Characterization of anti-EBNA-1 antibodies and exploration of their molecular mimicry potential in an EBV-infected Sjögren's syndrome patient.

There is a potential link between autoimmune diseases and Epstein-Barr virus (EBV) infections, with EBV playing a substantial role in the onset of Sjögren's syndrome (SjS). Some EBV proteins could mimic host self-antigens post-infection, leading to molecular mimicry. This similarity may cause the immune system to attack its tissues mistakenly. Among the various proteins associated with EBV, nuclear antigen 1 (EBNA-1) is essential for the latent replication of infected cells and is prevalent in all EBV-related diseases. In the study, single-chain variable fragment (scFv) antibodies targeting EBNA-1 were isolated using phage display technology from a primary SjS patient who also had a chronic active EBV infection. The specific clones were enriched after panning, and the binding activity of selected scFvs targeting EBNA-1 was confirmed. Sequence analysis indicated that the scFvs exhibiting positive signals could be grouped into five clones, all of which used homologous heavy chain V regions derived from germline Vh4-39, and two types of light chain V regions stemming from germline Vλ1-44 and Vλ3-15. These scFvs were found to exhibit a high degree of somatic mutations, likely indicative of antigen selection. Of the scFvs, P1-3 demonstrated the strongest binding affinity to EBNA-1, exhibiting a determined value of 7.3 x 10-8 M, and showed cross-reactivity to the SjS associated La/SSB self-antigen. The experimental results combined with AlphaFold 3 predictions revealed a potential epitope for scFv P1-3 binding to EBNA-1. Additionally, scFv P1-3 could also cross-bind to the modeled structure of La/SSB. We inferred a possible structural correlation between EBNA-1 and La/SSB involving an X2AX6PG epitope motif. This research contributes to our understanding of the structural basis of the interactions between antibodies and EBNA-1, shedding light on the VH and VL gene usage of anti-EBNA-1 antibodies in EBV-infected SjS patients and the potential origins of autoantibodies.

An auristatin-based antibody-drug conjugate targeting EphA2 in pancreatic cancer treatment.

Pancreatic adenocarcinoma, a highly aggressive form of cancer with a poor prognosis, necessitates the development of innovative treatment strategies. Our prior research showcased the growth-inhibiting effects of the anti-EphA2 antibody drug hSD5 on pancreatic cancer tumors. This antibody targets and induces the degradation of the EphA2 receptor while also prompting the antibody's internalization. A deeper dive into the hSD5 Fab crystallographic structure and docking studies revealed that hSD5's CDRH3 drives the primary interaction between hSD5 and the EphA2 active site. In this study, we developed a novel antibody-drug conjugate (ADC)-the auristatin-based hSD5-vedotin specifically targeting EphA2 in pancreatic cancer cells. This ADC aims at the tumor-specific antigen EphA2, triggering endocytosis and releasing the conjugated payload molecule Monomethyl auristatin E (MMAE), amplifying the tumor-killing effect. Upon cellular entry, hSD5-vedotin demonstrated an impressive tumor-killing response, inhibiting tumor cell growth and promoting apoptosis even at lower antibody concentrations. In a pancreatic cancer xenograft animal model, hSD5-vedotin showcased the potential to suppress tumor growth entirely. Notably, potential immune resistance responses were also observed in recurrent pancreatic cancer tumors. Our empirical results underscore the possibility of developing hSD5-vedotin further, which we anticipate will have a broader and more potent therapeutic impact on pancreatic cancer and other EphA2-related cancers.

Effectiveness of anti-erythropoietin producing Hepatocellular receptor Type-A2 antibody in pancreatic cancer treatment.

Erythropoietin-producing hepatocyte receptor type A2 (EphA2) is a tyrosine kinase that binds to ephrins (e.g., ephrin-A1) to initiate bidirectional signaling between cells. The binding of EphA2 and ephrin-A1 leads to the inhibition of Ras-MAPK activity and tumor growth. During tumorigenesis, the normal interaction between EphA2 and ephrin-A1 is hindered, which leads to the overexpression of EphA2 and induces cancer. The overexpression of EphA2 has been identified as a notable tumor marker in diagnosing and treating pancreatic cancer. In this study, we used phage display to isolate specific antibodies against the active site of EphA2 by using a discontinuous recombinant epitope for immunization. The therapeutic efficacy and inhibition mechanism of the generated antibody against pancreatic cancer was validated and clarified. The generated antibodies were bound to the conformational epitope of endogenous EphA2 on cancer cells, thus inducing cellular endocytosis and causing EphA2 degradation. Molecule signals pAKT, pERK, pFAK, and pSTAT3 were weakened, inhibiting the proliferation and migration of pancreatic cancer cells. The humanized antibody hSD5 could effectively inhibit the growth of the xenograft pancreatic cancer tumor cells BxPc-3 and Mia PaCa-2 in mice, respectively. When antibody hSD5 was administered with gemcitabine, significantly improved effects on tumor growth inhibition were observed. Based on the efficacy of the IgG hSD5 antibodies, clinical administration of the hSD5 antibodies is likely to suppress tumors in patients with pancreatic cancer and abnormal activation or overexpression of EphA2 signaling.

Single-chain fragment antibody disrupting the EphA4 function as a therapeutic drug for gastric cancer.

Studies have shown that the high expression of EphA4 in gastric cancer tissues may correlate with unfavorable clinical pathological characteristics. Therefore, EphA4 may be an effective target for treating gastric cancer in addition to HER-2/neu. In this study, generated scFv S3 can bind endogenous EphA4 of gastric cancer cells and has significant membrane staining. Additionally, scFv S3 binding to EphA4 inhibits the growth and migration of cancer cells and the growth induction that ephrinA1 generates in gastric cancer cells. We found that EphA4 molecules may degrade through antibody treatment of cells, and the increase in LAMP1 and LAMP2 indicates that lysosome is involved in the degradation. The scFv S3 administration leads to the signals pAKT, pERK, and pSTAT3 decrease in cancer cells. The xenograft model of HER-2/neu low expressing gastric cancer cell SNU-16 exhibits better therapeutic effects by scFv S3 than trastuzumab scFv. The scFv S3 administration in vivo can degrade EphA4 molecules in tumor tissues, decreasing Ki67 and increasing cleaved C3 molecule expression. Furthermore, we identified and validated that scFv S3 generates essential ionic bonding with R162 on EphA4. The antibody may provide effective treatment for patients with gastric cancer and abnormal activation or overexpression of EphA4 signaling.

Highly Selective Electrocatalytic Oxidation of Amines to Nitriles Assisted by Water Oxidation on Metal-Doped α-Ni(OH)2.

Selective oxidation to synthesize nitriles is critical for feedstock manufacturing in the chemical industry. Current strategies typically involve substitutions of alkyl halides with toxic cyanides or the use of strong oxidation reagents (oxygen or peroxide) under ammoxidation/oxidation conditions, setting considerable challenges in energy efficiency, sustainability, and production safety. Herein, we demonstrate a facile, green, and safe electrocatalytic route for selective oxidation of amines to nitriles under ambient conditions, assisted by the anodic water oxidation on metal-doped α-Ni(OH)2 (a typical oxygen evolution reaction catalyst). By controlling the balance between co-adsorption of the amine molecule and hydroxyls on the catalyst surface, we demonstrate that Mn doping significantly promotes the subsequent chemical oxidation of amines, resulting in Faradaic efficiencies of 96% for nitriles under ≥99% conversion. This anodic oxidation is further coupled with cathodic hydrogen evolution for overall atomic economy and additional green energy production.

Boron Carbon Oxynitride as a Novel Metal-Free Photocatalyst.

Boron-based nanomaterials are emerging as non-toxic, earth-abundant (photo)electrocatalyst materials in solar energy conversion for the production of solar hydrogen fuel and environmental remediation. Boron carbon oxynitride (BCNO) is a quaternary semiconductor with electronic, optical, and physicochemical properties that can be tuned by varying the composition of boron, nitrogen, carbon, and oxygen. However, the relationship between BCNO's structure and -photocatalytic activity relationship has yet to be explored. We performed an in-depth spectroscopic analysis to elucidate the effect of using two different nitrogen precursors and the effect of annealing temperatures in the preparation of BCNO. BCNO nanodisks (D = 6.7 ± 1.1 nm) with turbostratic boron nitride diffraction patterns were prepared using guanidine hydrochloride as the nitrogen source precursor upon thermal annealing at 800°C. The X-ray photoelectron spectroscopy (XPS) surface elemental analysis of the BCNO nanodisks revealed the B, C, N, and O compositions to be 40.6%, 7.95%, 37.7%, and 13.8%, respectively. According to the solid-state 11B NMR analyses, the guanidine hydrochloride-derived BCNO nanodisks showed the formation of various tricoordinate BNx(OH)3-x species, which also served as one of the photocatalytic active sites. The XRD and in-depth spectroscopic analyses corroborated the preparation of BCNO-doped hexagonal boron nitride nanodisks. In contrast, the BCNO annealed at 600 °C using melamine as the nitrogen precursor consisted of layered nanosheets composed of B, C, N, and O atoms covalently bonded in a honeycomb lattice as evidence by the XRD, XPS, and solid-state NMR analysis (11B and 13C) analyses. The XPS surface elemental composition of the melamine-derived BCNO layered structures consisted of a high carbon composition (75.1%) with a relatively low boron (5.24%) and nitrogen (7.27%) composition, which indicated the formation of BCNO-doped graphene oxides layered sheet structures. This series of melamine-derived BCNO-doped graphene oxide layered structures were found to exhibit the highest photocatalytic activity, exceeding the photocatalytic activity of graphitic carbon nitride. In this layered structure, the formation of the tetracoordinate BNx(OH)3-x(CO) species and the rich graphitic domains were proposed to play an important role in the photocatalytic activity of the BCNO-doped graphene oxides layered structures. The optical band gap energies were measured to be 5.7 eV and 4.2 eV for BCNO-doped hexagonal boron nitride nanodisks and BCNO-doped graphene oxides layered structures, respectively. Finally, BCNO exhibited an ultralong photoluminescence with an average decay lifetime of 1.58, 2.10, 5.18, and 8.14 µs for BGH01, BGH03, BMH01, BMH03, respectively. This study provides a novel metal-free photocatalytic system and provides the first structural analysis regarding the origin of BCNO-based photocatalyst.

Polymer-Coated Nanoparticles for Therapeutic and Diagnostic Non-10B Enriched Polymer-Coated Boron Carbon Oxynitride (BCNO) Nanoparticles as Potent BNCT Drug.

Boron neutron capture therapy (BNCT) is a powerful and selective anti-cancer therapy utilizing 10B-enriched boron drugs. However, clinical advancement of BCNT is hampered by the insufficient loading of B-10 drugs throughout the solid tumor. Furthermore, the preparation of boron drugs for BNCT relies on the use of the costly B-10 enriched precursor. To overcome these challenges, polymer-coated boron carbon oxynitride (BCNO) nanoparticles, with ~30% of boron, were developed with enhanced biocompatibility, cell uptake, and tumoricidal effect via BNCT. Using the ALTS1C1 cancer cell line, the IC50 of the PEG@BCNO, bare, PEI@BCNO were determined to be 0.3 mg/mL, 0.1 mg/mL, and 0.05 mg/mL, respectively. As a proof-of-concept, the engineered non-10B enriched polymer-coated BCNO exhibited excellent anti-tumor effect via BNCT due to their high boron content per nanoparticle and due to the enhanced cellular internalization and retention compared to small molecular 10B-BPA drug. The astrocytoma ALTS1C1 cells treated with bare, polyethyleneimine-, and polyethylene glycol-coated BCNO exhibited an acute cell death of 24, 37, and 43%, respectively, upon 30 min of neutron irradiation compared to the negligible cell death in PBS-treated and non-irradiated cells. The radical approach proposed in this study addresses the expensive and complex issues of B-10 isotope enrichment process; thus, enabling the preparation of boron drugs at a significantly lower cost, which will facilitate the development of boron drugs for BNCT.

Generation and Characterization of Single Chain Variable Fragment against Alpha-Enolase of Candida albicans.

Candida albicans (C. albicans) is an opportunistic human pathogen responsible for approximately a half of clinical candidemia. The emerging Candida spp. with resistance to azoles is a major challenge in clinic, suggesting an urgent demand for new drugs and therapeutic strategies. Alpha-enolase (Eno1) is a multifunctional protein and represents an important marker for invasive candidiasis. Thus, C. albicans Eno1 (CaEno1) is believed to be an important target for the development of therapeutic agents and antibody drugs. Recombinant CaEno1 (rCaEno1) was first used to immunize chickens. Subsequently, we used phage display technology to construct two single chain variable fragment (scFv) antibody libraries. A novel biopanning procedure was carried out to screen anti-rCaEno1 scFv antibodies, whose specificities were further characterized. The polyclonal IgY antibodies showed binding to rCaEno1 and native CaEno1. A dominant scFv (CaS1) and its properties were further characterized. CaS1 attenuated the growth of C. albicans and inhibited the binding of CaEno1 to plasminogen. Animal studies showed that CaS1 prolonged the survival rate of mice and zebrafish with candidiasis. The fungal burden in kidney and spleen, as well as level of inflammatory cytokines were significantly reduced in CaS1-treated mice. These results suggest CaS1 has potential of being immunotherapeutic drug against C. albicans infections.

Generation and characterization of avian-derived anti-human CD19 single chain fragment antibodies.

The human cluster of differentiation 19 (CD19) is highly expressed in most leukemia, rendering is a promising therapeutic target. In this study, we generated anti-CD19 single-chain variable fragments (scFv) from immunized chickens by phage display technology. After constructing a scFv antibody library with 2.5 × 108 compositional diversity for panning, one representative scFv clone S2 which can specifically recognize to the CD19 protein was isolated and characterized. The binding reactivity of the scFv S2 to the endogenous CD19 protein of the ARH-77 leukemia cancer cell was verified through flow cytometry and the binding affinity of scFv S2 is 6.9 × 10-8 M determined by the surface plasmon resonance system. Compared with the chicken germline, hyper mutation in the complementarity-determining regions (CDRs) suggested that scFv S2 could be generated through an antigen-driven humoral response. By molecular modeling, the possible CDR configurations of scFv S2 were constructed rationally. Furthermore, the characteristics of chicken antibodies of a protein database were investigated. The findings in this study contribute to antibody development and engineering because they reveal the geometric structures and properties of the CDRs in chicken antibodies.

Interaction of S17 Antibody with the Functional Binding Region of the Hepatitis B Virus Pre-S2 Epitope.

To understand the mechanism for inhibition of hepatitis B virus (HBV) infection is important. In this study, single-chain variable fragment (scFv) antibodies were generated and directed to the pre-S2 epitope of HBV surface antigen (HBsAg). These human scFvs were isolated from a person with history of HBV infection by phage display technology. An evaluation of panning efficiency revealed that the eluted phage titer was increased, indicating that specific clones were enriched after panning. Selected scFvs were characterized with the recombinant HBsAg through Western blotting and enzyme-linked immunosorbent assay to confirm the binding ability. Flow cytometry analysis and immunocytochemical staining revealed that one scFv, S17, could recognize endogenous HBsAg expressed on the HepG2215 cell membrane. Moreover, the binding affinity of scFv S17 to the pre-S2 epitope was determined to be 4.2 × 10-8 M. Two ion interactions were observed as the major driving forces for scFv S17 interacting with pre-S2 by performing a rational molecular docking analysis. This study provides insights into the structural basis to understand the interactions between an antibody and the pre-S2 epitope. The functional scFv format can potentially be used in future immunotherapeutic applications.