Development of nanobodies targeting hepatocellular carcinoma and application of nanobody-based CAR-T technology.

BACKGROUND: Chimeric antigen receptor T (CAR-T) cell therapy, as an emerging anti-tumor treatment, has garnered extensive attention in the study of targeted therapy of multiple tumor-associated antigens in hepatocellular carcinoma (HCC). However, the suppressive microenvironment and individual heterogeneity results in downregulation of these antigens in certain patients' cancer cells. Therefore, optimizing CAR-T cell therapy for HCC is imperative. METHODS: In this study, we administered FGFR4-ferritin (FGFR4-HPF) nanoparticles to the alpaca and constructed a phage library of nanobodies (Nbs) derived from alpaca, following which we screened for Nbs targeting FGFR4. Then, we conducted the functional validation of Nbs. Furthermore, we developed Nb-derived CAR-T cells and evaluated their anti-tumor ability against HCC through in vitro and in vivo validation. RESULTS: Our findings demonstrated that we successfully obtained high specificity and high affinity Nbs targeting FGFR4 after screening. And the specificity of Nbs targeting FGFR4 was markedly superior to their binding to other members of the FGFR family proteins. Furthermore, the Nb-derived CAR-T cells, targeting FGFR4, exhibited significantly enhanced anti-tumor efficacy in both experiments when in vitro and in vivo. CONCLUSIONS: In summary, the results of this study suggest that the CAR-T cells derived from high specificity and high affinity Nbs, targeting FGFR4, exhibited significantly enhanced anti-tumor efficacy in vitro and in vivo. This is an exploration of FGFR4 in the field of Nb-derived CAR-T cell therapy for HCC, holding promise for enhancing safety and effectiveness in the clinical treatment of HCC in the future.

The receptor binding domain of SARS-CoV-2 Omicron subvariants targets Siglec-9 to decrease its immunogenicity by preventing macrophage phagocytosis.

The development of a vaccine specific to severe acute respiratory syndrome coronavirus 2 Omicron has been hampered due to its low immunogenicity. Here, using reverse mutagenesis, we found that a phenylalanine-to-serine mutation at position 375 (F375S) in the spike protein of Omicron to revert it to the sequence found in Delta and other ancestral strains significantly enhanced the immunogenicity of Omicron vaccines. Sequence FAPFFAF at position 371-377 in Omicron spike had a potent inhibitory effect on macrophage uptake of receptor-binding domain (RBD) nanoparticles or spike-pseudovirus particles containing this sequence. Omicron RBD enhanced binding to Siglec-9 on macrophages to impair phagocytosis and antigen presentation and promote immune evasion, which could be abrogated by the F375S mutation. A bivalent F375S Omicron RBD and Delta-RBD nanoparticle vaccine elicited potent and broad nAbs in mice, rabbits and rhesus macaques. Our research suggested that manipulation of the Siglec-9 pathway could be a promising approach to enhance vaccine response.

A Mosaic Nanoparticle Vaccine Elicits Potent Mucosal Immune Response with Significant Cross-Protection Activity against Multiple SARS-CoV-2 Sublineages.

Because of the rapid mutation and high airborne transmission of SARS-CoV-2, a universal vaccine preventing the infection in the upper respiratory tract is particularly urgent. Here, a mosaic receptor-binding domain (RBD) nanoparticle (NP) vaccine is developed, which induces more RBD-targeted type IV neutralizing antibodies (NAbs) and exhibits broad cross-protective activity against multiple SARS-CoV-2 sublineages including the newly-emerged BF.7, BQ.1, XBB. As several T-cell-reactive epitopes, which are highly conserved in sarbecoviruses, are displayed on the NP surface, it also provokes potent and cross-reactive cellular immune responses in the respiratory tissue. Through intranasal delivery, it elicits robust mucosal immune responses and full protection without any adjuvants. Therefore, this intranasal mosaic NP vaccine can be further developed as a pan-sarbecovirus vaccine to block the viral entrance from the upper respiratory tract.

Dopamine Signaling Promotes Tissue-Resident Memory Differentiation of CD8+ T Cells and Antitumor Immunity.

Tissue-resident memory CD8+ T (TRM) cells have been associated with robust protective antitumor immune responses and improved prognosis of patients with cancer. Therefore, therapeutic strategies that modulate either the production or activity of TRM cells could be effective for treating cancer. Using a high-throughput drug screen, we showed that the neurotransmitter dopamine drives differentiation of CD8+ T cells into CD103+ TRM cells. In murine syngeneic tumor xenograft models and clinical human colon cancer samples, DRD5 served as the major functional dopamine receptor on CD8+ T cells and positively correlated with TRM cell density. DRD5 deficiency led to a failure of CD8+ T cells to accumulate in tissues, resulting in impaired TRM cell formation, reduced effector function, and uncontrolled disease progression. Moreover, dopamine treatment promoted the antitumor activity of CD8+ T cells and suppressed colorectal cancer growth in immunocompentent mouse models, and ex vivo preconditioning with dopamine enhanced the in vivo efficacy of chimeric antigen receptor (CAR)-T cells. Finally, in a patient with colorectal cancer cohort, dopamine expression was positively associated with patient survival and CD8+ T-cell infiltration. These findings suggest that dopaminergic immunoregulation plays an important role in the differentiation of CD8+ cells into CD103+ TRM cells and thereby modulates TRM-elicited antitumor immunity in colorectal cancer. SIGNIFICANCE: Identification of an immunostimulatory function of dopamine signaling by promoting tissue-resident memory T-cell differentiation and sustaining T-cell effector functions reveals potential therapeutic strategies and prognostic biomarkers for colorectal cancer.

MicroRNA-150 functions as a tumor suppressor and sensitizes osteosarcoma to doxorubicin-induced apoptosis by targeting RUNX2.

Osteosarcoma (OS) is the most common form of bone malignancy in children and adolescents. MicroRNAs (miRNAs) have been associated with the development and progression of OS. In the present study, reverse transcription-quantitative PCR, western blotting, Cell Counting Kit-8, luciferase and Transwell assays were performed to investigate the biological function of microRNA-150 (miR-150) in OS. The results revealed that miR-150 was significantly downregulated in OS cell lines (HOS, SAOS2, MG-63 and U2OS) in comparison with the normal osteoblast cells (hFOB1.19). Overexpression of miR-150 significantly inhibited cell proliferation in OS cells. miR-150 could sensitize OS cells to chemotherapy treatment of doxorubicin. Runt-related transcription factor 2 (RUNX2) was identified as a target gene of miR-150. RUNX2 knockdown exhibited similar inhibitory effects on both OS cell proliferation and chemotherapy sensitivity. Restoration of RUNX2 reversed the biological function of miR-150. Finally, miR-150 overexpression and RUNX2 knockdown enhanced caspase-3 cleavage. Taken together, the present study established a novel molecular mechanism, in that miR-150 plays tumor suppressor and chemoprotective roles by targeting RUNX2 in OS, indicating that miR-150 may be a potential therapeutic target for OS therapy in the future.