Cancer-oocyte SAS1B protein is expressed at the cell surface of multiple solid tumors and targeted with antibody-drug conjugates.

BACKGROUND: Sperm acrosomal SLLP1 binding (SAS1B) protein is found in oocytes, which is necessary for sperm-oocyte interaction, and also in uterine and pancreatic cancers. Anti-SAS1B antibody-drug conjugates (ADCs) arrested growth in these cancers. However, SAS1B expression in cancers and normal tissues has not been characterized. We hypothesized that SAS1B is expressed on the surface of other common solid cancer cells, but not on normal tissue cells, and might be selectively targeted therapeutically. METHODS: SAS1B expression in human normal and cancer tissues was determined by immunohistochemistry, and complementary DNA (cDNA) libraries were employed to PCR amplify human SAS1B and its transcripts. Monoclonal antibodies (mAbs) to human SAS1B were generated using mouse hybridomas. SAS1B deletion constructs were developed to map SAS1B's epitope, enabling the creation of a blocking peptide. Indirect immunofluorescence (IIF) of human transfected normal and cancer cells was performed to assess SAS1B expression. SAS1B intracellular versus surface expression in normal and tumor tissues was evaluated by flow cytometry after staining with anti-SAS1B mAb, with specificity confirmed with the blocking peptide. Human cancer lines were treated with increasing mAb and ADC concentrations. ATP was quantitated as a measure of cell viability. RESULTS: SAS1B expression was identified in a subset of human cancers and the cytoplasm of pancreatic islet cells. Two new SAS1B splice variants were deduced. Monoclonal antibodies were generated to SAS1B splice variant A. The epitope for mAbs SB2 and SB5 is between SAS1B amino acids 32-39. IIF demonstrated intracellular SAS1B expression in transfected kidney cells and on the cell surface of squamous cell lung carcinoma. Flow cytometry demonstrated intracellular SAS1B expression in all tumors and some normal cells. However, surface expression of SAS1B was identified only on cancer cells. SB2 ADC mediated dose-dependent cytotoxic killing of multiple human cancer lines. CONCLUSION: SAS1B is a novel cancer-oocyte antigen with cell surface expression restricted to cancer cells. In vitro, it is an effective target for antibody-mediated cancer cell lysis. These findings support further exploration of SAS1B as a potential therapeutic cancer target in multiple human cancers, either with ADC or as a chimeric antigen receptor-T (CAR-T) cell target.

Aggregation-Inhibiting scFv-Based Therapies Protect Mice against AAV1/2-Induced A53T-α-Synuclein Overexpression.

To date, there is no cure for Parkinson's disease (PD). There is a pressing need for anti-neurodegenerative therapeutics that can slow or halt PD progression by targeting underlying disease mechanisms. Specifically, preventing the build-up of alpha-synuclein (αSyn) and its aggregated and mutated forms is a key therapeutic target. In this study, an adeno-associated viral vector loaded with the A53T gene mutation was used to induce rapid αSyn-associated PD pathogenesis in C57BL/6 mice. We tested the ability of a novel therapeutic, a single chain fragment variable (scFv) antibody with specificity only for pathologic forms of αSyn, to protect against αSyn-induced neurodegeneration, after unilateral viral vector injection in the substantia nigra. Additionally, polyanhydride nanoparticles, which provide sustained release of therapeutics with dose-sparing properties, were used as a delivery platform for the scFv. Through bi-weekly behavioral assessments and across multiple post-mortem immunochemical analyses, we found that the scFv-based therapies allowed the mice to recover motor activity and reduce overall αSyn expression in the substantia nigra. In summary, these novel scFv-based therapies, which are specific exclusively for pathological aggregates of αSyn, show early promise in blocking PD progression in a surrogate mouse PD model.

Yeast Surface Display Platform for Rapid Selection of an Antibody Library via Sequential Counter Antigen Flow Cytometry.

Yeast surface display techniques have been increasingly employed as a tool for both the discovery and affinity maturation of antibodies. In this study, we describe the use of yeast surface display for the selection and affinity maturation of antibodies targeted to small molecules (haptens). In this approach, we coupled 4 to 15 sequential cycles of error-prone PCR to introduce heterogeneity into the sequence of an 12F6 scFv antibody that binds to chelated uranium; the resulting full-length constructs were combined to create a yeast-displayed scFv-library with high diversity. We also developed a stringent selection technique utilizing fluorescence-activated cell sorting; this was based on sequentially dropping the target antigen concentration, while concomitantly increasing the concentration of potential cross-reactive haptens in subsequent selection cycles. As a proof of the efficacy this approach, we confirmed that the antibodies identified via this approach retained binding to the target antigen (UO22+ complexed to a chelator), while binding with lesser affinity than the parental scFv to a structurally related haptens (the same chelator complexed to other metal ions). As will be described in this report, these scFv variants perform more efficiently in sensor-based assay than the parental 12F6 antibody. Combining the generation of scFv libraries via error-prone PCR with selection of yeast-displayed antibodies by fluorescence activated cell sorting will provide an efficient new method for the isolation of scFvs and other binding proteins with high affinity and specificity.

Novel chimeric monoclonal antibodies that block fentanyl effects and alter fentanyl biodistribution in mice.

The prevalence and societal impact of opioid use disorder (OUD) is an acknowledged public health crisis that is further aggravated by the current pandemic. One of the devastating consequences of OUD is opioid overdose deaths. While multiple medications are now available to treat OUD, given the prevalence and societal burden, additional well-tolerated and effective therapies are still needed. To this point, we have developed chimeric monoclonal antibodies (mAb) that will specifically complex with fentanyl and its analogs in the periphery, thereby preventing them from reaching the central nervous system. Additionally, mAb-based passive immunotherapy offers a high degree of specificity to drugs of abuse and does not interfere with an individual's ability to use any of the medications used to treat OUD. We hypothesized that sequestering fentanyl and its analogs in the periphery will mitigate their negative effects on the brain and peripheral organs. This study is the first report of chimeric mAb against fentanyl and its analogs. We have discovered, engineered the chimeric versions, and identified the selectivity of these antibodies, through in vitro characterization and in vivo animal challenge studies. Two mAb candidates with very high (0.1-1.3 nM) binding affinities to fentanyl and its analogs were found to be effective in engaging fentanyl in the periphery and blocking its effects in challenged animals. Results presented in this work constitute a major contribution in the field of novel therapeutics targeting OUD.

Enzyme Immunoassay-Based Platform for Accurate Detection of Serum Pathological α-Synuclein in Parkinson's Disease Patients.

An assay for accurately diagnosing early stage Parkinson's Disease (PD) is currently unavailable, and therefore, there is an urgent and unmet need. Such a diagnostic assay will enable prompt institution of appropriate supportive management measures to prevent rapid deterioration of disease and improve both quality of life and life expectancy of PD patients. A reliable assay platform will also be of great benefit to drug discovery and drug development in the area of PD. To this end, we describe the development of two indirect, competitive, semiquantitative enzyme immunoassays (EIAs), each employing a disparate singularly specific mouse monoclonal antibody (ssMAb) against pathological aggregates of human α-Synuclein (αSynagg), a well-established biomarker pathognomonic of PD. Our results demonstrate that these EIAs in tandem accurately discriminated between αSynagg serum concentrations from PD patients and age-matched healthy control (HC) individuals (PD = 1700 ± 220 ng/mL; HC = 870 ± 120 ng/mL with an overall sensitivity of 56%, specificity of 63%, positive predictive value of 60%, and negative predictive value of 59%). The limits of detection of αSynagg were 400 and 300 pg/mL for ssMAbs 3C5 and 5H6, respectively. These tandem EIAs have the potential to add to the repertoire of tools for earlier diagnosis of this debilitating disorder, as well as for drug development strategies.

Oxidation of Cytochrome 605 Is the Rate-Limiting Step when Ferrimicrobium acidiphilum Respires Aerobically on Soluble Iron.

Proteins that oxidize extracellular substrates in Gram-positive bacteria are poorly understood. Ferrimicrobium acidiphilum is an actinobacterium that respires aerobically on extracellular ferrous ions at pH 1.5. In situ absorbance measurements were conducted on turbid suspensions of intact Fm. acidiphilum using an integrating cavity absorption meter designed for that purpose. Initial velocity kinetic studies monitored the appearance of product ferric ions in the presence of catalytic quantities of cells. Cell-catalyzed iron oxidation obeyed the Michaelis-Menten equation with Km and Vmax values of 71 µM and 0.29 fmol/min/cell, respectively. Limited-turnover kinetic studies were conducted with higher concentrations of cells to detect and monitor changes in the absorbance properties of cellular redox proteins when the cells were exposed to limited quantities of soluble reduced iron. A single a-type cytochrome with reduced absorbance peaks at 448 and 605 nm was the only redox-active chromophore that was visible as the cells respired aerobically on iron. The reduced cytochrome 605 exhibited mathematical and correlational properties that were consistent with the hypothesis that oxidation of the cytochrome constituted the rate-limiting step in the aerobic respiratory process, with a turnover number of 35 ± 2 s-1 Genomic and proteomic analyses showed that Fm. acidiphilum could and did express only two a-type heme copper terminal oxidases. Cytochrome 605 was associated with the terminal oxidase gene that is located between nucleotides 31,090 and 33,039, inclusive, in the annotated circular genome of this bacterium.IMPORTANCE The identities and functions of proteins involved in aerobic respiration on extracellular ferrous ions at acidic pH are poorly understood in the four phyla of Gram-positive eukaryotes and archaea where such activities occur. In situ absorbance measurements were conducted on Fm. acidiphilum as it respired on extracellular iron using an integrating cavity absorption meter that permitted accurate optical measurements in turbid suspensions of the intact bacterium under physiological conditions. The significance of these measurements is that they permitted a direct spectrophotometric examination of the extents and rates of biological electron transfer events in situ under noninvasive physiological conditions without disrupting the complexity of the live cellular environment. One thing is certain: one way to understand how a protein functions in an intact organism is to actually observe that protein as it functions in the intact organism. This paper provides an example of just such an observation.

Optimization of Methods for the Production and Refolding of Biologically Active Disulfide Bond-Rich Antibody Fragments in Microbial Hosts.

Antibodies have been used for basic research, clinical diagnostics, and therapeutic applications. Escherichia coli is one of the organisms of choice for the production of recombinant antibodies. Variable antibody genes have canonical and non-canonical disulfide bonds that are formed by the oxidation of a pair of cysteines. However, the high-level expression of an antibody is an inherent problem to the process of disulfide bond formation, ultimately leading to mispairing of cysteines which can cause misfolding and aggregation as inclusion bodies (IBs). This study demonstrated that fragment antibodies are either secreted to the periplasm as soluble proteins or expressed in the cytoplasm as insoluble inclusion bodies when expressed using engineered bacterial host strains with optimal culture conditions. It was observed that moderate-solubilization and an in vitro matrix that associated refolding strategies with redox pairing more correctly folded, structured, and yielded functionally active antibody fragments than the one achieved by a direct dilution method in the absence of a redox pair. However, natural antibodies have canonical and non-canonical disulfide bonds that need a more elaborate refolding process in the presence of optimal concentrations of chaotropic denaturants and redox agents to obtain correctly folded disulfide bonds and high yield antibodies that retain biological activity.

Novel Autoimmune IgM Antibody Attenuates Atherosclerosis in IgM Deficient Low-Fat Diet-Fed, but Not Western Diet-Fed Apoe-/- Mice.

OBJECTIVE: Oxidized phospholipids (OxPL), such as the oxidized derivatives of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine, and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, have been shown to be the principal biologically active components of minimally oxidized LDL (low-density lipoprotein). The role of OxPL in cardiovascular diseases is well recognized, including activation of inflammation within vascular cells. Atherosclerotic Apoe-/- mice fed a high-fat diet develop antibodies to OxPL, and hybridoma B-cell lines producing natural anti-OxPL autoantibodies have been successfully generated and characterized. However, as yet, no studies have been reported demonstrating that treatment with OxPL neutralizing antibodies can be used to prevent or reverse advanced atherosclerosis. Approach and Results: Here, using a screening against 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine/1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, we generated a novel IgM autoantibody, 10C12, from the spleens of Apoe-/- mice fed a long-term Western diet, that demonstrated potent OxPL neutralizing activity in vitro and the ability to inhibit macrophage accumulation within arteries of Apoe-/- mice fed a Western diet for 4 weeks. Of interest, 10C12 failed to inhibit atherosclerosis progression in Apoe-/- mice treated between 18 and 26 weeks of Western diet feeding likely due at least in part to high levels of endogenous anti-OxPL antibodies. However, 10C12 treatment caused a 40% decrease in lipid accumulation within aortas of secreted IgM deficient, sIgM-/-Apoe-/-, mice fed a low-fat diet, when the antibody was administrated between 32-40 weeks of age. CONCLUSIONS: Taken together, these results provide direct evidence showing that treatment with a single autoimmune anti-OxPL IgM antibody during advanced disease stages can have an atheroprotective outcome.

Inhibition of Ebola Virus by a Molecularly Engineered Banana Lectin.

Ebolaviruses cause an often rapidly fatal syndrome known as Ebola virus disease (EVD), with average case fatality rates of ~50%. There is no licensed vaccine or treatment for EVD, underscoring the urgent need to develop new anti-ebolavirus agents, especially in the face of an ongoing outbreak in the Democratic Republic of the Congo and the largest ever outbreak in Western Africa in 2013-2016. Lectins have been investigated as potential antiviral agents as they bind glycans present on viral surface glycoproteins, but clinical use of them has been slowed by concerns regarding their mitogenicity, i.e. ability to cause immune cell proliferation. We previously engineered a banana lectin (BanLec), a carbohydrate-binding protein, such that it retained antiviral activity but lost mitogenicity by mutating a single amino acid, yielding H84T BanLec (H84T). H84T shows activity against viruses containing high-mannose N-glycans, including influenza A and B, HIV-1 and -2, and hepatitis C virus. Since ebolavirus surface glycoproteins also contain many high-mannose N-glycans, we assessed whether H84T could inhibit ebolavirus replication. H84T inhibited Ebola virus (EBOV) replication in cell cultures. In cells, H84T inhibited both virus-like particle (VLP) entry and transcription/replication of the EBOV mini-genome at high micromolar concentrations, while inhibiting infection by transcription- and replication-competent VLPs, which measures the full viral life cycle, in the low micromolar range. H84T did not inhibit assembly, budding, or release of VLPs. These findings suggest that H84T may exert its anti-ebolavirus effect(s) by blocking both entry and transcription/replication. In a mouse model, H84T partially (maximally, ~50-80%) protected mice from an otherwise lethal mouse-adapted EBOV infection. Interestingly, a single dose of H84T pre-exposure to EBOV protected ~80% of mice. Thus, H84T shows promise as a new anti-ebolavirus agent with potential to be used in combination with vaccination or other agents in a prophylactic or therapeutic regimen.

Combining Yeast Display and Competitive FACS to Select Rare Hapten-Specific Clones from Recombinant Antibody Libraries.

The development of antibodies to low molecular weight haptens remains challenging due to both the low immunogenicity of many haptens and the cross-reactivity of the protein carriers used to generate the immune response. Recombinant antibodies and novel display technologies have greatly advanced antibody development; however, new techniques are still required to select rare hapten-specific antibodies from large recombinant libraries. In the present study, we used a combination of phage and yeast display to screen an immune antibody library (size, 4.4 × 10(6)) against hapten markers for petroleum contamination (phenanthrene and methylphenanthrenes). Selection via phage display was used first to enrich the library between 20- and 100-fold for clones that bound to phenanthrene-protein conjugates. The enriched libraries were subsequently transferred to a yeast display system and a newly developed competitive FACS procedure was employed to select rare hapten-specific clones. Competitive FACS increased the frequency of hapten-specific scFvs in our yeast-displayed scFvs from 0.025 to 0.005% in the original library to between 13 and 35% in selected pools. The presence of hapten-specific scFvs was confirmed by competitive ELISA using periplasmic protein. Three distinct antibody clones that recognize phenanthrene and methylphenanthrenes were selected, and their distinctive binding properties were characterized. To our knowledge, these are first antibodies that can distinguish between methylated (petrogenic) versus unmethylated (pyrogenic) phenanthrenes; such antibodies will be useful in detecting the sources of environmental contamination. This selection method could be generally adopted in the selection of other hapten-specific recombinant antibodies.

The Multicenter Aerobic Iron Respiratory Chain of Acidithiobacillus ferrooxidans Functions as an Ensemble with a Single Macroscopic Rate Constant.

Electron transfer reactions among three prominent colored proteins in intact cells of Acidithiobacillus ferrooxidans were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scattered light. The concentrations of proteins in the periplasmic space were estimated to be 350 and 25 mg/ml for rusticyanin and cytochrome c, respectively; cytochrome a was present as one molecule for every 91 nm(2) in the cytoplasmic membrane. All three proteins were rapidly reduced to the same relative extent when suspensions of live bacteria were mixed with different concentrations of ferrous ions at pH 1.5. The subsequent molecular oxygen-dependent oxidation of the multicenter respiratory chain occurred with a single macroscopic rate constant, regardless of the proteins' in vitro redox potentials or their putative positions in the aerobic iron respiratory chain. The crowded electron transport proteins in the periplasm of the organism constituted an electron conductive medium where the network of protein interactions functioned in a concerted fashion as a single ensemble with a standard reduction potential of 650 mV. The appearance of product ferric ions was correlated with the reduction levels of the periplasmic electron transfer proteins; the limiting first-order catalytic rate constant for aerobic respiration on iron was 7,400 s(-1). The ability to conduct direct spectrophotometric studies under noninvasive physiological conditions represents a new and powerful approach to examine the extent and rates of biological events in situ without disrupting the complexity of the live cellular environment.

A pharmacodynamic comparison study of different botulinum toxin type A preparations.

BACKGROUND: Because more botulinum toxin (BoNT) preparations have become available worldwide, there is a clinical need to compare the pharmacologic profiles of these products. OBJECTIVE: We compared three different preparations: onabotulinumtoxinA (ona-BoNT/A), abobotulinumtoxinA (abo-BoNT/A), and Neuronox (neu-BoNT/A), in a mouse model using a digit abduction scoring (DAS) assay. METHODS: The efficacy, duration of effect, and safety margin of each preparation was determined after delivering a single injection to the right gastrocnemius (0-240 U/kg body weight of neu-BoNT/A or ona-BoNT/A; 0-600 Speywood Units/kg body weight of abo-BoNT/A). RESULTS: Neu-BoNT/A (intramuscular (IM) median effective dose (ED(50) ) 11.2 ± 2.7 U/kg) and ona-BoNT/A (IM ED(50) 11.9 ± 2.4 U/kg) had similar effects in terms of muscle weakness at significantly lower doses than abo-BoNT/A (IM ED(50) 41.2 ± 2.4 U/kg; p < .001). The safety margin (ratio between IM ED(50) and IM median lethal dose (LD(50) )) of neu-BoNT/A (10.7 ± 2.6 U/kg) was also similar to that of ona-BoNT/A (10.3 ± 1.3 U/kg) but significantly higher than that of abo-BoNT/A (5.9 ± 0.4 U/kg; p < .02). Neu-BoNT/A and ona-BoNT/A also produced comparable patterns of DAS response and body weight recovery by day 29. CONCLUSION: Neu-BoNT/A and ona-BoNT/A may be interchangeable based on a simple dose ratio.