Repolarization of Tumor-Associated Macrophages in a Genetically Engineered Nonsmall Cell Lung Cancer Model by Intraperitoneal Administration of Hyaluronic Acid-Based Nanoparticles Encapsulating MicroRNA-125b.

Tumor-associated macrophages (TAMs) acquire a pro-tumor (M2) phenotype, which promotes tumor growth, angiogenesis, and metastasis. Certain microRNAs (miRs), such as miR-125b, can reprogram TAMs into an antitumor/pro-inflammatory (M1) phenotype. Using CD44 targeting hyaluronic acid-poly(ethylenimine) (HA-PEI)-based nanoparticles encapsulating miR-125b, we have herein shown macrophage-specific delivery and transfection upon intraperitoneal (i.p.) administration. We have exploited the inherent ability of peritoneal macrophages to migrate toward the inflammation/injury and demonstrated that following intraperitoneal administration of HA-PEI nanoparticles, there is an accumulation of HA-PEI nanoparticles in the macrophage-ablated lung tissues of both naïve and KRAS/p53 double mutant genetically engineered (KP-GEM) nonsmall cell lung cancer (NSCLC) mouse model. Additionally, upon transfection with miR-125b, we observed a >6-fold increase in the M1 to M2 macrophage ratio and 300-fold increase in the iNOS (M1 marker)/Arg-1 (M2 marker) ratio in TAMs as compared to the untreated control group. The results of these studies show that i.p. administered macrophage-specific HA-PEI nanoparticles can successfully transfect TAMs in lung tissues of both naïve mice and a KP-GEM NSCLC mouse model. Successful TAM repolarization toward the M1 phenotype has significant implication in anticancer immunotherapy.

A Choice-Based Design Approach to Influence Beneficial Use of a Green Space by Family Caregivers at a Tertiary Care Hospital in India.

OBJECTIVES, PURPOSES, AND AIMS: Principles of behavioral economics are now being used across fields in changing human behavior toward perceived benefit. We studied the effectiveness of simple, cost-effective design cues based on these principles, in encouraging use of a neglected outdoor space in a hospital. BACKGROUND: Benefits of access to nature and green spaces are established in healthcare; however, we found utilization of an outdoor space to be suboptimal. Presenting choices in different ways is known to influence user choice. METHODS: We designed four design interventions based to nudge relative-caregivers waiting outside the operation theater toward utilizing an adjoining outdoor balcony. These included making the terrace prominent, reducing indoor sensory stimulation, adding stereotypical elements, and improving usability. We measured usage via video camera data and satisfaction using questionnaires. RESULTS: The number of users and average time per person spent on the balcony increased over the consecutive intervention periods as compared to baseline (trend p < .01 for both), mainly driven by the addition of stereotypical elements. There were no adverse events in the balcony. There was no difference in the satisfaction related outcomes on questionnaire; a larger percentage of positive comments tended to be related to the balcony in the final intervention. CONCLUSIONS: Cost-effective design cues nudged patient-caregivers toward spending more time with nature, thus changing behavior toward that with perceived benefits.

Conditional Control of Universal CAR T Cells by Cleavable OFF-Switch Adaptors.

As living drugs, engineered T cell therapies are revolutionizing disease treatment with their unique functional capabilities. However, they suffer from limitations of potentially unpredictable behavior, toxicities, and nontraditional pharmacokinetics. Engineering conditional control mechanisms responsive to tractable stimuli such as small molecules or light is thus highly desirable. We and others previously developed "universal" chimeric antigen receptors (CARs) that interact with coadministered antibody adaptors to direct target cell killing and T cell activation. Universal CARs are of high therapeutic interest due to their ability to simultaneously target multiple antigens on the same disease or different diseases by combining with adaptors to different antigens. Here, we further enhance the programmability and potential safety of universal CAR T cells by engineering OFF-switch adaptors that can conditionally control CAR activity, including T cell activation, target cell lysis, and transgene expression, in response to a small molecule or light stimulus. Moreover, in adaptor combination assays, OFF-switch adaptors were capable of orthogonal conditional targeting of multiple antigens simultaneously, following Boolean logic. OFF-switch adaptors represent a robust new approach for the precision targeting of universal CAR T cells with potential for enhanced safety.

Conditional control of universal CAR T cells by cleavable OFF-switch adaptors.

As living drugs, engineered T cell therapies are revolutionizing disease treatment with their unique functional capabilities. However, they suffer from limitations of potentially unpredictable behavior, toxicities, and non-traditional pharmacokinetics. Engineering conditional control mechanisms responsive to tractable stimuli such as small molecules or light is thus highly desirable. We and others previously developed "universal" chimeric antigen receptors (CARs) that interact with co-administered antibody adaptors to direct target cell killing and T cell activation. Universal CARs are of high therapeutic interest due to their ability to simultaneously target multiple antigens on the same disease or different diseases by combining with adaptors to different antigens. Here, we further enhance the programmability and potential safety of universal CAR T cells by engineering OFF-switch adaptors that can conditionally control CAR activity, including T cell activation, target cell lysis, and transgene expression, in response to a small molecule or light stimulus. Moreover, in adaptor combination assays, OFF-switch adaptors were capable of orthogonal conditional targeting of multiple antigens simultaneously following Boolean logic. OFF-switch adaptors represent a robust new approach for precision targeting of universal CAR T cells with potential for enhanced safety.

Post-translational covalent assembly of CAR and synNotch receptors for programmable antigen targeting.

Chimeric antigen receptors (CARs) and synthetic Notch (synNotch) receptors are engineered cell-surface receptors that sense a target antigen and respond by activating T cell receptor signaling or a customized gene program, respectively. Here, to expand the targeting capabilities of these receptors, we develop "universal" receptor systems for which receptor specificity can be directed post-translationally via covalent attachment of a co-administered antibody bearing a benzylguanine (BG) motif. A SNAPtag self-labeling enzyme is genetically fused to the receptor and reacts with BG-conjugated antibodies for covalent assembly, programming antigen recognition. We demonstrate that activation of SNAP-CAR and SNAP-synNotch receptors can be successfully targeted by clinically relevant BG-conjugated antibodies, including anti-tumor activity of SNAP-CAR T cells in vivo in a human tumor xenograft mouse model. Finally, we develop a mathematical model to better define the parameters affecting universal receptor signaling. SNAP receptors provide a powerful strategy to post-translationally reprogram the targeting specificity of engineered cells.

BOXR1030, an anti-GPC3 CAR with exogenous GOT2 expression, shows enhanced T cell metabolism and improved anti-cell line derived tumor xenograft activity.

PURPOSE: The solid tumor microenvironment (TME) drives T cell dysfunction and inhibits the effectiveness of immunotherapies such as chimeric antigen receptor-based T cell (CAR T) cells. Early data has shown that modulation of T cell metabolism can improve intratumoral T cell function in preclinical models. EXPERIMENTAL DESIGN: We evaluated GPC3 expression in human normal and tumor tissue specimens. We developed and evaluated BOXR1030, a novel CAR T therapeutic co-expressing glypican-3 (GPC3)-targeted CAR and exogenous glutamic-oxaloacetic transaminase 2 (GOT2) in terms of CAR T cell function both in vitro and in vivo. RESULTS: Cell surface expression of tumor antigen GPC3 was observed by immunohistochemical staining in tumor biopsies from hepatocellular carcinoma, liposarcoma, squamous lung cancer, and Merkel cell carcinoma patients. Compared to control GPC3 CAR alone, BOXR1030 (GPC3-targeted CAR T cell that co-expressed GOT2) demonstrated superior in vivo efficacy in aggressive solid tumor xenograft models, and showed favorable attributes in vitro including an enhanced cytokine production profile, a less-differentiated T cell phenotype with lower expression of stress and exhaustion markers, an enhanced metabolic profile and increased proliferation in TME-like conditions. CONCLUSIONS: Together, these results demonstrated that co-expression of GOT2 can substantially improve the overall antitumor activity of CAR T cells by inducing broad changes in cellular function and phenotype. These data show that BOXR1030 is an attractive approach to targeting select solid tumors. To this end, BOXR1030 will be explored in the clinic to assess safety, dose-finding, and preliminary efficacy (NCT05120271).