Acidity-Activated Charge-Convertible Silver Nanocomposites for Enhanced Bacteria-Specific Aggregation and Antibacterial Activity.

Bacterial infections has become an intractable problem to human health. To overcome this challenge, we developed an antimicrobial agent (AgNPs@PDPE) via the conjugation of a pH-responsive copolymer of PDMAEMA-b-PPEGMA onto AgNPs surface. The AgNPs@PDPE underwent an acidity-induced surface charge conversion that favored bacteria-specific aggregation and antibacterial activity improvement. The specific interaction between AgNPs@PDPE and bacteria under acidic conditions was confirmed via an electrochemical method using AgNPs@PDPE modified glassy carbon electrode as the working electrode. AgNPs@PDPE could efficiently aggregate and inhibit the growth of both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) under acidic condition. The AgNPs@PDPE could also selectively distinguish pathogenic bacteria from host cells, and this characteristic is benefical for reducing the damage to surrounding tissues in the host. Moreover, AgNPs@PDPE could promote the healing of E. coli- and S. aureus-induced infection, as proven by the histological and TNF-α immunohistochemical analyses of rat dermal wounds. The proposed antimicrobial agent could to be an alternative treatment strategy for the safe treatment of treat bacteria-induced infections in clinics.

Anti-CD24 Antibody-Nitric Oxide Conjugate Selectively and Potently Suppresses Hepatic Carcinoma.

Nitric oxide (NO) has a wide range of potential applications in tumor therapy. However, a targeted delivery system for NO donors has remained elusive, creating a bottleneck that limits its druggability. The antibody-drug conjugate (ADC) is a targeted drug delivery system composed of an antibody linked to an active cytotoxic drug. This design may compensate for the weak targeting ability and various biological functions of the NO donor. In this study, we designed the NO donor HL-2, which had a targeted, cleaved disulfide bond and an attachable maleimide terminal. We conjugated HL-2 with an antibody that targeted CD24 through a thioether bond to generate an ADC-like immunoconjugate, antibody-nitric oxide conjugate (ANC), which we named HN-01. HN-01 showed efficient internalization and significantly increased the release of NO in hepatic carcinoma cells in vitro. HN-01 induced apoptosis of tumor cells and suppressed tumor growth in hepatic carcinoma-bearing nude mice through antibody-dependent co-toxicity; HN-01 also increased NO levels in tumor cells. Collectively, this study expands the concept of ADC and provides an innovative NO donor and ANC to address current challenges in targeted delivery of NO. This new inspiration for an ANC design can also be used in future studies for other molecules with intracellular targets. SIGNIFICANCE: This study is the first to expand the concept of ADC with an antibody-nitric oxide conjugate that suppresses hepatic carcinoma in vitro and in vivo.

Engineering a high-affinity humanized anti-CD24 antibody to target hepatocellular carcinoma by a novel CDR grafting design.

Cluster of differentiation 24 (CD24) is a specific surface marker involved in the tumorigenesis and progression of hepatocellular carcinoma (HCC). However, all reported anti-CD24 antibodies are murine ones with inevitable immunogenicity. To address this, a method using both molecular structure and docking-based complementarity determining region (CDR) grafting was employed for humanization. After xenogeneic CDR grafting into a human antibody, three types of canonical residues (in the VL/VH interface core, in the loop foundation, and interaction with loop residues) that support loop conformation and residues involved in the antigen-binding interface were back-mutated. Four engineered antibodies were produced, among which hG7-BM3 has virtually identical 3-D structure and affinity parameters with the parental chimeric antibody cG7. In vitro, hG7-BM3 demonstrated superior immunogenicity and serum stability to cG7. Antibody-dependent cellular cytotoxicity (ADCC), tumor cell internalization and in vivo targeting assays indicate that hG7-BM3 has the potential for development as an antibody-drug conjugate (ADC). We therefore generated the hG7-BM3-VcMMAE conjugate, which was shown to induce tumor cell apoptosis and effectively suppress nude mice bearing HCC xenografts. In conclusion, our study provides new inspiration for antibody humanization and an ADC candidate for laboratory study and clinical applications.

Selective targeted delivery of doxorubicin via conjugating to anti-CD24 antibody results in enhanced antitumor potency for hepatocellular carcinoma both in vitro and in vivo.

PURPOSE: Antibody-drug conjugates (ADCs) represent a promising therapeutic approach for clinical application. Cluster of differentiation 24 (CD24) is over-expressed in several human malignancies, especially in hepatocellular carcinoma (HCC). We aimed to develop a new class of CD24-targeted ADCs for HCC. METHODS: DOX was conjugated with G7mAb by a heterobifunctional cross-linker GMBS (N-[gamma-maleimido butyryloxy] succinimide ester) and further analyzed using HPLC. The targeting specificity and endocytosis of the newly generated ADC, G7mAb-DOX, were characterized using flow cytometry assay, near-infrared fluorescence imaging and laser scanning confocal microscope. The antitumor effects were evaluated in nude mice bearing HCC xenografts. RESULTS: G7mAb-DOX with average two drug molecules per antibody was selectively captured and endocytosed by CD24 (+) tumor cells in vitro. In vivo, the ADC was proved to target tumor tissues, suppress tumor growth and prolong the survival of HCC-bearing nude mice with improved efficacy and less systemic toxicity compared with either G7mAb or DOX single-agent treatment. CONCLUSION: These studies provide proof of concept for development of DOX-based ADCs which provide a novel approach for HCC-targeted immune therapy in clinical application.

[Transglutaminase-based antibody-drug conjugation: antibody site-specific mutation and identification].

Transglutaminase (TG) posttranslational modification of antibody permits more precisely conjugating. Based on the amino acid sequence of an anti-CD24 antibody (cG7), this article is aimed to generate a deglycosylated cG7 mutant (cG7Q). Firstly, we introduced additional glutamines at position 297 (N297Q) by site-directed mutagenesis, and then transfected the recombinant plasmids into CHO-s cells via electroporation method and screened by Dot blot assay. Subsequently, cG7Q was expressed and purified through Protein A affinity chromatography, further identified by SDS-PAGE electrophoresis and Western blot. Its affinity was detected with surface plasmon resonance and flow cytometry assay, and ADCC effect was determined by lactate dehydrogenase (LDH) release. Eventually, a cG7 mutant, cG7Q was successfully expressed with sequence-specific conjugation sites for further study.

A novel antibody targeting CD24 and hepatocellular carcinoma in vivo by near-infrared fluorescence imaging.

Liver cancer is one of the most common malignant cancers worldwide. The poor response of liver cancer to chemotherapy has whipped up the interest in targeted therapy with monoclonal antibodies because of its potential efficiency. One promising target is cluster of differentiation 24 (CD24), which is known to beover-expressed on hepatocellular carcinoma (HCC), providing prospect for HCC targeted diagnosis and therapy. In this study we developed a novel CD24 targeted monoclonal antibody G7mAb based on hybridoma technology and then generated a single-chain antibodyfragment (scFv) G7S. Firstly, ELISA, western blot, and flow cytometry assays demonstrated specific binding of CD24 by G7mAb and G7S. Further, G7mAb was demonstrated to have similar binding capacity as ML5 (a commercial Anti-CD24 Mouse Antibody) inimmunohistochemical assay. Further more, a near-infrared fluorescent dye multiplex probe amplification (MPA) was conjugated to G7mAb and G7S to form G7mAb-MPA and G7S-MPA. The near-infrared fluorescence imaging revealed that G7mAb and G7S aggregate in CD24+Huh7 hepatocellular carcinoma xenograft tissuevia specific binding to CD24 in vivo. In conclussion, G7mAb and G7S were tumor targeted therapeutic and diagnostic potentials in vitro and in vivo as anticipated.