Chimeric antigen receptors with mutated IgG4 Fc spacer avoid fc receptor binding and improve T cell persistence and antitumor efficacy.

The success of adoptive therapy using chimeric antigen receptor (CAR)-expressing T cells partly depends on optimal CAR design. CARs frequently incorporate a spacer/linker region based on the constant region of either IgG1 or IgG4 to connect extracellular ligand-binding with intracellular signaling domains. Here, we evaluated the potential for the IgG4-Fc linker to result in off-target interactions with Fc gamma receptors (FcγRs). As proof-of-principle, we focused on a CD19-specific scFv-IgG4-CD28-zeta CAR and found that, in contrast to CAR-negative cells, CAR+ T cells bound soluble FcγRs in vitro and did not engraft in NSG mice. We hypothesized that mutations to avoid FcγR binding would improve CAR+ T cell engraftment and antitumor efficacy. Thus, we generated CD19-specific CARs with IgG4-Fc spacers that had either been mutated at two sites (L235E; N297Q) within the CH2 region (CD19R(EQ)) or incorporated a CH2 deletion (CD19Rch2Δ). These mutations reduced binding to soluble FcγRs without altering the ability of the CAR to mediate antigen-specific lysis. Importantly, CD19R(EQ) and CD19Rch2Δ T cells exhibited improved persistence and more potent CD19-specific antilymphoma efficacy in NSG mice. Together, these studies suggest that optimal CAR function may require the elimination of cellular FcγR interactions to improve T cell persistence and antitumor responses.

T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.

Induction treatments for acute myeloid leukemia (AML) have remained largely unchanged for nearly 50 years, and AML remains a disease of poor prognosis. Allogeneic hematopoietic cell transplantation can achieve cures in select patients and highlights the susceptibility of AML to donor-derived immunotherapy. The interleukin-3 receptor α chain (CD123) has been identified as a potential immunotherapeutic target because it is overexpressed in AML compared with normal hematopoietic stem cells. Therefore, we developed 2 chimeric antigen receptors (CARs) containing a CD123-specific single-chain variable fragment, in combination with a CD28 costimulatory domain and CD3-ζ signaling domain, targeting different epitopes on CD123. CD123-CAR-redirected T cells mediated potent effector activity against CD123+ cell lines as well as primary AML patient samples. CD123 CAR T cells did not eliminate granulocyte/macrophage and erythroid colony formation in vitro. Additionally, T cells obtained from patients with active AML can be modified to express CD123 CARs and are able to lyse autologous AML blasts in vitro. Finally, CD123 CAR T cells exhibited antileukemic activity in vivo against a xenogeneic model of disseminated AML. These results suggest that CD123 CAR T cells are a promising immunotherapy for the treatment of high-risk AML.

Review of Rodent Euthanasia Methods.

The optimal choice of euthanasia method for laboratory rodents depends on a number of factors, including the scientific goals of the study, the need to minimize animal pain and/or distress, applicable guidelines and laws, the training and proficiency of personnel, and the safety and emotional needs of the personnel performing the euthanasia. This manuscript aims to provide guidance to researchers so they may select the method of euthanasia that results in minimal experimental confounds, such as the creation of artifact and alteration of tissues and analytes. Specific situations addressed include euthanasia of large numbers of rodents and euthanasia of neonates. Recent literature supports the notion of significant strain-dependent differences in response to euthanasia methods such as CO2 inhalation. To assist researchers in selecting a strain-appropriate method of euthanasia, the authors present a summary of methodologies for assessing the effectiveness of euthanasia techniques, including elements and parameters for a scoring rubric to assess them.

An intact microbiota is required for the gastrointestinal toxicity of the immunosuppressant mycophenolate mofetil.

BACKGROUND: Mycophenolate mofetil (MMF) is commonly prescribed after transplantation and has major advantages over other immunosuppressive drugs, but frequent gastrointestinal (GI) side-effects limit its use. The mechanism(s) underlying MMF-related GI toxicity have yet to be elucidated. METHODS: To investigate MMF-related GI toxicity, experimental mice were fed chow containing MMF (0.563%) and multiple indices of toxicity, including weight loss and colonic inflammation, were measured. Changes in intestinal microbial composition were detected using 16S rRNA Illumina sequencing, and downstream PICRUSt analysis was used to predict metagenomic pathways involved. Germ-free (GF) mice and mice treated with orally administered broad-spectrum antibiotics (ABX) were utilized to interrogate the importance of the microbiota in MMF-induced GI toxicity. RESULTS: Mice treated with MMF exhibited significant weight loss, related to loss of body fat and muscle, and marked colonic inflammation. MMF exposure was associated with changes in gut microbial composition, as demonstrated by a loss of overall diversity, expansion of Proteobacteria (specifically Escherichia/Shigella), and enrichment of genes involved in lipopolysaccharide (LPS) biosynthesis, which paralleled increased levels of LPS in the feces and serum. MMF-related GI toxicity was dependent on the intestinal microbiota, as MMF did not induce weight loss or colonic inflammation in GF mice. Furthermore, ABX prevented and reversed MMF-induced weight loss and colonic inflammation. CONCLUSIONS: An intact intestinal microbiota is required to initiate and sustain the GI toxicity of MMF. MMF treatment causes dynamic changes in the composition of the intestinal microbiota that may be a targetable driver of the GI side-effects of MMF.

Comparison of a Supraglottic Airway Device (v-gel®) with Blind Orotracheal Intubation in Rabbits.

INTRODUCTION: Achieving a secure airway in rabbits is generally considered more difficult than in cats or dogs. Their relatively large tongue, small oropharyngeal cavity and glottis limit direct visualization. A rabbit-specific supraglottic airway device (SGAD) may offer benefits over blind orotracheal intubation. ANIMALS AND METHODS: Fifteen adult New Zealand white rabbits were randomized to SGAD or orotracheal intubation (ETT). All animals were sedated with dexmedetomidine (0.1 mg kg-1 IM) and midazolam (0.5 mg kg-1 IM), followed by induction with alfaxalone (0.3 mg kg-1 IV). Two CT scans of the head and neck were performed, following sedation and SGAD/ETT placement. The following were recorded: time to successful device insertion, smallest cross-sectional airway area, airway sealing pressure, and histological score of tracheal tissue. Data were analyzed with a Mann-Whitney test. RESULTS: Two rabbits were excluded following failed ETT. Body masses were similar [ETT; n = 6, 2.6 (2.3-4.5) kg, SGAD; n = 7, 2.7 (2.4-5.0) kg]. SGAD placement was significantly faster [33 (14-38) s] than ETT [59 (29-171) s]. Cross-sectional area (CSA) was significantly reduced from baseline [12.2 (6.9-3.4) mm2] but similar between groups [SGAD; 2.7 (2.0-12.3) mm2, ETT; 3.8 (2.3-6.6) mm2]. In the SGAD group, the device tip migrated into the laryngeal vestibule in 6/7 rabbits, reducing the CSA. ETT airway seals were higher [15 (10-20) cmH2O], but not significant [SGAD; 5 (5-20) cmH2O, p = 0.06]. ETT resulted in significantly more mucosal damage [histological score 3.3 (1.0-5.0)], SGAD; 0.67 (0.33-3.67). CONCLUSION: The SGAD studied was faster to place and caused less damage than orotracheal intubation, but resulted in a similar CSA.

Engineering human T cells for resistance to methotrexate and mycophenolate mofetil as an in vivo cell selection strategy.

Gene transfer and drug selection systems that enforce ongoing transgene expression in vitro and in vivo which are compatible with human pharmaceutical drugs are currently underdeveloped. Here, we report on the utility of incorporating human enzyme muteins that confer resistance to the lymphotoxic/immunosuppressive drugs methotrexate (MTX) and mycophenolate mofetil (MMF) in a multicistronic lentiviral vector for in vivo T lymphocyte selection. We found that co-expression of human dihydrofolate reductase (DHFR(FS); L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2(IY); T333I, S351Y) conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically (up to 0.1 µM MTX and 1.0 µM MPA). Furthermore, using a immunodeficient mouse model that supports the engraftment of central memory derived human T cells, in vivo selection studies demonstrate that huEGFRt(+)DHFR(FS+)IMPDH2(IY+) T cells could be enriched following adoptive transfer either by systemic administration of MTX alone (4.4 -fold), MMF alone (2.9-fold), or combined MTX and MMF (4.9-fold). These findings demonstrate the utility of both DHFR(FS)/MTX and IMPDH2(IY)/MMF for in vivo selection of lentivirally transduced human T cells. Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients.

Effects of cholesterol-tagged small interfering RNAs targeting 12/15-lipoxygenase on parameters of diabetic nephropathy in a mouse model of type 1 diabetes.

We previously showed that the 12/15-lipoxygenase (12/15-LO) pathway of arachidonate acid metabolism is involved in multiple events related to diabetic nephropathy (DN), including glomerular hypertrophy and extracellular matrix deposition (Kang SW, Adler SG, Nast CC, LaPage J, Gu JL, Nadler JL, Natarajan R. Kidney Int 59: 1354-1362, 2001; Kang SW, Natarajan R, Shahed A, Nast CC, LaPage J, Mundel P, Kashtan C, Adler SG. J Am Soc Nephrol 14: 3178-3187, 2003; Kim YS, Lanting L, Adler SG, Natarajan R. Kindney Int 64: 1702-1714, 2003; Reddy MA, Adler SG, Kim YS, Lanting L, Rossi JJ, Kang SW, Nadler JL, Shahed A, Natarajan R. Am J Physiol Renal Physiol 283: F985-F994, 2002). In this study, we investigated whether in vivo delivery of small interfering RNAs (siRNAs) targeting 12/15-LO can ameliorate renal injury and DN in a streptozotocin-injected mouse model of type 1 diabetes. To achieve greater in vivo access and siRNA expression in the kidney, we used double-stranded 12/15-LO siRNA oligonucleotides conjugated with cholesterol. Diabetic DBA/2J mice were injected subcutaneously with either cholesterol-tagged 12/15-LO siRNA, mismatched control siRNA, or vehicle alone, twice weekly for 7 wk. Relative to controls, mice that received 12/15-LO siRNA showed significant reduction in albuminuria, kidney-to-body weight ratios, glomerular mesangial matrix expansion, renal structural damage, and monocyte/macrophage infiltration. These effects were associated with lower renal cortical or glomerular levels of profibrotic markers transforming growth factor-beta, connective tissue growth factor, type I and type IV collagens, plasminogen activator inhibitor 1, and fibronectin. The diabetes-induced increase in glomerular cyclin-dependent kinase inhibitors that are associated with hypertrophy was also prevented by siRNA administration. Our results show for the first time that systemic delivery of cholesterol-tagged siRNAs targeting 12/15-LO has renoprotective effects under diabetic conditions and therefore could be a novel therapeutic approach for DN.