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 construction of modular universal chimeric antigen receptor T (MU-CAR-T) cells by covalent linkage of allogeneic T cells and various antibody fragments.

BACKGROUND: Chimeric antigen receptor-T (CAR-T) cells therapy is one of the novel immunotherapeutic approaches with significant clinical success. However, their applications are limited because of long preparation time, high cost, and interpersonal variations. Although the manufacture of universal CAR-T (U-CAR-T) cells have significantly improved, they are still not a stable and unified cell bank. METHODS: Here, we tried to further improve the convenience and flexibility of U-CAR-T cells by constructing novel modular universal CAR-T (MU-CAR-T) cells. For this purpose, we initially screened healthy donors and cultured their T cells to obtain a higher proportion of stem cell-like memory T (TSCM) cells, which exhibit robust self-renewal capacity, sustainability and cytotoxicity. To reduce the alloreactivity, the T cells were further edited by double knockout of the T cell receptor (TCR) and class I human leukocyte antigen (HLA-I) genes utilizing the CRISPR/Cas9 system. The well-growing and genetically stable universal cells carrying the CAR-moiety were then stored as a stable and unified cell bank. Subsequently, the SDcatcher/GVoptiTag system, which generate an isopeptide bond, was used to covalently connect the purified scFvs of antibody targeting different antigens to the recovered CAR-T cells. RESULTS: The resulting CAR-T cells can perform different functions by specifically targeting various cells, such as the eradication of human immunodeficiency virus type 1 (HIV-1)-latenly-infected cells or elimination of T lymphoma cells, with similar efficiency as the traditional CAR-T cells did. CONCLUSION: Taken together, our strategy allows the production of CAR-T cells more modularization, and makes the quality control and pharmaceutic manufacture of CAR-T cells more feasible.

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.

Nanobody-derived bispecific CAR-T cell therapy enhances the anti-tumor efficacy of T cell lymphoma treatment.

T cell lymphoma (TCL) is a highly heterogeneous group of diseases with a poor prognosis and low 5-year overall survival rate. The current therapeutic regimens have relatively low efficacy rates. Clinical studies of single-target chimeric antigen receptor T cell (CAR-T cell) therapy in T lymphocytes require large and multiple infusions, increasing the risks and cost of treatment; therefore, optimizing targeted therapy is a way to improve overall prognosis. Despite significant advances in bispecific CAR-T cell therapy to avoid antigen escape in treatment of B cell lymphoma, applying this strategy to TCL requires further investigation. Here, we constructed an alpaca nanobody (Nb) phage library and generated high-affinity and -specificity Nbs targeting CD30 and CD5, respectively. Based on multiple rounds of screening, bispecific NbCD30-CD5-CAR T cells were constructed, and their superior anti-tumor effect against TCL was validated in vitro and in vivo. Our findings demonstrated that Nb-derived bispecific CAR-T cells significantly improved anti-tumor efficacy in TCL treatment compared with single-target CAR-T cells and bispecific single chain variable fragment (scFv)-derived CAR-T cells. Because Nbs are smaller and less immunogenic, the synergistic effect of Nb-based bispecific CAR-T cells may improve their safety and efficacy in future clinical applications.

Enhancement of CAR-T cell activity against cholangiocarcinoma by simultaneous knockdown of six inhibitory membrane proteins.

BACKGROUND: Existing treatments for cholangiocarcinoma have poor efficacy. However, chimeric antigen receptor-T (CAR-T) cells are emerging as a potential therapeutic strategy. Solid tumors possess multiple adverse factors in an immunosuppressive microenvironment that impair CAR-T cell infiltration and function. This study aimed to improve the function of CAR-T cells through knock down immune checkpoints and immunosuppressive molecular receptors. METHODS: We evaluated the expression of epidermal growth factor receptor (EGFR) and B7 homolog 3 protein (B7H3) antigens in cholangiocarcinoma tissues using immunohistochemistry and screened specific immune checkpoints in the cholangiocarcinoma microenvironment via flow cytometry. Subsequently, we engineered CAR-T cells targeting EGFR and B7H3 antigens. We simultaneously knocked down immune checkpoints and immunosuppressive molecular receptors in CAR-T cells by constructing two clusters of small hairpin RNAs and evaluated the engineered CAR-T cells for antitumor activity both in vitro, using tumor cell lines and cholangiocarcinoma organoid models, and in vivo, using humanized mouse models. RESULTS: We observed high expression of EGFR and B7H3 antigens in cholangiocarcinoma tissues. EGFR-CAR-T and B7H3-CAR-T cells demonstrated specific anti-tumor activity. We found an abundance of programmed cell death protein 1 (PD-1), T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), and T cell immunoglobulin and ITIM domain (Tigit) on infiltrated CD8+ T cells in the cholangiocarcinoma microenvironment. We then decreased the expression of these 3 proteins on the surface of CAR-T cells, named PTG-scFV-CAR-T cells. Furthermore, we knocked-down the expression of transforming growth factor beta receptor (TGFßR), interleukin-10 receptor (IL-10R), and interleukin-6 receptor (IL-6R) of PTG-scFV-CAR-T cells. Those cells, named PTG-T16R-scFV-CAR-T cells, potently killed tumor cells in vitro and promoted apoptosis of tumor cells in a cholangiocarcinoma organoid model. Finally, the PTG-T16R-scFv-CAR-T cells showed greater inhibitory effect on tumor growth in vivo, and were superior in prolonging the survival of mice. CONCLUSIONS: Our results revealed that PTG-T16R-scFV-CAR-T cells with knockdown of sextuplet inhibitory molecules exhibited strong immunity against cholangiocarcinoma and long-term efficacy both in vitro and in vivo. This strategy provides an effective and personalized immune cell therapy against cholangiocarcinoma.

Intranasal delivery of berberine via in situ thermoresponsive hydrogels with non-invasive therapy exhibits better antidepressant-like effects.

The efficacy of antidepressant therapy is frequently limited by challenges related to the potential to reach the brain. The development of new strategies to deliver more antidepressants to the brain so as to bypass the blood-brain barrier (BBB) is beneficial for the treatment of nervous system diseases, especially depression. Here, we have reported an unconventional strategy by the intranasal delivery of berberine with an in situ thermoresponsive hydrogel as the holder in the nasal cavity to improve its antidepressant-like activity. A berberine/hydroxylpropyl-ß-cyclodextrin (HP-ß-CD) inclusion complex was first prepared to improve the solubility of berberine and loaded into a thermoresponsive hydrogel system of poloxamers. A radioactive tracer of 125I-labeled berberine was used to investigate brain targeting. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed to study the pharmacokinetic change in the hippocampus. Monoamine neurotransmitters were analyzed in a reserpine-induced depression model, and metabolomic analysis of the hippocampus was performed in a chronic unpredictable mild stress (CUMS)-induced depression model. The radioactive tracer analysis manifested that the thermoresponsive hydrogel administered intranasally could maintain a high concentration gradient of berberine to the brain, and the relative bioavailability of berberine was enhanced approximately by 110 times that of the oral berberine/HP-ß-CD inclusion complex in the hippocampus. The thermoresponsive hydrogel system resulted in similar or better antidepressant-like efficacy even with a lower dosage in reserpine and CUMS-induced depression in rats. The pharmacometabolomics analysis revealed that in addition to increasing the hippocampal monoamine levels, berberine via intranasal administration exhibited a unique mechanism by restoring the mitochondrial dysfunction as well as phospholipid and sphingolipid abnormalities as compared to intragastric (IG) administration. We consider this a safer and more effective strategy with a lower dosage than traditional oral drugs for the treatment of depression.

Mesenchymal stem cells promote human melanocytes proliferation and resistance to apoptosis through PTEN pathway in vitiligo.

BACKGROUND: Vitiligo is an acquired chronic and recurrent skin disease that causes a depigmentation disorder, resulting in selective destruction of melanocytes (MC). However, the mechanism that leads to melanocyte dysfunction and death remains unclear. METHODS: We performed RNA sequencing, immunohistochemistry, and immunoblotting to characterize the patterns of phosphatase and tensin homolog (PTEN)/phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway activation in vitiligo. We also cocultured primary melanocytes with mesenchymal stem cells (MSCs) in a Transwell system to explore how MSCs inhibit the PTEN/PI3K/AKT pathway in melanocytes. RESULTS: We identified that vitiligo normal-lesional junction skin presented with high expression of PTEN, which led to the inhibition of AKT phosphorylation (p-AKT) at S-473. Furthermore, PTEN overexpression led to oxidative stress-induced apoptosis in melanocytes. Coculturing with MSCs enhanced the cell proliferation of human melanocytes and repressed PTEN expression, which inhibited oxidative stress-induced apoptosis. CONCLUSION: We report that vitiligo patients present with high PTEN expression, which may play a role in the impairment of melanocytes. Furthermore, our study provides evidence that MSCs target the PTEN/PI3K/AKT pathway to regulate cell proliferation and apoptosis in human melanocytes, indicating that MSCs may serve as a promising therapy for vitiligo.

MDS-associated somatic mutations and clonal hematopoiesis are common in idiopathic cytopenias of undetermined significance.

Establishing a diagnosis in patients suspected of having a myelodysplastic syndrome (MDS) can be challenging and could be informed by the identification of somatic mutations. We performed a prospective study to examine the frequency and types of mutations encountered in 144 patients with unexplained cytopenias. Based on bone marrow findings, 17% were diagnosed with MDS, 15% with idiopathic cytopenias of undetermined significance (ICUS) and some evidence of dysplasia, and 69% with ICUS and no dysplasia. Bone marrow DNA was sequenced for mutations in 22 frequently mutated myeloid malignancy genes. Somatic mutations were identified in 71% of MDS patients, 62% of patients with ICUS and some dysplasia, and 20% of ICUS patients and no dysplasia. In total, 35% of ICUS patients carried a somatic mutation or chromosomal abnormality indicative of clonal hematopoiesis. We validated these results in a cohort of 91 lower-risk MDS and 249 ICUS cases identified over a 6-month interval. Mutations were found in 79% of those with MDS, in 45% of those with ICUS with dysplasia, and in 17% of those with ICUS without dysplasia. The spectrum of mutated genes was similar with the exception of SF3B1 which was rarely mutated in patients without dysplasia. Variant allele fractions were comparable between clonal ICUS (CCUS) and MDS as were mean age and blood counts. We demonstrate that CCUS is a more frequent diagnosis than MDS in cytopenic patients. Clinical and mutational features are similar in these groups and may have diagnostic utility once outcomes in CCUS patients are better understood.

Changes in the intestinal absorption mechanism of icariin in the nanocavities of cyclodextrins.

Icariin is a bioactive herbal ingredient isolated from Herba epimedii, which has been widely used for the treatment of osteoporosis and male sexual dysfunction in traditional Chinese medicine. The major objective of this work is to investigate the different enhancing effects of ß-cyclodextrin (ß-CD) and hydroxypropyl-ß-cyclodextrin (HP-ß-CD) on the intestinal absorption of icariin, and to identify the molecular mechanisms of this action. Host-guest-type interactions of icariin with cyclodextrins nanocavities were unambiguously demonstrated by the phase-solubility diagram, ultraviolet spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, X-ray powder diffractometry, and two dimensional proton nuclear magnetic resonance rotating-frame Overhauser effect spectroscopy. These results were further supported using molecular modeling studies. The rat single-pass intestinal perfusion model showed that the absorption of icariin was affected by P-glycoprotein (Pgp). The icariin/HP-ß-CD inclusion complex provided greater enhancement in the intestinal absorption than the icariin/ß-CD inclusion complex. Therefore, the enhancing effect was involved in a solubilizing effect and/or Pgp inhibitory effect. Finally, fluorescence anisotropy measurements and Pgp adenosine triphosphatase (ATPase) assay demonstrated that ß-CD exhibited no effect on Pgp, while HP-ß-CD showed inhibition by restraining the Pgp ATPase activity rather than changing the fluidity of cell membrane.

Dietary blue pigments derived from genipin, attenuate inflammation by inhibiting LPS-induced iNOS and COX-2 expression via the NF-κB inactivation.

BACKGROUND AND PURPOSE: The edible blue pigments produced by gardenia fruits have been used as value-added colorants for foods in East Asia for 20 years. However, the biological activity of the blue pigments derived from genipin has not been reported. METHODOLOGY/PRINCIPAL FINDINGS: The anti-inflammatory effect of blue pigments was studied in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophage in vitro. The secretions of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) were inhibited in concentration-dependent manner by blue pigments. Real-time reverse-transcription polymerase chain reaction (Real-time RT-PCR) analyses demonstrated that the mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-6, and tumor necrosis factor alpha (TNF-α) was inhibited, moreover, ELISA results showed that the productions of IL-6 and TNF-α were inhibited. Cell-based ELISA revealed the COX-2 protein expression was inhibited. The proteome profiler array showed that 12 cytokines and chemokines involved in the inflammatory process were down-regulated by blue pigments. Blue pigments inhibited the nuclear transcription factor kappa-B (NF-κB) activation induced by LPS, and this was associated with decreasing the DNA-binding activity of p65 and p50. Furthermore, blue pigments suppressed the degradation of inhibitor of κB (IκB) α, Inhibitor of NF-κB Kinase (IKK) α, IKK-ß, and phosphorylation of IκB-α. The anti-inflammatory effect of blue pigments in vivo was studied in carrageenan-induced paw edema and LPS-injecting ICR mice. Finally, blue pigments significantly inhibited paw swelling and reduced plasma TNF-α and IL-6 production in vivo. CONCLUSIONS AND IMPLICATIONS: These results suggest that the anti-inflammatory properties of blue pigments might be the results from the inhibition of iNOS, COX-2, IL-6, IL-1ß, and TNF-α expression through the down-regulation of NF-κB activation, which will provide strong scientific evidence for the edible blue pigments to be developed as a new health-enhancing nutritional food for the prevention and treatment of inflammatory diseases.

Ethanol extract from a Chinese herbal formula, "Zuojin Pill", inhibit the expression of inflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 mouse macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Zuojin Pill (ZJP), a traditional Chinese medicinal decoction that has been used in treating gastritis, gastric ulcer since 15th century, contains two herbs: Rhizoma Coptidis and Fructus Evodiae in the ratio of 6:1 (w/w). Alkaloids are the main active principles contributing to ZJP's efficacy, but anti-inflammatory mechanism has not been fully clarified. AIM OF THE STUDY: The objective of the study is to reveal anti-inflammatory molecular mechanism of ethanol extract from ZJP, which would form an additional proof to the traditional experience of ZJP in clinical administration. MATERIALS AND METHODS: Seven alkaloids were determined from the ethanol extract of ZJP using high performance liquid chromatography (HPLC) with the gradient mobile phase. The ethanol extract from ZJP were used to evaluate the anti-inflammatory action in murine macrophage cell line RAW 264.7 treated with lipopolysaccharide (LPS). Production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) were measured by the Griess colorimetric method and enzyme-linked immunosorbent assay (ELISA), respectively. Proteome profiler array was analyzed to evaluate 40 cytokines at protein level. In addition, interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) synthesis were analyzed using ELISA to confirm the result of the Proteome profiler array. The gene expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-6, and interleukin 1ß (IL-1ß) were detected by quantitative real-time reverse-transcription polymerase chain reaction (real-time RT-PCR). Furthermore, the nuclear translocation of the NF-κB p50 and p65 subunits was detected with ELISA. RESULTS: The secretions of NO, PGE(2) and the mRNA expression of iNOS, COX-2 were significantly inhibited, moreover, the protein and mRNA expressions of IL-6, IL-1ß and TNF-α were inhibited by preventing the nuclear translocation of the NF-κB p50 and p65 subunits. The proteome profiler array showed that 15 cytokines and chemokines involved in the inflammatory process were down-regulated by ZJP. CONCLUSION: These results suggest that the anti-inflammatory properties of ethanol extract from ZJP might be the results from the inhibition of iNOS, COX-2, IL-6, IL-1ß, and TNF-α expression through preventing the nuclear translocation of the NF-κB p50 and p65 subunits in RAW 264.7 cells. In addition, these results provided evidence to understand the therapeutic effects of ZJP on gastritis, gastric ulcer, and other inflammatory diseases in clinic.

Parenteral iron therapy is associated with a characteristic pattern of iron staining on bone marrow aspirate smears.

We developed anecdotal evidence that parenteral iron therapy is associated with a characteristic pattern of iron staining in bone marrow aspirate smears. In this pattern, uniform blue granules are observed within reticuloendothelial cells/stromal cells with Prussian blue staining, many times in curvilinear arrays. To test this hypothesis, marrow cases submitted for morphologic evaluation to our laboratory during an approximate 2-month period in 2006 were assessed for this pattern, and, when it was observed, clinical information was sought to determine if the patient had received parenteral iron. Fourteen cases were identified that displayed the pattern of interest. In 10 of these cases, the pattern was widespread (numerous granules present within virtually all marrow spicules), whereas in 4 cases, the pattern was seen only focally. In all cases in which the pattern was widespread, patients were found to have received parenteral iron at some point before the aspiration procedure. Our findings indicate that parenteral iron therapy is associated with a characteristic pattern of iron staining in bone marrow aspirate smears.