Extracellular vesicles (EVs) have emerged as essential mediators of intercellular communication, transporting a complex repertoire of lipids, proteins, and nucleic acids that mirror the physiological and pathological status of their parent cells. This review provides a comprehensive overview of EVs from their biogenesis and molecular composition to their translational potential in human disease. This review outlines the major classes of EVs, including exosomes, microvesicles, apoptotic bodies, and oncosomes, together with recent developments in their isolation, molecular characterization, and omics-based profiling. Special focus is given to the role of EVs in viral infection, inflammation, and immune regulation, as well as their contribution to disease development and cancer biology. Moreover, we highlight the emerging clinical applications of mesenchymal stem cell-derived EVs (MSC-EVs) in regenerative medicine and oncology, alongside the therapeutic modulation of EV signaling by photobiomodulation (PBM). Finally, we address key translational challenges related to standardization, scalability, and regulatory validation. As exosome-based therapeutics fall under strict FDA and EMA oversight, their translation further depends on harmonized quality controls and robust safety evaluation. By integrating molecular mechanisms with clinical applications, this review emphasizes the transformative potential of EVs as next-generation diagnostic and therapeutic tools in precision medicine.

Extracellular vesicles (EVs) are membrane-bound nanoparticles released by almost all cell types into the extracellular space, acting as important mediators of intercellular communication by transferring proteins, lipids, and nucleic acids horizontally. EVs are generally classified into small EVs (200 nm), microvesicles, and apoptotic bodies, with current classification methods focusing on physical properties, molecular composition, and cellular origin, as detailed in the MISEV2023 guidelines. EVs are highly promising for diagnostic and therapeutic applications due to their intrinsic biocompatibility, stability in biological fluids, capacity to carry diverse molecular cargo, and potential for drug delivery and functionalization to enable targeted delivery and tissue repair. This narrative review discusses the emerging roles of EVs across various medical fields, including obstetrics and gynecology, ophthalmology, otorhinolaryngology, urology, oncology, orthopedics, neurology, immunology, wound healing, chronic pain management, dermatology, and cardiology. In each discipline, EVs show potential as biomarkers for diagnosing physiological or pathological conditions and as carriers for targeted drug delivery and regenerative treatments. Exosomes, a major type of small EVs, have especially attracted attention as versatile nanocarriers for precision medicine. However, translation into clinical practice requires addressing key pitfalls, including the standardization of isolation and characterization protocols, dose definition, GMP-compliant large-scale production, and regulatory approval. Ongoing interdisciplinary collaboration across disciplines and thorough clinical testing will be essential to unlock the full biomedical potential of EVs and establish them as transformative tools in personalized healthcare.

Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance. An intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (dbBiTES, 300 kDa). dbBiTEs were tested in vitro by EM, flow cytometry and cell cytoxicity and in vivo by PET tumor imaging and redirected T-cell therapy. The interlocked hinge regions are compatible with a structural model that fits the electron micrographs of 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES exhibit high in vitro cytotoxicity, high in vivo tumor targeting as demonstrated by PET imaging, and redirected dbBiTE coated T-cells (1 microgram/10 million cells) that kill CEA+ target cells in vivo in CEA transgenic mice. dbBiTE redirected T-cell therapy is a promising, efficient approach for targeting and killing cancer cells.

Background: Immunocytokines (ICKs) are antibody directed cytokines produced by genetic fusion of an antibody to a cytokine.Methods: We now show that antibodies conjugated by click chemistry to interleukin‐2 (IL‐2)‐Fc form fully active conjugates, and in one example, equivalent activity to a genetically produced ICK.Results: An IL‐2‐Fc fusion protein was optimized for click chemistry at hinge cysteines using protein stabilizing IL‐2 mutations at Lys35 and Cys125 and Fc hinge mutations at Cys142 and Cys148. The IL‐2‐Fc fusion protein with K35E and C125S mutations with 3 intact hinge cysteines, designated as IL‐2‐Fc Par, was selected based on its minimal tendency to aggregate. IL‐2‐Fc‐antibody clicked conjugates retained high IL‐2 activity and bound target antigens comparable to parent antibodies. An IL‐2‐Fc‐anti‐CEA click conjugate showed comparable anti‐tumor activity to an anti‐CEA‐IL‐2 ICK in immunocompetent CEA transgenic mice bearing CEA positive orthotopic breast tumors. Significant increases in IFNγ⁺/CD8⁺ and decreases in FoxP3⁺/CD4⁺ T‐cells were found for the clicked conjugate and ICK therapies, suggesting a common mechanism of tumor reduction.Conclusion: The production of antibody targeted IL‐2 therapy via a click chemistry approach is feasible with comparable activity to genetically produced ICKs with the added advantage of multiplexing with other monoclonal antibodies.

Background: The potent immune effects of interleukin‐2 (IL‐2) for cancer therapy can be increased by genetic fusion of IL‐2 to the Fc domain of an antibody (IL‐2‐Fc) or tumor targeted by genetic fusion to a whole antibody known as an immunocytokine (ICK).Methods: An anti‐CEA ICK (M5A‐IL‐2) was compared to an IL‐2‐Fc fusion protein using tumor therapy and PET imaging in CEA transgenic immunocompetent mice bearing CEA positive colon or breast tumors. Combination with stereotactic radiation therapy (SRT) was performed with either ICK or IL‐2‐Fc.Results: ICK and IL‐2‐Fc had comparable antitumor effects in both tumor models, although ICK had higher tumor uptake and slower blood clearance than an IL‐2‐Fc. Analysis of IFNγ⁺/CD8⁺ and FoxP3⁺/CD4⁺ T cells revealed higher levels of IFNγ‐producing CD8⁺ T cells in ICK treated mice versus more efficient Treg elimination in IL‐2‐Fc treated mice. No significant or lasting toxicity was detected for either agent. Combination therapies with SRT revealed comparable efficacy and induction of immune memory for both ICK and IL‐2‐Fc when mice were rechallenged post‐therapy.Conclusions: IL‐2‐Fc had comparable antitumor efficacy to CEA‐targeted M5A‐IL‐2 ICK, while both fusion proteins induced immune memory when combined with SRT. Differences in the therapeutic mechanisms of both agents were observed.

Background/Objectives: Immunocytokines (ICKs) are antibody–cytokine fusion proteins that deliver cytokines directly to tumors to increase immune responses, which are otherwise absent or limited by the delivery of antibodies alone. Tumor-associated glycoprotein-72 (TAG72) is overexpressed in numerous solid tumors. Methods: An anti-TAG72-IL-2 fusion protein was expressed in mammalian cells and tested in vitro for binding and bioactivity, and in vivo in two models. Results: In vitro studies showed high antigen specificity against TAG-72-positive tumor cell lines and IL-2 activity in CD25 (IL-2R)-positive reporter cells. To study the anti-tumor activity of huCC49-IL-2 in an immunocompetent model, the TAG-72 expression was established in murine mammary and colorectal cells by transfection with murine st6galnac-I gene (mSTn). Four daily doses of anti-TAG72-IL-2 monotherapy for TAG-72-expressing orthotopic murine mammary tumors in immunocompetent mice resulted in minimal whole-body toxicity and significant tumor growth reduction mediated by tumor infiltration of IFNγ⁺ CD8⁺ T cells. When mammary tumors were pretreated with image-guided fractionated radiation therapy (IGRT) followed by anti-TAG72-IL-2 therapy, an improved tumor growth inhibition was observed along with an increased tumor infiltration of IFNγ⁺ CD8⁺ T cells and a significant reduction in Foxp3⁺ CD4⁺ cells. Anti-TAG72-IL-2 monotherapy in TAG-72⁺ colorectal tumors led to a significant tumor reduction but also cures (4/7), with a rejection of rechallenges with both TAG-72-positive and -negative MC38 cells, thus demonstrating evidence of immune memory and antigen spreading. Conclusions: antiTAG-72-IL-2 therapy showed strong anti-tumor effects driven by activated CD8⁺ T cells making it a promising approach to the treatment of TAG-72⁺ tumors.