Preferential Localization of MUC1 Glycoprotein in Exosomes Secreted by Non-Small Cell Lung Carcinoma Cells.

Lung cancer remains to be the leading cause of cancer-related mortality worldwide. Finding new noninvasive biomarkers for lung cancer is still a significant clinical challenge. Exosomes are membrane-bound, nano-sized vesicles that are released by various living cells. Studies on exosomal proteomics may provide clues for developing clinical assays. In this study, we performed semi-quantitative proteomic analysis of proteins that were purified from exosomes of NCI-H838 non-small cell lung cancer cell line, with total cellular membrane proteins as control. In the exosomes, LC-MS/MS by data-independent analysis mode identified 3235 proteins. THBS1, ANXA6, HIST1H4A, COL18A1, MDK, SRGN, ENO1, TUBA4A, SLC3A2, GPI, MIF, MUC1, TALDO1, SLC7A5, ICAM1, HSP90AA1, G6PD, and LRP1 were found to be expressed in exosomes at more than 5-fold higher level as compared to total cellular membrane proteins. A well-known cancer biomarker, MUC1, is expressed at 8.98-fold higher in exosomes than total cellular membrane proteins. Subsequent analysis of plasma exosomes from non-small cell lung cancer (NSCLC) patients by a commercial electrochemiluminescence immunoassay showed that exosomal MUC1 level is 1.5-fold higher than healthy individuals (mean value 1.55 ± 0.16 versus mean value 1.05 ± 0.06, p = 0.0213). In contrast, no significant difference of MUC1 level was found between NSCLC patients and healthy individuals' plasma (mean value 5.48 ± 0.65 versus mean value 4.16 ± 0.49). These results suggest that certain proteins, such as MUC1, are selectively enriched in the exosome compartment. The mechanisms for their preferential localization and their biological roles remain to be studied.

Potent Neutralization Antibodies Induced by a Recombinant Trimeric Spike Protein Vaccine Candidate Containing PIKA Adjuvant for COVID-19.

The structures of immunogens that elicit the most potent neutralization antibodies to prevent COVID-19 infection are still under investigation. In this study, we tested the efficacy of a recombinant trimeric Spike protein containing polyI:C (PIKA) adjuvant in mice immunized by a 0-7-14 day schedule. The results showed that a Spike protein-specific antibody was induced at Day 21 with titer of above 50,000 on average, as measured by direct binding. The neutralizing titer was above 1000 on average, as determined by a pseudo-virus using monoclonal antibodies (40592-MM57 and 40591-MM43) with IC50 at 1 µg/mL as standards. The protein/peptide array-identified receptor-binding domain (RBD) was considered as immunodominant. No linear epitopes were found in the RBD, although several linear epitopes were found in the C-terminal domain right after the RBD and heptad repeat regions. Our study supports the efficacy of a recombinant trimeric Spike protein vaccine candidate for COVID-19 that is safe and ready for storage and distribution in developing countries.

An antibody-drug conjugate targeting a GSTA glycosite-signature epitope of MUC1 expressed by non-small cell lung cancer.

Antibodies targeting aberrantly glycosylated proteins are ineffective in treating cancer. Antibody-drug conjugates have emerged as effective alternatives, facilitating tumor-specific drug delivery. Previous studies have assessed the aberrantly glycosylated tandem repeat region of MUC1 glycoprotein as three site-specific glycosylated neoantigen peptide motifs (PDTR, GSTA, and GVTS) for binding with a monoclonal antibody. This study aimed to develop an antibody-drug conjugate for cancer treatment based on monoclonal antibodies against the aforementioned three neoantigen peptide motifs. Internalization of monoclonal antibodies was assessed via immunofluorescence staining and colocalization with lysosomal markers in live cells. Antibody positivity in tumor and peritumoral tissue samples was assessed via immunohistochemistry. The efficacy of anti-MUC1 ADCs was evaluated using various cancer cell lines and a mouse tumor xenograft model. An anti-MUC1 ADC was synthesized by conjugating GSTA neoantigen-specific 16A with monomethyl auristatin E (MMAE), which displayed potent antitumoral efficacy with an IC50 ranging 0.2-49.4 nM toward various cancer cells. In vivo, 16A-MMAE inhibited tumor growth in a dose-dependent manner in a mouse xenograft model established using the NCI-H838 NSCLC cell line, at a minimum effective dose of 1 mg/kg. At 3 mg/kg, 16A-MMAE did not cause significant toxicity in a transgenic mouse expressing human MUC1. The high antitumoral efficacy of 16A-MMAE suggests that aberrant glycosylated MUC1 neoantigen is a potential target for the development of ADCs for treating various cancers. Personalized therapy may be achieved through such glycosite-specific ADCs.