrieval depend on the activation of β2 (but not β1)-adrenergic receptors. Offering an explanation for the opposing functions of these two receptors, the impairing effects of stress, glucocorticoids and β2 agonists on retrieval are blocked by pertussis toxin, which inactivates signaling by Gi/o-coupled receptors. In hippocampal slices, β2 signaling decreases cAMP levels and greatly reduces the increase in cAMP mediated by β1 signaling. Finally, augmenting cAMP signaling in the hippocampus prevents the impairment of retrieval by systemic β2 agonists or glucocorticoids. These results demonstrate that the β2 receptor can be a critical effector of acute stress, and that β1 and β2 receptors can have quite distinct roles in CNS signaling and cognition.

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We previously described a role for adrenergic signaling in the hippocampus to promote contextual and spatial memory retrieval. A subsequent study performing expression analysis of the immediate-early gene (IEG) Arc suggested that activation of CA1 but not CA3 pyramidal neurons during memory retrieval is impaired in the absence of NE. The current study sought to confirm and extend those observations by performing expression analysis of a second IEG product, Fos, following a much greater variety of testing conditions. In mutant mice lacking NE, induction of Fos was normal in all regions of the hippocampus and amygdala shortly after fear conditioning. In contrast, when testing contextual fear one day after training, induction of Fos in CA1 and the central nucleus of the amygdala (CeA), but not CA3, the dentate gyrus or other amygdaloid nuclei, was impaired in the mutant mice. This pattern corresponded to the memory retrieval deficit exhibited by these mice. On the other hand, induction was normal in CA1 and CeA when testing cued fear one day after training, or contextual fear one week or one month after training, conditions in which retrieval is normal in the absence of NE. Acute restoration of NE in the mutant mice before testing but not before training rescued retrieval of contextual fear and restored Fos induction in CA1 and CeA. Because NE facilitates retrieval through the activation of β1-adrenergic receptors, β1 knockout mice were also examined and found to exhibit reduced induction of Fos in CA1 and CeA following retrieval. Based on these and previous results, we hypothesize that adrenergic signaling is critical for the full activation of CA1 pyramidal neurons in response to excitatory input from CA3 pyramidal neurons conveying retrieved contextual information.

Using lentiviral technology, we recently demonstrated that incorporation of CD27 costimulation into CARs greatly improves antitumor activity and T cell persistence. Still, virus-mediated gene transfer is expensive, laborious and enables long-term persistence, creating therapies which cannot be easily discontinued if toxic. To address these concerns, we utilized a non-integrating RNA platform to engineer human T cells to express FRα-specific, CD27 CARs and tested their capacity to eliminate human FRα+ cancer. Novel CARs comprised of human components were constructed, C4-27z and C4opt-27z, a codon-optimized variant created for efficient expression. Following RNA electroporation, C4-27z and C4opt-27z CAR expression is initially ubiquitous but progressively declines across T cell populations. In addition, C4-27z and C4opt-27z RNA CAR T cells secrete high levels of Th-1 cytokines and display strong cytolytic function against human FRα+ cancers in a time- and antigen-dependent manner. Further, C4-27z and C4opt-27z CAR T cells exhibit significant proliferation in vivo, facilitate the complete regression of fully disseminated human ovarian cancer xenografts in mice and reduce the progression of solid ovarian cancer. These results advocate for rapid progression of C4opt-27z RNA CAR to the clinic and establish a new paradigm for preclinical optimization and validation of RNA CAR candidates destined for clinical translation.

Xamoterol, a partial b 1 -adrenergic receptor agonist, has been reported to impair the retrieval of hippocampus-dependent spatial reference memory in rats. In contrast, xamoterol restores memory retrieval in gene-targeted mice lacking norepinephrine (NE) and in a transgenic mouse model of Down syndrome in which NE levels are reduced. Restoration of retrieval by xamoterol in these two models complements the observation that NE and b 1 signaling are required for hippocampusdependent retrieval of contextual and spatial reference memory in wild-type mice and rats. Additional evidence indicates that cAMP-mediated PKA and Epac signaling are required for the retrieval of hippocampus-dependent memory. As a result, we hypothesized that xamoterol has effects in addition to the stimulation of b 1 receptors that, at higher doses, act to counter the effects of b 1 signaling. Here we report that xamoterol-induced disruption of memory retrieval depends on b 2 -adrenergic receptor signaling. Interestingly, the impairment of memory retrieval by xamoterol is blocked by pretreatment with pertussis toxin, an uncoupling agent for G i/o signaling, suggesting that b 2 signaling opposes b 1 signaling during memory retrieval at the level of G protein and cAMP signaling. Finally, similar to the time-dependent roles for NE, b 1 , and cAMP signaling in hippocampus-dependent memory retrieval, xamoterol only impairs retrieval for several days after training, indicating that its effects are also limited by the age of the memory. We conclude that the disruption of memory retrieval by xamoterol is mediated by G i/o -coupled b 2 signaling, which opposes the G s -coupled b 1 signaling that is transiently required for hippocampus-dependent emotional memory retrieval.

A single intramuscular application of the live but not UV-inactivated recombinant rabies virus (RABV) variant TriGAS in mice induces the robust and sustained production of RABV-neutralizing antibodies that correlate with long-term protection against challenge with an otherwise lethal dose of the wild-type RABV. To obtain insight into the mechanism by which live TriGAS induces long-lasting protective immunity, quantitative PCR (qPCR) analysis of muscle tissue, draining lymph nodes, spleen, spinal cord, and brain at different times after TriGAS inoculation revealed the presence of significant copy numbers of RABV-specific RNA in muscle, lymph node, and to a lesser extent, spleen for several days postinfection. Notably, no significant amounts of RABV RNA were detected in brain or spinal cord at any time after TriGAS inoculation. Differential qPCR analysis revealed that the RABV-specific RNA detected in muscle is predominantly genomic RNA, whereas RABV RNA detected in draining lymph nodes is predominantly mRNA. Comparison of genomic RNA and mRNA obtained from isolated lymph node cells showed the highest mRNA-to-genomic-RNA ratios in B cells and dendritic cells (DCs), suggesting that these cells represent the major cell population that is infected in the lymph node. Since RABV RNA declined to undetectable levels by 14 days postinoculation of TriGAS, we speculate that a transient infection of DCs with TriGAS may be highly immunostimulatory through mechanisms that enhance antigen presentation. Our results support the superior efficacy and safety of TriGAS and advocate for its utility as a vaccine. Rabies is a zoonotic disease that continues to be an important public health problem causing an estimated 55,000 human deaths each year globally, the majority of which occur in Africa and Asia (1). In Asia, parts of America, and large parts of Africa, canines remain the principal host of rabies virus (RABV), and more than 95% of all human rabies cases are caused by exposure to infected dogs (2, 3). The most effective means to protect humans and livestock against rabies is by prophylactic immunization with an RABV vaccine. Although inactivated tissue culture RABV vaccines are safe, they induce protective immunity only when administered in multiple doses and generally require repeat booster doses to provide long-lasting protection. Repeated prophylactic immunization is cost-prohibitive and operationally unrealistic for both people and animals in developing countries. A single-dose RABV vaccine capable of inducing robust, enduring immunity would be highly advantageous in controlling rabies in dogs, livestock, and humans worldwide.A hallmark of wild-type RABV is its neuroinvasiveness-a unique ability to invade the central nervous system (CNS) from peripheral sites of inoculation (4, 5). Important aspects of this property include the capacity to preserve the integrity of the neuronal network and the ability to evade host immunity (6, 7). One of the important mechanisms that contribute to both of these features of rabies pathogenes...