Department of Molecular and Medical Pharmacology

Targeted delivery of protein therapeutics remains challenging for translating biologics into effective treatments. Here, we introduce a universal strategy leveraging elevated glycolysis, a hallmark of many pathological states, and its resulting extracellular acidification as a navigational cue. Therapeutic proteins are encapsulated within pH-responsive polymer shells that remain near-neutral at physiological pH but gradually gain positive charge under acidic conditions. This dynamic charge modulation allows nanocapsules to sense pH gradients between healthy and diseased tissues, directing them toward pathological sites. Unlike receptor-mediated targeting that operates over nanometer scales, this receptor-independent approach enables long-range targeting. In vivo models of cancer, chronic inflammation, and acute injury demonstrate selective accumulation of encapsulated proteins at diseased sites, enhancing therapeutic efficacy while reducing systemic toxicity. By transforming a ubiquitous metabolic signature into a directional driving force, this lactate acid gradient-mediated targeting (LaGET) platform offers a previously underexplored paradigm for targeted delivery of protein therapeutics.

ConspectusExtracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by virtually all cells, carrying protected lipids, nucleic acids, proteins, and active enzymes that faithfully reflect the physiological and pathological states of their cellular origins. Tumor- and neuron-derived EVs are abundantly present in peripheral blood, even at early disease stages, and thus represent highly attractive substrates for liquid biopsy. However, the clinical translation of EV-based diagnostics has been constrained by a central challenge: the inability to selectively enrich disease-relevant EVs from a vast background of normal EVs with sufficient specificity, efficiency, and compatibility for seamless integration with downstream molecular and functional analyses. Conventional physical isolation approaches generate heterogeneous EV mixtures that dilute disease-specific signals, whereas traditional immunoaffinity capture often suffers from nonspecific interactions and low recovery due to sparse and heterogeneous antigen density on EV membranes.To overcome these limitations, our laboratory has developed a chemical biology solution utilizing the bioorthogonal inverse-electron-demand Diels-Alder reaction between trans-cyclooctene (TCO) and tetrazine (Tz). By labeling tumor or neuronal EVs in plasma with TCO-grafted antibodies and covalently immobilizing them onto Tz-functionalized substrates, our three EV enrichment platforms, namely, EV Click Chips, EV Click Beads, and EV Click MagBeads, enable rapid, irreversible, and highly specific capture of defined EV subpopulations. These click chemistry-mediated enrichment strategies reduce nonspecific binding, markedly improve capture efficiency, and preserve EV integrity, providing a robust foundation for downstream genetic, proteomic, and functional analyses. Building on this chemical biology solution, we established three complementary EV assay modalities. Platform #1, the EV Digital Scoring Assay, couples click chemistry-mediated EV enrichment with RT-digital PCR to quantify tumor-specific mRNAs or oncogenic mutations. This "enrich-then-count" strategy has demonstrated strong clinical utility in early detection of hepatocellular carcinoma (HCC), molecular staging of prostate cancer, and detection of actionable gene alterations in pancreatic cancer and Ewing sarcoma. A refined version enables real-time HCC treatment-response monitoring, outperforming serum AFP and radiographic criteria in monitoring treatment responses. Platform #2, the EV Surface Protein Assay, uses antibody-directed click enrichment followed by immuno-PCR or RT-qPCR to quantify tumor-specific EV subpopulations. Analogous to tissue immunohistochemistry but executed in a liquid-biopsy format, this assay has shown accuracy in early detection of HCC, pancreatic ductal adenocarcinoma, and epithelial ovarian cancer and supports longitudinal monitoring in prostate and thyroid cancers. Platform #3, the EV Protease Activity Assay, extends EV analysis into functional biology by measuring enzymatic activities preserved within enriched EVs. In osteosarcoma, matrix metalloproteinase activity profiles stratified localized versus metastatic disease and tracked therapeutic response. In neurology, quantifying β-secretase activity in neuronal EVs enabled highly accurate detection of early Alzheimer's disease and correlated with cognitive performance.Together, these TCO-Tz click chemistry-enabled platforms provide a modular, robust, and clinically adaptable toolkit for noninvasive EV-based diagnostics. By uniting chemical precision with biological and clinical relevance, this framework advances the broader vision of real-time, disease-specific liquid biopsy across oncology and neurodegeneration, laying the foundation for next-generation integrated diagnostic systems.

Fluorine-18 is often considered an ideal positron emitter owing to its excellent chemical, physiological, and nuclear properties. Consequently, the development of rapid, simple, and reliable ¹⁸F-labeling strategies remains critically important for synthesizing new radiopharmaceuticals for PET molecular imaging. A common approach involves the synthesis of ¹⁸F-labeled prosthetic groups that subsequently undergo bioconjugation with peptides or other biomolecules to generate ¹⁸F-labeled imaging probes. However, conventional synthetic methods for these prosthetic groups are often lengthy, require large quantities of precursor and solvent, and typically rely on elevated reaction temperatures. Herein, we report a droplet-based microscale synthetic methodology for the preparation of the [¹⁸F]FVSB prosthetic group that minimizes precursor and solvent usage, proceeds rapidly, and operates at relatively low temperatures. Conditions were optimized using a platform for performing droplet reactions in parallel, enabling high-throughput study of multiple reaction parameters within a short period of time. Additionally, we introduce a simple micro-cartridge purification technique that affords purified [¹⁸F]FVSB in small volumes. Furthermore, we describe an efficient bioconjugation that requires substantially lower reagent amounts than the previously reported macroscale method. The microscale process we report could facilitate wider use of this ¹⁸F-labeling strategy and can be extended to label other thiol-bearing peptides or biomolecules.

Ewing Sarcoma (ES) is a rare but aggressive malignancy of bone tissue in adolescents and young adults, where early detection of progression and real-time treatment monitoring remain unmet clinical needs. Tumor extracellular vesicles (EVs) carry surface markers and nucleic acid cargo that can serve as minimally invasive biomarkers, but single-marker EV assays often lack specificity, and colocalized-marker approaches may suffer from low sensitivity. Here, we report the ES EV Capture-Release-Capture (CaReCa) assay, a two-step enrichment strategy that combines desthiobiotin (DTB)-mediated capture/release of CD99⁺ EVs with click chemistry-mediated recapture of CD99⁺/B7-H3⁺ EVs, introducing molecular specificity to suppress background signals. To overcome limited yield from EVs with colocalized markers, we incorporated RT-digital PCR quantification of encapsulated ACTB mRNA, a stable housekeeping transcript, as a sensitive proxy for EV abundance. Using only 100 µL of plasma, the ES EV CaReCa assay distinguished ES patients (n = 20) from healthy donors (n = 20) with an AUROC of 0.98. Longitudinal analysis further demonstrated that dynamic changes in the assay readouts paralleled disease progression and treatment response, consistent with PET/CT findings. Together, these results establish CaReCa as a sensitive, specific, and scalable liquid biopsy platform with translational potential for noninvasive monitoring of ES patients.

PurposeThe prognosis for pediatric high-grade gliomas associated with mutations in the H3-3A gene is very poor. To investigate whether tumor lysate-pulsed dendritic cells (DC) together with checkpoint blockade might be a potential treatment modality for diffuse hemispheric glioma H3 G34-mutant (DHG), we have developed a novel syngeneic mouse model.MethodsWe used the RCAS/tv-A system to target the expression of H3G34R and PDGFβ and knock out p53 in neural progenitors in C57BL/6 neonatal mice. Three independent cell lines were obtained that expressed transcripts associated with oligodendrocyte and interneuron lineages. Lethal tumor developed following intracranial injection.ResultsTwo cycles of DC vaccination with PD-1 blockade decreased tumor burden and increased survival. In treatment resistant tumors we found higher expression of several genes involved in remodeling the extracellular matrix compared with tumors from untreated animals, suggesting a causal link to resistance to immunotherapy in this tumor model.ConclusionImmunotherapy involving autologous dendritic cells pulsed with tumor lysate and combined with anti-PD-1 antibody might be an effective treatment for DHG. Treatment failure in our tumor model is associated with increased expression of genes implicated in remodeling extracellular matrix in the tumor microenvironment.

Chronic granulomatous disease (CGD) is a severe monogenic immunodeficiency caused by damaging variants in genes required for microbicidal NADPH oxidase activity. Autosomal recessive p47^phox-deficient CGD (p47-CGD) is predominantly caused by a two-nucleotide deletion in exon 2 (delGT) of NCF1. We developed PM359, an autologous CD34+ hematopoietic stem-cell therapy in which prime editing is used to correct delGT. Two participants received PM359 after myeloid conditioning with busulfan: neutrophils and platelets engrafted promptly in both patients. Adverse events were consistent with myeloid conditioning with busulfan. NADPH oxidase activity was observed in neutrophils within 1 month and was maintained for 6 months and 4 months as of the last follow-up visit in Participants 1 and 2, respectively. These results support further investigation of prime editing of CD34+ cells to treat p47-CGD. (Funded by Prime Medicine; ClinicalTrials.gov number, NCT06559176.).

BackgroundPediatric high-grade gliomas (pHGG) are among the most aggressive childhood brain tumors, with limited treatment options and poor prognosis. Vaccine-based immunotherapy offers a promising strategy by leveraging tumor-specific or associated antigens to stimulate durable anti-tumor immune responses with minimal toxicity.DiscussionThis review outlines the scientific rationale for vaccine therapies in pHGG, detailing key targets such as glioma-associated antigens (EphA2, IL-13Rα2, survivin), driver mutation–derived neoantigens (H3.3K27M, TP53, IDH1), and viral antigens (CMV pp65). We evaluate current vaccine platforms, including peptide vaccines, dendritic cell vaccines, mRNA-based vaccines, and neoantigen-personalized approaches, highlighting early-phase clinical trial results that demonstrate safety and immunogenicity. Despite encouraging preliminary data, several challenges hinder clinical translation, including the distinct immune environment in the central nervous system, intratumoral heterogeneity, low mutational burden, immunosuppressive microenvironments, steroid use, and logistical hurdles in vaccine production and trial design. Future research must address these barriers through optimized antigen selection, combinatorial therapies, novel delivery systems, and pediatric-specific immune profiling.ConclusionWith continued multidisciplinary collaboration, vaccine therapies may emerge as a meaningful addition to the therapeutic arsenal for children with pHGG.

B cells play a crucial role in humoral immunity, acting as sentinels against viral infections by using their B cell receptors (BCRs) to recognize viral proteins. This recognition typically triggers a response leading to the production of neutralizing antibodies against viral surface proteins, such as the viral envelope proteins. However, recent studies have revealed a surprising dual role for BCRs, showing that some enveloped viruses and viral vectors, such as Dengue virus and lentiviral vectors, can exploit anti-viral BCRs as their attachment and entry receptors to infect/transduce B cells. While these viruses use a simple low-pH-dependent fusion mechanism for entry, it remained unclear whether BCRs could facilitate the entry of viruses with more complex fusion requirements, such as HIV-1 and SARS-CoV-2, which rely on their cognate receptors to activate their fusion machinery. In this study, we investigated the ability of BCRs to mediate viral entry for HIV-1 and SARS-CoV-2, which require specific host receptors (CD4 and ACE2, respectively) to activate their fusion machinery. We found that while anti-HIV-1 envelope protein BCRs can mediate viral attachment, they are unable to facilitate viral fusion and entry. In contrast, anti-SARS-CoV-2 Spike (S) protein BCRs not only mediate attachment but also enable viral entry in the absence of the ACE2 receptor. Our findings demonstrate that the ability of anti-viral BCRs to mediate viral fusion/entry is not universal but depends on the specific viral envelope protein. This novel entry pathway has important implications for both viral replication and the development of B cell-mediated immunity.

Fibroblast activation protein (FAP) has been proposed as a pan-tumor target for PET imaging using FAP-targeted tracers. Here, we explore the potential value of FAP PET in renal tumors. Methods: Six patients with renal tumors (4 with clear cell renal cell carcinoma, 1 with papillary renal cell carcinoma, and 1 with renal oncocytoma) who were included in a prospective imaging study (NCT04147494) underwent [⁶⁸Ga]Ga-FAPI-46 PET before nephrectomy. FAP PET radiotracer uptake and FAP expression by immunohistochemistry were assessed in the tumors and surrounding renal parenchyma. Results: Tumoral FAP radiotracer uptake was highest in clear cell renal cell carcinoma (median SUV_max, 3.1; range, 2.5-5.3), followed by renal oncocytoma (SUV_max, 1.9) and papillary renal cell carcinoma (SUV_max, 1.1). The FAP PET signal strongly correlated with FAP expression by immunohistochemistry (SUV_max; r = 0.93; P = 0.007). Conclusion: FAP expression in different renal tumors, including renal cell carcinoma, was lower when compared with cancers with known FAP expression, such as sarcoma. Although our data do not favor FAP-based theranostic approaches in renal cell carcinoma, studies in larger cohorts are warranted for conclusive evidence.