Sanaee, M. | bioRxiv Science

bioRxiv2025

A neoantigen-microbead platform for personalized T cell cancer vaccination

Colorectal cancer (CRC) is a leading cause of cancer mortality and is characterized by a high tumor mutational burden, making it responsive to immunotherapy. We developed a bioinformatic and manufacturing pipeline for personalized cancer vaccines and evaluated it in the MC38 colon adenocarcinoma mouse model. Thirty-six high-scoring neoantigens (NAGs) were identified by exome and transcriptome analysis, produced as six purified polypeptides, and coupled to paramagnetic beads. Intralymphatic vaccination of C57BL/6 mice with NAG beads induced robust NAG-specific T cell and antibody responses, resulting in significant inhibition of MC38 tumor growth. Treated tumors displayed increased necrosis and CD8+ T cell infiltration. Compared with soluble peptides, bead-coupled antigens elicited superior protection. Studies in T cell-deficient and antibody-depleted mice confirmed that both CD4+ and CD8+ T cells mediated the antitumor effect. These findings highlight the potential of NAG bead vaccination as an effective immunotherapy for CRC.

cancer biology↗

bioRxiv2021

Coincident Fluorescence Burst Analysis of dUTP-Loaded Exosome-Mimetic Nanovesicles

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/463914v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@13a85b2org.highwire.dtl.DTLVardef@11f6cd3org.highwire.dtl.DTLVardef@219a73org.highwire.dtl.DTLVardef@232f95_HPS_FORMAT_FIGEXP M_FIG C_FIG The targeting functionality and low immunogenicity of exosomes and exosome-mimetic nanovesicles make them promising as drug-delivery carriers. To tap into this potential, accurate non-destructive methods to load them and characterize their contents are of utmost importance. However, their small size, polydispersity and aggregation in solution make quantitative characterizations of their loading particularly challenging. Here we develop an ad-hoc methodology based on a burst analysis of dual-color confocal fluorescence microscopy experiments, suited for quantitative characterizations of exosome-like nanovesicles and of their loading. We apply it to study bioengineered nanovesicles, loaded with dUTP cargo molecules, synthetized from detergent-resistant membranes of animal extracellular vesicles and human red blood cells. For both classes of bioengineered nanovesicles we prove, by means of dual-color fluorescence cross-correlation spectroscopy (FCCS), successful loading. Furthermore, by a dual-color coincident fluorescence burst (DC-CFB) analysis of the experimental data, we retrieve size and loading statistics for both types of nanovesicles. The procedure affords single-vesicle characterizations, which are essential for reliable quantitative studies of loading processes in exosomes and exosome-mimetic nanovesicles, especially in light of the typically high heterogeneity of their populations. Moreover, the method implementation can be easily adapted to the investigation of a variety of combinations of different cargo molecules and biological nanovesicles besides the proof-of-principle demonstrations considered in this study. The results provide a powerful characterization tool, well-suited for the optimization of loading processes of biomimetic nanovesicles and their advanced engineering for therapeutic drug delivery.

biophysics↗