Anti-Acinetobacter Baumannii single-chain variable fragments provide therapeutic efficacy in an immunocompromised mouse pneumonia model.

BACKGROUND: The emergence of carbapenem-resistant and extensively drug-resistant (XDR) Acinetobacter baumannii as well as inadequate effective antibiotics calls for an urgent effort to find new antibacterial agents. The therapeutic efficacy of two human scFvs, EB211 and EB279, showing growth inhibitory activity against A. baumannii in vitro, was investigated in immunocompromised mice with A. baumannii pneumonia. RESULTS: The data revealed that infected mice treated with EB211, EB279, and a combination of the two scFvs showed better survival, reduced bacterial load in the lungs, and no marked pathological abnormalities in the kidneys, liver, and lungs when compared to the control groups receiving normal saline or an irrelevant scFv. CONCLUSIONS: The results from this study suggest that the scFvs with direct growth inhibitory activity could offer promising results in the treatment of pneumonia caused by XDR A. baumannii.

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.

Astragalus membranaceus-Derived Anti-Programmed Death-1 Monoclonal Antibodies with Immunomodulatory Therapeutic Effects against Tumors.

Astragalus membranaceus polysaccharide (APS) components are main ingredients of TCM and have proven efficacy to activate T cells and B cells, enhancing immunity in humans. In this study, elevated cytokine and anti-PD-1 antibody titers were found in mice after immunization with APS. Therefore, phage-display technology was utilized to isolate specific anti-programmed death-1 (PD-1) antibodies from mice stimulated by APS and to confirm whether the isolated anti-PD-1 antibody could inhibit the interaction of PD-1 with the programmed death-ligand 1 (PD-L1), resulting in tumor growth inhibition. The isolated single-chain fragment variable (scFv) S12 exhibited the highest binding affinity of 20 nM to PD-1, completed the interaction between PD-1 and PD-L1, and blocked the effect of PD-L1-induced T cell exhaustion in peripheral blood mononuclear cells in vitro. In the animal model, the tumor growth inhibition effect after scFv S12 treatment was approximately 48%. However, meaningful synergistic effects were not observed when scFv S12 was used as a cotreatment with ixabepilone. Moreover, this treatment caused a reduction in the number of tumor-associated macrophages in the tumor tissue. These experimental results indirectly indicate the ability of APS to induce specific antibodies associated with the immune checkpoint system and the potential benefits for improving immunity in humans.

[Human selenium-containing single-chain variable fragment with glutathione peroxidase activity protects NIH3T3 fibroblast against oxidative damage].

Ultraviolet B (UVB medium wave, 280-315 nm) induces cellular oxidative damage and apoptosis by producing reactive oxygen species (ROS). Glutathione peroxidase functions as an antioxidant by catalyzing the reduction of hydrogen peroxide, the more important member of reactive oxygen species. A human selenium-containing single-chain variable fragment (se-scFv-B3) with glutathione peroxidase activity of 1288 U/µmol was generated and investigated for its antioxidant effects in UVB-induced oxidative damage model. In particular, cell viability, lipid peroxidation extent, cell apoptosis, the change of mitochondrial membrane potential, caspase-3 activity and the levels of intracellular reactive oxygen species were assayed. Human se-scFv-B3 protects NIH3T3 cells against ultraviolet B-induced oxidative damage and subsequent apoptosis by prevention of lipid peroxidation, inhibition of the collapse of mitochondrial membrane potential as well as the suppression of the caspase-3 activity and the level of intracellular ROS. It seems that antioxidant effects of human se-scFv-B3 are mainly associated with its capability to scavenge reactive oxygen species, which is similar to that of the natural glutathione peroxidase.

Human scFv antibodies (Afribumabs) against Africanized bee venom: Advances in melittin recognition.

Africanized Apis mellifera bees, also known as killer bees, have an exceptional defensive instinct, characterized by mass attacks that may cause envenomation or death. From the years 2000-2013, 77,066 bee accidents occurred in Brazil. Bee venom comprises several substances, including melittin and phospholipase A2 (PLA2). Due to the lack of antivenom for bee envenomation, this study aimed to produce human monoclonal antibody fragments (single chain fragment variable; scFv), by using phage display technology. These fragments targeted melittin and PLA2, the two major components of bee venom, to minimize their toxic effects in cases of mass envenomation. Two phage antibody selections were performed using purified melittin. As the commercial melittin is contaminated with PLA2, phages specific to PLA2 were also obtained during one of the selections. Specific clones for melittin and PLA2 were selected for the production of soluble scFvs, named here Afribumabs: prefix: afrib- (from Africanized bee); stem/suffix: -umab (fully human antibody). Afribumabs 1 and 2 were tested in in vitro and in vivo assays to assess their ability to inhibit the toxic actions of purified melittin, PLA2, and crude bee venom. Afribumabs reduced hemolysis caused by purified melittin and PLA2 and by crude venom in vitro and reduced edema formation in the paws of mice and prolonged the survival of venom-injected animals in vivo. These results demonstrate that Afribumabs may contribute to the production of the first non-heterologous antivenom treatment against bee envenomation. Such a treatment may overcome some of the difficulties associated with conventional immunotherapy techniques.

Development of single chain variable fragment (scFv) antibodies against surface proteins of 'Ca. Liberibacter asiaticus'.

'Candidatus Liberibacter asiaticus' is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vector, pKM19. The antibody population is enriched for antibodies that bind antigens of 'Ca. Liberibacter asiaticus'. The primary library has more than 10(7) unique antibodies and the genes that encode them. We have screened this library for antibodies that bind to specifically-chosen proteins that are present on the surface of 'Ca. Liberibacter asiaticus'. These proteins were used as targets for affinity-based selection of scFvs that bind to the major outer membrane protein, OmpA; the polysaccharide capsule protein KpsF; a protein component of the type IV pilus (CapF); and, two flagellar proteins FlhA and FlgI. These scFvs have been used in ELISA and dot blot assays against purified protein antigens and 'Ca. Liberibacter asiaticus' infected plant extracts. We have also recloned many of these scFvs into a plasmid expression vector designed for the production of scFvs. Screening of these scFvs was more efficient when phage-bound, rather than soluble scFvs, were used. We have demonstrated a technology to produce antibodies at will and against any protein target encoded by 'Ca. Liberibacter asiaticus'. Applications could include advanced diagnostic methods for huanglongbing and the development of immune labeling reagents for in planta applications.

The protective effects and underlying mechanism of an anti-oligomeric Aß42 single-chain variable fragment antibody.

Oligomeric Aß42 aggregates have been identified as one of the major neurotoxic components of Alzheimer's disease (AD). Immunotherapy targeted against these Aß42 aggregates has been proposed as an appropriate therapeutic approach for the treatment of AD. Here, we report an anti-oligomeric Aß42 single-chain variable fragment (scFv) antibody, named MO6, obtained from the human antibody library of a healthy donor. ScFv MO6 specifically recognized and bound to the oligomeric Aß42 (Aß42 oligomers and immature protofibrils; 18-37 kDa), and reduced their levels mainly by blocking their formation, although scFv MO6 also induced disaggregation of Aß42 aggregates. More importantly, scFv MO6 ameliorated or attenuated Aß42-induced cytotoxicity and increased cell viability by up to 33%. Furthermore, scFv MO6 efficiently passed through an in vitro blood-brain barrier (BBB) model with a delivery efficiency of 66% after 60 min post-administration. ScFv MO6 is a monovalent antibody with an affinity constant (KD) of 5.2×10(-6) M for Aß42 oligomers. Molecular docking simulations of Aß42 to scFv MO6 revealed that the approach and specific binding of scFv MO6 to oligomeric Aß42 aggregates was achieved by conformational recognition and directed induction, which resulted in a more dynamic adaptation of Aß42 to scFv MO6, occurring mainly in the N-terminal (3-4), middle (12-19) and C-terminal (34-42) regions of Aß42. This binding mode of scFv MO6 to Aß42 explains its protective effects against oligomeric Aß42. Our findings may be applied for the design of a smaller antibody specific for Aß42 oligermers.

Therapeutic Strategy for the Prevention of Pseudorabies Virus Infection in C57BL/6 Mice by 3D8 scFv with Intrinsic Nuclease Activity.

3D8 single chain variable fragment (scFv) is a recombinant monoclonal antibody with nuclease activity that was originally isolated from autoimmune-prone MRL mice. In a previous study, we analyzed the nuclease activity of 3D8 scFv and determined that a HeLa cell line expressing 3D8 scFv conferred resistance to herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). In this study, we demonstrate that 3D8 scFv could be delivered to target tissues and cells where it exerted a therapeutic effect against PRV. PRV was inoculated via intramuscular injection, and 3D8 scFv was injected intraperitoneally. The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays. Intraperitoneal injection of 5 and 10 µg 3D8 scFv resulted in no detectable toxicity. The survival rate in C57BL/6 mice was 9% after intramuscular injection of 10 LD50 PRV. In contrast, the 3D8 scFv-injected C57BL/6 mice showed survival rates of 57% (5 µg) and 47% (10 µg). The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.

Novel EGFR-specific immunotoxins based on panitumumab and cetuximab show in vitro and ex vivo activity against different tumor entities.

PURPOSE: The epidermal growth factor receptor (EGFR) is overexpressed in many solid tumors. EGFR-specific monoclonal antibodies (mAbs), such as cetuximab and panitumumab, have been approved for the treatment of colorectal and head and neck cancer. To increase tissue penetration, we constructed single-chain fragment variable (scFv) antibodies derived from these mAbs and evaluated their potential for targeted cancer therapy. The resulting scFv-based EGFR-specific immunotoxins (ITs) combine target specificity of the full-size mAb with the cell-killing activity of a toxic effector domain, a truncated version of Pseudomonas exotoxin A (ETA'). METHODS: The ITs and corresponding imaging probes were tested in vitro against four solid tumor entities (rhabdomyosarcoma, breast, prostate and pancreatic cancer). Specific binding and internalization of the ITs scFv2112-ETA' (from cetuximab) and scFv1711-ETA' (from panitumumab) were demonstrated by flow cytometry and for the scFv-SNAP-tag imaging probes by live cell imaging. Cytotoxic potential of the ITs was analyzed in cell viability and apoptosis assays. Binding of the ITs was proofed ex vivo on rhabdomyosarcoma, prostate and breast cancer formalin-fixed paraffin-embedded biopsies. RESULTS: Both novel ITs showed significant pro-apoptotic and anti-proliferative effects toward the target cells, achieving IC50 values of 4 pM (high EGFR expression) to 460 pM (moderate EGFR expression). Additionally, rapid internalization and specific in vitro and ex vivo binding on patient tissue were confirmed. CONCLUSIONS: These data demonstrate the potent therapeutic activity of two novel EGFR-specific ETA'-based ITs. Both molecules are promising candidates for further development toward clinical use in the treatment of various solid tumors to supplement the existing therapeutic regimes.

Bispecific antibody platforms for cancer immunotherapy.

Over the past decades advances in bioengineering and expanded insight in tumor immunology have resulted in the emergence of novel bispecific antibody (bsAb) constructs that are capable of redirecting immune effector cells to the tumor microenvironment. (Pre-) clinical studies of various bsAb constructs have shown impressive results in terms of immune effector cell retargeting, target dependent activation and the induction of anti-tumor responses. This review summarizes recent advances in the field of bsAb-therapy and limitations that were encountered. Furthermore, we will discuss potential future developments that can be expected to take the bsAb approach successfully forward.

Protection from diabetes development by single-chain antibody-mediated delivery of a NF-κB inhibitor specifically to ß-cells in vivo.

Recently, we reported the generation of single-chain antibodies (SCAs) highly specific for rodent and human ß-cells. Our current report describes the generation of a fusion protein of one of these SCAs (SCA B1) with a NF-κB essential modifier (NEMO)-binding domain (NBD) peptide, thereby creating a selective inhibitor of NF-κB activation in ß-cells. The SCA B1-NBD fusion protein was cloned in the pIRES-EGFP, expressed in bacteria, and purified by metal affinity chromatography; the newly generated complex was then administered intravenously to rodents and evaluated for its ability to protect ß-cells against cytokines in vitro and diabetogenic agents in vivo. First, it was shown clearly that our SCA B1-NBD fusion protein binds highly selective to CD rat ß-cells in vivo. Second, we observed that SCA B1-mediated in vivo delivery of the NBD peptide completely blocked IL-1ß + IFNγ- and TNFα + IFNγ-mediated induction of NF-κB as well as islet dysfunction in culture. Finally, repeated intravenous injection of SCA B1-NBD prior to multiple low-dose administration of streptozotocin in CD mice not only induced a striking resistance to diabetes development but also preserved ß-cell mass. In conclusion, our data show for the first time that a SCA B1-NBD fusion peptide reliably protects ß-cells against cytokines in vitro and allows protection from diabetes development in CD mice in vivo.

PAA-derived gold nanorods for cellular targeting and photothermal therapy.

A single-step LbL procedure to functionalize CTAB-capped GNRs via electrostatic self-assembly is reported. This approach allows for consistent biomolecule/GNR coupling using standard carboxyl-amine conjugation chemistry. The focus is on cancer-targeting biomolecule/GNR conjugates and selective photothermal destruction of cancer cells by GNR-mediated hyperthermia and NIR light. GNRs were conjugated to a single-chain antibody selective for colorectal carcinoma cells and used as probes to demonstrate photothermal therapy. Selective targeting and GNR uptake in antigen-expressing SW 1222 cells were observed using fluorescence microscopy. Selective photothermal therapy is demonstrated using SW 1222 cells, where >62% cell death was observed after cells are treated with targeted A33scFv-GNRs.