Switchable control over in vivo CAR T expansion, B cell depletion, and induction of memory.

Chimeric antigen receptor (CAR) T cells with a long-lived memory phenotype are correlated with durable, complete remissions in patients with leukemia. However, not all CAR T cell products form robust memory populations, and those that do can induce chronic B cell aplasia in patients. To address these challenges, we previously developed a switchable CAR (sCAR) T cell system that allows fully tunable, on/off control over engineered cellular activity. To further evaluate the platform, we generated and assessed different murine sCAR constructs to determine the factors that afford efficacy, persistence, and expansion of sCAR T cells in a competent immune system. We find that sCAR T cells undergo significant in vivo expansion, which is correlated with potent antitumor efficacy. Most importantly, we show that the switch dosing regimen not only allows control over B cell populations through iterative depletion and repopulation, but that the "rest" period between dosing cycles is the key for induction of memory and expansion of sCAR T cells. These findings introduce rest as a paradigm in enhancing memory and improving the efficacy and persistence of engineered T cell products.

Ex Vivo Cell-Based Screening Platform for Modulators of Hepatosteatosis.

Nonalcoholic fatty liver disease (NAFLD) is the result of the ectopic accumulation of lipids in hepatic cells and is the early stage of liver diseases including fibrosis, cirrhosis, and hepatocellular carcinoma. While some mechanisms of aberrant lipid storage are understood, unbiased phenotypic drug screening holds the potential to identify new therapeutic small molecule mechanisms that reverse lipid accumulation in hepatic cells and prevent disease progression. Immortalized hepatocyte cell lines are often used as in vitro models of hepatocyte function, including in the study of lipid accumulation. However, mechanisms and therapeutic agents studied in these systems suffer from poor translation to primary cells and animal models of disease. Herein, we report an ex vivo high-throughput screening platform using primary mouse hepatocytes with a physiologically relevant lipid-laden phenotype isolated from mice that are administered a choline-methionine deficient diet. This screening platform using primary diseased hepatocytes may help to overcome a major hurdle in liver disease drug discovery and could lead to the development of new therapeutics for hepatosteatosis.

Engineering a long-acting, potent GLP-1 analog for microstructure-based transdermal delivery.

Antidiabetic treatments aiming to reduce body weight are currently gaining increased interest. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist administered twice daily via s.c. injection, improves glycemic control, often with associated weight reduction. To further improve the therapeutic efficacy of exendin-4, we have developed a novel peptide engineering strategy that incorporates a serum protein binding motif onto a covalent side-chain staple and applied to the peptide to enhance its helicity and, as a consequence, its potency and serum half-life. We demonstrated that one of the resulting peptides, E6, has significantly improved half-life and glucose tolerance in an oral glucose tolerance test in rodents. Chronic treatment of E6 significantly decreased body weight and fasting blood glucose, improved lipid metabolism, and also reduced hepatic steatosis in diet-induced obese mice. Moreover, the high potency of E6 allowed us to administer this peptide using a dissolvable microstructure-based transdermal delivery system. Pharmacokinetic and pharmacodynamic studies in guinea pigs showed that a single 5-min application of a microstructure system containing E6 significantly improved glucose tolerance for 96 h. This delivery strategy may offer an effective and patient-friendly alternative to currently marketed GLP-1 injectables and can likely be extended to other peptide hormones.

Design of Potent and Proteolytically Stable Oxyntomodulin Analogs.

Incretin-based peptides are effective therapeutics for treating type 2 diabetes mellitus (T2DM). Oxyntomodulin (OXM), a dual agonist of GLP-1R and GCGR, has shown superior weight loss and glucose lowering effects, compared to single GLP-1R agonists. To overcome the short half-life and rapid renal clearance of OXM, which limit its therapeutic potential, both lipid and PEG modified OXM analogs have been reported. However, these approaches often result in reduced potency or PEG-associated toxicity. Herein, we report a new class of cross-linked OXM analogs that show increased plasma stability and higher potency in activating both GLP-1R and GCGR. Moreover, the extended in vivo half-life results in superior antihyperglycemic activity in mice compared to the wild-type OXM.

A Small Molecule Inhibits Deregulated NRF2 Transcriptional Activity in Cancer.

NRF2 serves as the master regulator of oxidative stress resistance in mammalian cells. Although NRF2 activation decreases tumorigenic events in normal cells, accumulating evidence suggests that cancers have broadly selected for NRF2-activating mutations to promote anabolic growth and chemoresistance. Small molecules which inhibit NRF2 activity may therefore offer promise as an alternative anticancer treatment in NRF2 dependent cancers. We have used a high throughput screen to identify small molecules which decrease NRF2 transcriptional activity at antioxidant response element sites. One such molecule, termed AEM1, is capable of broadly decreasing the expression of NRF2 controlled genes, sensitizing A549 cells to various chemotherapeutic agents, and inhibiting the growth of A549 cells in vitro and in vivo. Profiling of multiple cell lines for their responsiveness to AEM1 revealed that AEM1's activities are restricted to cell lines harboring mutations which render NRF2 constitutively active.

Redirection of genetically engineered CAR-T cells using bifunctional small molecules.

Chimeric antigen receptor (CAR)-engineered T cells (CAR-Ts) provide a potent antitumor response and have become a promising treatment option for cancer. However, despite their efficacy, CAR-T cells are associated with significant safety challenges related to the inability to control their activation and expansion and terminate their response. Herein, we demonstrate that a bifunctional small molecule "switch" consisting of folate conjugated to fluorescein isothiocyanate (folate-FITC) can redirect and regulate FITC-specific CAR-T cell activity toward folate receptor (FR)-overexpressing tumor cells. This system was shown to be highly cytotoxic to FR-positive cells with no activity against FR-negative cells, demonstrating the specificity of redirection by folate-FITC. Anti-FITC-CAR-T cell activation and proliferation was strictly dependent on the presence of both folate-FITC and FR-positive cells and was dose titratable with folate-FITC switch. This novel treatment paradigm may ultimately lead to increased safety for CAR-T cell immunotherapy.

Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists.

On the basis of the 3D structure of a bovine antibody with a well-folded, ultralong complementarity-determining region (CDR), we have developed a versatile approach for generating human or humanized antibody agonists with excellent pharmacological properties. Using human growth hormone (hGH) and human leptin (hLeptin) as model proteins, we have demonstrated that functional human antibody CDR fusions can be efficiently engineered by grafting the native hormones into different CDRs of the humanized antibody Herceptin. The resulting Herceptin CDR fusion proteins were expressed in good yields in mammalian cells and retain comparable in vitro biological activity to the native hormones. Pharmacological studies in rodents indicated a 20- to 100-fold increase in plasma circulating half-life for these antibody agonists and significantly extended in vivo activities in the GH-deficient rat model and leptin-deficient obese mouse model for the hGH and hLeptin antibody fusions, respectively. These results illustrate the utility of antibody CDR fusions as a general and versatile strategy for generating long-acting protein therapeutics.