Local delivery of cell surface-targeted immunocytokines programs systemic antitumor immunity.

Systemically administered cytokines are potent immunotherapeutics but can cause severe dose-limiting toxicities. To overcome this challenge, cytokines have been engineered for intratumoral retention after local delivery. However, despite inducing regression of treated lesions, tumor-localized cytokines often elicit only modest responses at distal untreated tumors. In the present study, we report a localized cytokine therapy that safely elicits systemic antitumor immunity by targeting the ubiquitous leukocyte receptor CD45. CD45-targeted immunocytokines have lower internalization rates relative to wild-type counterparts, leading to sustained downstream cis and trans signaling between lymphocytes. A single intratumoral dose of αCD45-interleukin (IL)-12 followed by a single dose of αCD45-IL-15 eradicated treated tumors and untreated distal lesions in multiple syngeneic mouse tumor models without toxicity. Mechanistically, CD45-targeted cytokines reprogrammed tumor-specific CD8+ T cells in the tumor-draining lymph nodes to have an antiviral transcriptional signature. CD45 anchoring represents a broad platform for protein retention by host immune cells for use in immunotherapy.

Surfactant-Mediated Assembly of Precision-Size Liposomes.

Liposomes can greatly improve the pharmacokinetics of therapeutic agents due to their ability to encapsulate drugs and accumulate in target tissues. Considerable effort has been focused on methods to synthesize these nanocarriers in the past decades. However, most methods fail to controllably generate lipid vesicles at specific sizes and with low polydispersity, especially via scalable approaches suitable for clinical product manufacturing. Here, we report a surfactant-assisted liposome assembly method enabling the precise production of monodisperse liposomes with diameters ranging from 50 nm to 1 µm. To overcome scalability limitations, we used tangential flow filtration, a scalable size-based separation technique, to readily concentrate and purify the liposomal samples from more than 99.9% of detergent. Further, we propose two modes of liposome self-assembly following detergent dilution to explain the wide range of liposome size control, one in which phase separation into lipid-rich and detergent-rich phases drives the formation of large bilayer liposomes and a second where the rate of detergent monomer partitioning into solution controls bilayer leaflet imbalances that promote fusion into larger vesicles. We demonstrate the utility of controlled size assembly of liposomes by evaluating nanoparticle uptake in macrophages, where we observe a clear linear relationship between vesicle size and total nanoparticle uptake.

Local delivery of cell surface-targeted immunocytokines programs systemic anti-tumor immunity.

Cytokine therapies are potent immunotherapy agents but exhibit severe dose-limiting toxicities. One strategy to overcome this involves engineering cytokines for intratumoral retention following local delivery. Here, we develop a localized cytokine therapy that elicits profound anti-tumor immunity by engineered targeting to the ubiquitous leukocyte receptor CD45. We designed CD45-targeted immunocytokines (αCD45-Cyt) that, upon injection, decorated the surface of leukocytes in the tumor and tumor-draining lymph node (TDLN) without systemic exposure. αCD45-Cyt therapy eradicated both directly treated tumors and untreated distal lesions in multiple syngeneic mouse tumor models. Mechanistically, αCD45-Cyt triggered prolonged pSTAT signaling and reprogrammed tumor-specific CD8+ T cells in the TDLN to exhibit an anti-viral transcriptional signature. CD45 anchoring represents a broad platform for protein retention by host immune cells for use in immunotherapy.

Percutaneous Intratumoral Immunoadjuvant Gel Increases the Abscopal Effect of Cryoablation for Checkpoint Inhibitor Resistant Cancer.

Percutaneous cryoablation is a common clinical therapy for metastatic and primary cancer. There are rare clinical reports of cryoablation inducing regression of distant metastases, known as the "abscopal" effect. Intratumoral immunoadjuvants may be able to augment the abscopal rate of cryoablation, but existing intratumoral therapies suffer from the need for frequent injections and inability to confirm target delivery, leading to poor clinical trial outcomes. To address these shortcomings, an injectable thermoresponsive gel-based controlled release formulation is developed for the FDA-approved Toll-like-receptor 7 (TLR7) agonist imiquimod ("Imigel") that forms a tumor-resident depot upon injection and contains a contrast agent for visualization under computed tomography (CT). The poly-lactic-co-glycolic acid-polyethylene glycol-poly-lactic-co-glycolic acid (PLGA-PEG-PLGA)-based amphiphilic copolymer gel's underlying micellar nature enables high drug concentration and a logarithmic release profile that is additive with the neo-antigen release from cryoablation, requiring only a single injection. Rheological testing demonstrated the thermoresponsive increase in viscosity at body temperature and radio-opacity via microCT. Its ability to significantly augment the abscopal rate of cryoablation is demonstrated in otherwise immunotherapy resistant metastatic tumors in two aggressive colorectal and breast cancer dual tumor models with an all or nothing response, responders generally demonstrating complete regression of bilateral tumors in 90-day survival studies.

Characterizing residue transfer efficiencies using a fluorescent imaging technique.

To reduce the uncertainty associated with current estimates of children's exposure to pesticides by dermal contact and indirect ingestion, residue transfer data are required. Prior to conducting exhaustive studies, a screening study to identify the important parameters for characterizing these transfers was designed. A fluorescence imaging system was developed (Ivancic et al., in press) to facilitate collection of surface residue transfer data for repeated contacts. Next, parameters that affect residue transfer from surface-to-skin, skin-to-other objects, and skin-to-mouth were evaluated using the imaging system and the fluorescent tracer riboflavin as a surrogate for pesticide residues. Riboflavin was applied as a residue to surfaces of interest. Controlled transfer experiments were conducted by varying contact parameters with each trial. The mass of a tracer transferred was measured and the contact surface area estimated using video imaging techniques. Parameters evaluated included: surface type, surface loading, contact motion, pressure, duration, and skin condition. Transfers both onto, and off of, the hand were measured. To efficiently identify parameter changes resulting in significant effects, the Youden ruggedness test was used to select the combination of parameters varied in each contact trial. In this way, more than one parameter could be varied at a time and the number of trials required was minimized. Results of this study showed that surface loading and skin condition (significant at alpha=0.05) are among the important parameters for characterizing residue transfers of riboflavin. Duration of contact within the time range investigated does not have a significant effect on transfer of this tracer. Results of this study demonstrate the potential for collecting dermal transfer data using the Ivancic et al. fluorescence imaging system and provide preliminary data to reduce uncertainty associated with estimating dermal exposures resulting from contact with residue-contaminated surfaces. These data will also aid in determining what additional residue transfer data should be collected and what type of microactivity data are needed to estimate dermal and indirect ingestion exposure to residues on household surfaces.

Frequency of mouthing behavior in young children.

Young children may be more likely than adults to be exposed to pesticides following a residential application as a result of hand- and object-to-mouth contacts in contaminated areas. However, relatively few studies have specifically evaluated mouthing behavior in children less than 5 years of age. Previously unpublished data collected by the Fred Hutchinson Cancer Research Center (FHCRC) were analyzed to assess the mouthing behavior of 72 children (37 males/35 females). Total mouthing behavior data included the daily frequency of both mouth and tongue contacts with hands, other body parts, surfaces, natural objects, and toys. Eating events were excluded. Children ranged in age from 11 to 60 months. Observations for more than 1 day were available for 78% of the children. The total data set was disaggregated by gender into five age groups (10-20, 20-30, 30-40, 40-50, 50-60 months). Statistical analyses of the data were then undertaken to determine if significant differences existed among the age/gender subgroups in the sample. A mixed effects linear model was used to test the associations among age, gender, and mouthing frequencies. Subjects were treated as random and independent, and intrasubject variability was accounted for with an autocorrelation function. Results indicated that there was no association between mouthing frequency and gender. However, a clear relationship was observed between mouthing frequency and age. Using a tree analysis, two distinct groups could be identified: children < or = 24 and children >24 months of age. Children < or = 24 months exhibited the highest frequency of mouthing behavior with 81+/-7 events/h (mean+/-SE) (n=28 subjects, 69 observations). Children >24 months exhibited the lowest frequency of mouthing behavior with 42+/-4 events/h (n=44 subjects, 117 observations). These results suggest that children are less likely to place objects into their mouths as they age. These changes in mouthing behavior as a child ages should be accounted for when assessing aggregate exposure to pesticides in the residential environment.