Stepwise Ultra-pH-Sensitive Micelles Overcome a pKa Barrier for Systemic Lymph Node Delivery.

While local nanoparticle delivery to lymph nodes is well studied, there are few design criteria for intravenous delivery to the entire lymph node repertoire. In this study, we investigated the effect of NP pH transition on lymph node targeting by employing a series of ultra-pH-sensitive (UPS) polymeric micelles. The UPS library responds to pH thresholds (pKa 6.9, 6.2, and 5.3) over a range of physiological pH. We observed a dependence of intravenous lymph node targeting on micelle pH transition. UPS6.9 (subscript indicates pKa) shows poor lymph node delivery, while UPS5.3 delivers efficiently to lymph node sets. We investigated targeting mechanisms of UPS5.3, observing an accumulation among lymph node lymphatics and a dependence on lymph node-resident macrophages. To overcome the pH-threshold barrier, which limits UPS6.9, we rationally designed a nanoparticle coassembly of UPS6.9 with UPS5.3, called HyUPS. The HyUPS micelle retains the constitutive pH transitions of each polymer, showing stepwise responses to discrete pH thresholds. We demonstrate that HyUPS improves UPS6.9 delivery to lymph nodes, extending this platform for disease detection of lymph node metastasis.

Loss of p53 and mutational heterogeneity drives immune resistance in an autochthonous mouse lung cancer model with high tumor mutational burden.

The role of tumor mutational burden (TMB) in shaping tumor immunity is a key question that has not been addressable using genetically engineered mouse models (GEMMs) of lung cancer. To induce TMB in lung GEMMs, we expressed an ultra-mutator variant of DNA polymerase-E (POLE)P286R in lung epithelial cells. Introduction of PoleP286R allele into KrasG12D and KrasG12D; p53L/L (KP) models significantly increase their TMB. Immunogenicity and sensitivity to immune checkpoint blockade (ICB) induced by Pole is partially dependent on p53. Corroborating these observations, survival of NSCLC patients whose tumors have TP53truncating mutations is shorter than those with TP53WT with immunotherapy. Immune resistance is in part through reduced antigen presentation and in part due to mutational heterogeneity. Total STING protein levels are elevated in Pole mutated KP tumors creating a vulnerability. A stable polyvalent STING agonist or p53 induction increases sensitivity to immunotherapy offering therapeutic options in these polyclonal tumors.

Elucidation of Protonation Cooperativity of a STING-Activating Polymer.

Stimuli-responsive nanomaterials have the potential to improve the performance and overcome existing barriers of conventional nanotherapeutics. Molecular cooperativity design in stimuli-responsive nanomedicine can amplify physiological signals, enabling a cooperative response for improved diagnostic and therapeutic precision. Previously, this work reported an ultra-pH-sensitive polymer, PEG-b-PC7A, that possesses innate immune activating properties by binding to the stimulator of interferon genes (STING) through polyvalent phase condensation. This interaction enhances STING activation and synergizes with the endogenous STING ligand for robust cancer immunotherapy. Despite its successes in innate immune activation, the fundamental physicochemical and pH-responsive properties of PC7A require further investigation. Here, this study elucidates the protonation cooperativity driven by the phase transition of PC7A copolymer. The highly cooperative system displays an "all-or-nothing" proton distribution between highly charged unimer (all) and neutral micelle (nothing) states without gradually protonated intermediates. The binary protonation behavior is further illustrated in pH-precision-controlled release of a representative anticancer drug, ß-lapachone, by PC7A micelles over a noncooperative PE5A polymer. Furthermore, the bimodal distribution of protons is represented by a high Hill coefficient (nH  > 9), featuring strong positive cooperativity. This study highlights the nanoscale pH cooperativity of an immune activating polymer, providing insights into the physicochemical characterization and design parameters for future nanotherapeutics development.

Polyvalent design in the cGAS-STING pathway.

Polyvalent interactions mediate the formation of higher-order macromolecular assemblies to improve the sensitivity, specificity, and temporal response of biological signals. In host defense, innate immune pathways recognize danger signals to alert host of insult or foreign invasion, while limiting aberrant activation from auto-immunity and cellular senescence. Of recent attention are the unique higher-order assemblies in the cGAS-STING pathway. Natural stimulation of cGAS enzymes by dsDNA induces phase separation and enzymatic activation for switchlike production of cGAMP. Subsequent binding of cGAMP to STING induces oligomerization of STING molecules, offering a scaffold for kinase assembly and signaling transduction. Additionally, the discovery of PC7A, a synthetic polymer which activates STING through a non-canonical biomolecular condensation, illustrates the engineering design of agonists by polyvalency principles. Herein, we discuss a mechanistic and functional comparison of natural and synthetic agonists to advance our understanding in STING signaling and highlight the principles of polyvalency in innate immune activation. The combination of exogenous cGAMP along with synthetic PC7A stimulation of STING offers a synergistic strategy in spatiotemporal orchestration of the immune milieu for a safe and effective immunotherapy against cancer.

Nano-Immune-Engineering Approaches to Advance Cancer Immunotherapy: Lessons from Ultra-pH-Sensitive Nanoparticles.

Immunotherapy has transformed the field of oncology and patient care. By leveraging the immune system of the host, immunostimulatory compounds exert a durable, personalized response against the patient's own tumor. Despite the clinical success, the overall response rate from current therapies (e.g., immune checkpoint inhibitors) remains low (∼20%) because tumors develop multiple resistance pathways at molecular, cellular, and microenvironmental levels. Unlike other oncologic therapies, harnessing antitumor immunity requires precise activation of a complex immunological system with multiple levels of regulation over its function. This requires the ability to exert control over immune cells in both intracellular compartments and various extracellular sites, such as the tumor microenvironment, in a spatiotemporally coordinated fashion.The immune system has evolved to sense and respond to nano- and microparticulates (e.g., viruses, bacteria) as foreign pathogens. Through the versatile control of composition, size, shape, and surface properties of nanoparticles, nano-immune-engineering approaches are uniquely positioned to mount appropriate immune responses against cancer. This Account highlights the development and implementation of ultra-pH-sensitive (UPS) nanoparticles in cancer immunotherapy with an emphasis on nanoscale cooperativity. Nanocooperativity has been manifested in many biological systems and processes (e.g., protein allostery, biomolecular condensation), where the system can acquire emergent properties distinct from the sum of individual parts acting in isolation.Using UPS nanoparticles as an example, we illustrate how all-or-nothing protonation cooperativity during micelle assembly/disassembly can be leveraged to augment the cancer-immunity cycle toward antitumor immunity. The cooperativity behavior enables instant and pH-triggered payload release and dose accumulation in acidic sites (e.g., endocytic organelles of antigen presenting cells, tumor microenvironment), intercepting specific immunological and tumor pathophysiological processes for therapy. These efforts include T cell activation in lymph nodes by coordinating cytosolic delivery of tumor antigens to dendritic cells with simultaneous activation of stimulator of interferon genes (STING), or tumor-targeted delivery of acidotic inhibitors to reprogram the tumor microenvironment and overcome T cell retardation. Each treatment strategy represents a nodal intervention in the cancer-immunity cycle, featuring the versatility of UPS nanoparticles. Overall, this Account aims to highlight nanoimmunology, an emerging cross field that exploits nanotechnology's unique synergy with immunology through nano-immune-engineering, for advancing cancer immunotherapy.

Detection of Lymph Node Metastases by Ultra-pH-Sensitive Polymeric Nanoparticles.

Lymph node (LN) dissection followed by histological analysis is the current standard for diagnosis of LN metastasis but the method suffers from patient morbidity and low sensitivity of detection. Ultra-pH sensitive (UPS) nanoparticles show remarkable accuracy in the delineation of primary tumor margins for precision cancer surgery. Herein we investigate the effectiveness of UPS nanoparticles to detect cancer-involved LNs. Methods: We synthesized a series of indocyanine green (ICG) conjugated UPS nanoparticles with distinct pKa (UPS5.3, UPS6.1, and UPS6.9). Systemically administered UPS-ICG nanoparticles in the 4T1.2-BALB/cj mouse model were imaged with real-time, near-infrared fluorescence (NIRF) to guide removal of LNs. Ex vivo imaging of gross tissue enabled quantification of fluorescence intensity. Histological analysis was used as the gold standard diagnostic test. Results: Macrophage uptake of UPS nanoparticles elevates the background signal in benign LNs. However, cancer foci within LNs show distinctive clustering of UPS-ICG fluorescence. UPS5.3 achieves accurate detection of metastatic LNs as shown by a receiver operating characteristic (ROC) area under the curve (AUC) of 0.96 ± 0.03. UPS6.1 and UPS6.9 offer decreased discriminatory power at ROC AUC of 0.73 ± 0.1 and 0.88 ± 0.07, respectively. Conclusions: All UPS compositions show cancer-specific discrimination of metastatic LNs over benign LNs with the best outcomes from UPS5.3. Detection of micro-metastatic LNs (cancer foci < 2 mm) remains a challenge. This study provides information on the detection of LN status for image-guided resection of metastatic LNs.