Characterization of a Small Iron Protein, Pyrococcus Furiosus Rubredoxin, as a Potential Cancer Drug Delivery System

Background: Cancer is an elusive neoplastic disease that claims the lives of many people around the world every year. Though treatments have become more specific to the different types of cancer, the need for antineoplastic drugs that target cancer cells and leave normal cells unharmed, with little to no systemic toxicity remains, and rubredoxin might be such a tool. Rubredoxin is a small (53 amino acids), water soluble, non-heme iron electron transfer protein that contains an iron atom cofactor, which can be substituted with various cytotoxic transition metals such as nickel and cobalt with little or no effect on the protein. Rubredoxin from the hyperthermophile Pyrococcus furiosus is thermostable and appears to have low immunogenicity. The focus of this project is to incorporate tumor-specific binding sequences at several modifiable sites on the protein as well as substitute the iron-center with cytotoxic metals. Once a stable rubredoxin containing these characteristics is created, its effects and efficacy will be studied on specific cancer cells in vitro

Early detection of NSCLC with scFv selected against IgM autoantibody.

Survival of patients with lung cancer could be significantly prolonged should the disease be diagnosed early. Growing evidence indicates that the immune response in the form of autoantibodies to developing cancer is present before clinical presentation. We used a phage-displayed antibody library to select for recombinant scFvs that specifically bind to lung cancer-associated IgM autoantibodies. We selected for scFv recombinant antibodies reactive with circulating IgM autoantibodies found in the serum of patients with early stage lung adenocarcinoma but not matched controls. Discriminatory performance of 6 selected scFvs was validated in an independent set of serum from stage 1 adenocarcinoma and matching control groups using two independent novel methods developed for this application. The panel of 6 selected scFvs predicted cancer based on seroreactivity value with sensitivity of 0.8 and specificity of 0.87. Receiver Operative Characteristic curve (ROC) for combined 6 scFv has an AUC of 0.88 (95%CI, 0.76-1.0) as determined by fluorometric microvolume assay technology (FMAT) The ROC curve generated using a homogeneous bridging Mesa Scale Discovery (MSD) assay had an AUC of 0.72 (95% CI, 0.59-0.85). The panel of all 6 antibodies demonstrated better discriminative power than any single scFv alone. The scFv panel also demonstrated the association between a high score - based on seroreactivity - with poor survival. Selected scFvs were able to recognize lung cancer associated IgM autoantibodies in patient serum as early as 21 months before the clinical presentation of disease. The panel of antibodies discovered represents a potential unique non-invasive molecular tool to detect an immune response specific to lung adenocarcinoma at an early stage of disease

Characterization of the Native and Denatured Herceptin by Enzyme Linked Immunosorbent Assay and Quartz Crystal Microbalance Using a High-Affinity Single Chain Fragment Variable Recombinant Antibody

Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain a single chain fragment variable (scFv, designated 2B4) to a linear synthetic peptide representing Herceptin’s heavy chain CDR3. Enzyme linked immunosorbent assays (ELISAs) and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35–220.5 nM) dynamic range. Herceptin denatures and forms significant amounts of aggregates when heated. UV–vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 10¹³ M^–2. The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize nonspecific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of the use of QCM to characterize human therapeutic antibodies in samples are also discussed

Characterization of hyperthermophilic redox protein rubredoxin as a potential targeted cancer therapeutic

Background: Cancer is an elusive neoplastic disease that claims the lives of many people around the world every year. Though treatments have become more specific to the different types of cancer, the need for antineoplastic drugs that target cancer cells and leave normal cells unharmed, with little to no systemic toxicity remains, and rubredoxin might be such a tool. Rubredoxin is a small (53 amino acids), water soluble, non-heme iron electron transfer protein that contains an iron atom cofactor, which can be substituted with various cytotoxic transition metals such as nickel and cobalt with little or no effect on the protein. Rubredoxin from the hyperthermophile Pyrococcus furiosus is thermostable and appears to have low immunogenicity. The focus of this project is to incorporate tumor-specific binding sequences at several modifiable sites on the protein as well as substitute the iron-center with cytotoxic metals. Once a stable rubredoxin containing these characteristics is created, its effects and efficacy will be studied on specific cancer cells in vitro. Methods and results: Site-directed mutagenesis was used to incorporate a test epitope (E-tag) at the D20 position within rubredoxin. An RGD-sequence was previously cloned at the D20 position within rubredoxin. The mutant proteins were purified using anion-exchange DEAE, sizeexclusion G-75 Sephadex, and ceramic hydroxyapatite column chromatography. Proteins were analyzed using absorption spectroscopy, bicinchoninic acid (BCA) assay, SDS-PAGE, electrospray ionization mass spectroscopy, and thermostability. Binding studies for the D20-Etag mutant were done using dot blot. The metal content of the mutants was assessed using inductively-coupled plasma mass spectrometry. Lastly, integrin-stimulated Jurkat cancer cell lines were incubated with wild-type rubredoxin, D20-Etag, D20-RGD, and the effect of these mutants were assessed for apoptosis and necrosis at the 24 hour and 48 hour time points via gel electrophoresis. Conclusions: The epitope E-tag was successfully incorporated between the D20 and N21 amino acid residues using site directed mutagenesis. The D20-Etag and D20-RGD mutant rubredoxin proteins were successfully expressed, purified, and analyzed. There was an apoptotic effect of D20-RGD rubredoxin on the Jurkat cell line. These results provided a further understanding and appreciation of rubredoxin as a potential targeted therapeutic to cancer cells Cancer is an elusive neoplastic disease that claims the lives of many people around the world every year. Though treatments have become more specific to the different types of cancer, the need for antineoplastic drugs that target cancer cells and leave normal cells unharmed, with little to no systemic toxicity remains, and rubredoxin might be such a tool. Rubredoxin is a small (53 amino acids), water soluble, non-heme iron electron transfer protein that contains an iron atom cofactor, which can be substituted with various cytotoxic transition metals such as nickel and cobalt with little or no effect on the protein. Rubredoxin from the hyperthermophile Pyrococcus furiosus is thermostable and appears to have low immunogenicity. The focus of this project is to incorporate tumor-specific binding sequences at several modifiable sites on the protein as well as substitute the iron-center with cytotoxic metals. Once a stable rubredoxin containing these characteristics is created, its effects and efficacy will be studied on specific cancer cells in vitro. Methods and results: Site-directed mutagenesis was used to incorporate a test epitope (E-tag) at the D20 position within rubredoxin. An RGD-sequence was previously cloned at the D20 position within rubredoxin. The mutant proteins were purified using anion-exchange DEAE, sizeexclusion G-75 Sephadex, and ceramic hydroxyapatite column chromatography. Proteins were analyzed using absorption spectroscopy, bicinchoninic acid (BCA) assay, SDS-PAGE, electrospray ionization mass spectroscopy, and thermostability. Binding studies for the D20-Etag mutant were done using dot blot. The metal content of the mutants was assessed using inductively-coupled plasma mass spectrometry. Lastly, integrin-stimulated Jurkat cancer cell lines were incubated with wild-type rubredoxin, D20-Etag, D20-RGD, and the effect of these mutants were assessed for apoptosis and necrosis at the 24 hour and 48 hour time points via gel electrophoresis. Conclusions: The epitope E-tag was successfully incorporated between the D20 and N21 amino acid residues using site directed mutagenesis. The D20-Etag and D20-RGD mutant rubredoxin proteins were successfully expressed, purified, and analyzed. There was an apoptotic effect of D20-RGD rubredoxin on the Jurkat cell line. These results provided a further understanding and appreciation of rubredoxin as a potential targeted therapeutic to cancer cells

Heterogeneity among Helicobacter pylori Strains in Expression of the Outer Membrane Protein BabA

The BabA adhesin of Helicobacter pylori is an outer membrane protein that binds to the fucosylated Lewis b histo-blood group antigen on the surface of gastric epithelial cells. We screened a phage-displayed ScFv (single-chain fragment variable) recombinant antibody library for antibodies reactive with a recombinant BabA fragment and identified two such antibodies. Each antibody recognized an ∼75-kDa protein present in wild-type H. pylori strain J99 but absent from an isogenic babA mutant strain. An immunoreactive BabA protein was detected by at least one of the antibodies in 18 (46%) of 39 different wild-type H. pylori strains and was detected more commonly in cagA-positive strains than in cagA-negative strains. Numerous amino acid polymorphisms were detected among BabA proteins expressed by different strains, with the greatest diversity occurring in the middle region of the proteins. Among the 18 strains that expressed a detectable BabA protein, there was considerable variation in the level of binding to Lewis b in vitro. Heterogeneity among H. pylori strains in expression of the BabA protein may be a factor that contributes to differing clinical outcomes among H. pylori-infected humans

Approach used to determine scFv binding activity to serum IgM using fluorescent (FMAT) assay.

<p><b>A.</b> Anti-human IgM - specific antibodies immobilized onto beads were used to capture IgM from human serum samples. Recombinant scFv antibodies bound to human IgM were detected with fluorescent-labeled secondary antibodies (specific for the E- tag on the scFv) using a fluorescent (FMAT8100) plate reader. <b>B.</b> Binding activity of 384 scFv to control or cancer serum as determined using Fluorometric Microvolume Assay Technology (FMAT). Each bar represents the fluorescent signal obtained when scFv bound to beads bearing human serum IgM. ScFv demonstrating differential binding to IgM from cancer and control serum samples were selected for the further analysis (see arrows indicating the signals for scFv selected in this representative experiment: B6, J4 and E3).</p