Signaling pathway of globo-series glycosphingolipids and ß1,3-galactosyltransferase V (ß3GalT5) in breast cancer.

The globo-series glycosphingolipids (GSLs) SSEA3, SSEA4, and Globo-H specifically expressed on cancer cells are found to correlate with tumor progression and metastasis, but the functional roles of these GSLs and the key enzyme ß1,3-galactosyltransferase V (ß3GalT5) that converts Gb4 to SSEA3 remain largely unclear. Here we show that the expression of ß3GalT5 significantly correlates with tumor progression and poor survival in patients, and the globo-series GSLs in breast cancer cells form a complex in membrane lipid raft with caveolin-1 (CAV1) and focal adhesion kinase (FAK) which then interact with AKT and receptor-interacting protein kinase (RIP), respectively. Knockdown of ß3GalT5 disrupts the complex and induces apoptosis through dissociation of RIP from the complex to interact with the Fas death domain (FADD) and trigger the Fas-dependent pathway. This finding provides a link between SSEA3/SSEA4/Globo-H and the FAK/CAV1/AKT/RIP complex in tumor progression and apoptosis and suggests a direction for the treatment of breast cancer, as demonstrated by the combined use of antibodies against Globo-H and SSEA4.

Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy.

Vascular endothelial growth factor (VEGF) is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv) or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%-347% tumor growth ratio (% of Day 0 tumor volume) on Day 21 vs. 435% with Avastin). This finding suggests a potential use of these three antibodies for VEGF-targeted therapy.

Structural basis for the antibody neutralization of herpes simplex virus.

Glycoprotein D (gD) of herpes simplex virus (HSV) binds to a host cell surface receptor, which is required to trigger membrane fusion for virion entry into the host cell. gD has become a validated anti-HSV target for therapeutic antibody development. The highly inhibitory human monoclonal antibody E317 (mAb E317) was previously raised against HSV gD for viral neutralization. To understand the structural basis of antibody neutralization, crystals of the gD ectodomain bound to the E317 Fab domain were obtained. The structure of the complex reveals that E317 interacts with gD mainly through the heavy chain, which covers a large area for epitope recognition on gD, with a flexible N-terminal and C-terminal conformation. The epitope core structure maps to the external surface of gD, corresponding to the binding sites of two receptors, herpesvirus entry mediator (HVEM) and nectin-1, which mediate HSV infection. E317 directly recognizes the gD-nectin-1 interface and occludes the HVEM contact site of gD to block its binding to either receptor. The binding of E317 to gD also prohibits the formation of the N-terminal hairpin of gD for HVEM recognition. The major E317-binding site on gD overlaps with either the nectin-1-binding residues or the neutralizing antigenic sites identified thus far (Tyr38, Asp215, Arg222 and Phe223). The epitopes of gD for E317 binding are highly conserved between two types of human herpesvirus (HSV-1 and HSV-2). This study enables the virus-neutralizing epitopes to be correlated with the receptor-binding regions. The results further strengthen the previously demonstrated therapeutic and diagnostic potential of the E317 antibody.

Immunogenicity of a spike protein subunit-based COVID-19 vaccine with broad protection against various SARS-CoV-2 variants in animal studies.

SARS-CoV-2 pandemic has profound impacts on human life and global economy since the outbreak in 2019. With the new variants continue to emerge with greater immune escaping capability, the protectivity of the available vaccines is compromised. Therefore, development a vaccine that is capable of inducing immunity against variants including omicron strains is in urgent need. In this study, we developed a protein-based vaccine BCVax that is consisted of antigen delta strain spike protein and QS21-based adjuvant AB801 in nanoparticle immune stimulation complex format (AB801-ISCOM). Results from animal studies showed that high level of anti-S protein IgG was induced after two doses of BCVax and the IgG was capable of neutralizing multiple variants of pseudovirus including omicron BA.1 or BA.2 strains. In addition, strong Th1 response was stimulated after BCVax immunization. Furthermore, BCvax with AB801-ISCOM as the adjuvant showed significant stronger immunity compared with the vaccine using aluminum hydroxide plus CpG 1018 as the adjuvant. BCVax was also evaluated as a booster after two prior vaccinations, the IgG titers and pseudovirus neutralization activities against BA.2 or BA.4/BA.5 were further enhanced suggesting BCVax is a promising candidate as booster. Taken together, the pre-clinical data warrant BCVax for further development in clinic.

Preclinical Studies of OBI-999: A Novel Globo H-Targeting Antibody-Drug Conjugate.

Globo H (GH), a hexasaccharide, is expressed at low levels in normal tissues but is highly expressed in multiple cancer types, rendering it a promising target for cancer immunotherapy. OBI-999, a novel antibody-drug conjugate, is derived from a conjugation of a GH-specific mAb with a monomethyl auristatin E (MMAE) payload through a site-specific ThioBridge and a cleavable linker. OBI-999 high homogeneity with a drug-to-antibody ratio of 4 (>95%) was achieved using ThioBridge. OBI-999 displayed GH-dependent cellular internalization and trafficked to endosome and lysosome within 1 and 5 hours, respectively. Furthermore, OBI-999 showed low nanomolar cytotoxicity in the assay with high GH expression on tumor cells and exhibited a bystander killing effect on tumor cells with minimal GH expression. Tissue distribution indicated that OBI-999 and free MMAE gradually accumulated in the tumor, reaching maximum level at 168 hours after treatment, whereas OBI-999 and free MMAE decreased quickly at 4 hours after treatment in normal organs. Maximum MMAE level in the tumor was 16-fold higher than in serum, suggesting that OBI-999 is stable during circulation and MMAE is selectively released in the tumor. Excellent tumor growth inhibition of OBI-999 was demonstrated in breast, gastric, and pancreatic cancer xenograft or lung patient-derived xenograft models in a dose-dependent manner. The highest nonseverely toxic dose in cynomolgus monkeys is 10 mg/kg determined by a 3-week repeated-dose toxicology study demonstrating an acceptable safety margin. Taken together, these results support further clinical development of OBI-999, which is currently in a phase I/II clinical study in multiple solid tumors (NCT04084366). OBI-999, the first GH-targeting ADC, displayed excellent tumor inhibition in animal models across multiple cancer types, including breast, gastric, pancreatic, and lung cancers, warranting further investigation in the treatment of solid tumors.

GATA-1 and NF-Y cooperate to mediate erythroid-specific transcription of Gfi-1B gene.

Expression of Gfi (growth factor-independence)-1B, a Gfi-1-related transcriptional repressor, is restricted to erythroid lineage cells and is essential for erythropoiesis. We have determined the transcription start site of the human Gfi-1B gene and located its first non-coding exon approximately 7.82 kb upstream of the first coding exon. The genomic sequence preceding this first non-coding exon has been identified to be its erythroid-specific promoter region in K562 cells. Using gel-shift and chromatin immunoprecipitation (ChIP) assays, we have demonstrated that NF-Y and GATA-1 directly participate in transcriptional activation of the Gfi-1B gene in K562 cells. Ectopic expression of GATA-1 markedly stimulates the activity of the Gfi-1B promoter in a non-erythroid cell line U937. Interestingly, our results have indicated that this GATA-1-mediated trans-activation is dependent on NF-Y binding to the CCAAT site. Here we conclude that functional cooperation between GATA-1 and NF-Y contributes to erythroid-specific transcriptional activation of Gfi-1B promoter.

Simple SPION incubation as an efficient intracellular labeling method for tracking neural progenitor cells using MRI.

Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs.

Identification of a novel function of the clathrin-coated structure at the plasma membrane in facilitating GM-CSF receptor-mediated activation of JAK2.

It is well known that ligand binding to the high-affinity GM-CSF receptor (GMR) activates JAK2. However, how and where this event occurs in a cellular environment remains unclear. Here, we demonstrate that clathrin- but not lipid raft-mediated endocytosis is crucial for GMR signaling. Knockdown expression of clathrin heavy chain or intersectin 2 (ITSN2) attenuated GMR-mediated activation of JAK2, whereas inhibiting clathrin-coated pits or plagues to bud off the membrane by the dominant-negative mutant of dynamin enhanced such event. Moreover, unlike the wild-type receptor, an ITSN2-non-binding mutant of GMR defective in targeting to clathrin-coated pits or plagues [collectively referred to as clathrin-coated structures (CCSs) here] failed to activate JAK2 at such locations. Additional experiments demonstrate that ligand treatment not only enhanced JAK2/GMR association at CCSs, but also induced a conformational change of JAK2 which is required for JAK2 to be activated by CCS-localized CK2. Interestingly, ligand-independent activation of the oncogenic mutant of JAK2 (JAK2V617F) also requires the targeting of this mutant to CCSs. But JAK2V617F seems to be constitutively in an open conformation for CK2 activation. Together, this study reveals a novel functional role of CCSs in GMR signaling and the oncogenesis of JAK2V617F.