Analysis of sFlt Isoforms as Biomarkers for the Development of Preeclampsia

Preeclampsia is a multi-system disorder characterized by hypertension, edema and proteinuria affecting between 5-10% of pregnancies. A subset of cases progress to severe preeclampsia with exacerbated hypertension/proteinuria and evidence of nervous system, liver and/or kidney dysfunction, in addition to fetal growth restriction. Soluble fms-like tyrosine kinase-1 (sFlt) is minimally expressed in many tissues, including the placenta, and is a circulating antagonist to vascular endothelial growth factor. With progression of pregnancy, sFlt levels significantly rise, especially in women who develop preeclampsia. Diagnostic tests to predict preeclampsia in pregnant women are limited and current tests measure total sFlt in relationship to placental growth factor with varying sensitivity and specificity. We hypothesized that a pregnancy-specific splice variant of sFlt (sFlt1-14), almost exclusively expressed by the placenta, would serve as an improved serum biomarker for the development of preeclampsia. Monoclonal antibodies (mAbs) were developed that specifically bind the two predominant isoforms of sFlt (sFlt1 and sFlt1-14) by hybridoma generation from wild type mice immunized with c-terminal peptides of the two isoforms. Western blot, ELISA and affinity analysis indicated the mAbs were specific for sFlt1 or sFlt1-14 splice variants and recognized these proteins in biological fluids (amniotic fluid or serum). A quantitative capture ELISA was developed whereby total sFlt in biological fluid is captured by a unique human mAb and specific levels of sFlt1 or sFlt1-14 are detected by their respective mouse mAb, followed by anti-murine secondary antibody development. Using recombinant sFlt1 or sFlt1-14 as standards, these endogenous proteins were quantified in commercially available third trimester human pregnant sera. Future studies will measure these isoforms in sera prospectively collected from women with known outcomes of a healthy pregnancy or preeclampsia and the ability of absolute quantitation of the isoform(s) or a ratio of the two to predict the likely onset and severity of preeclampsia will be evaluated

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Circulating Levels of sFlt1 Splice Variants as Predictive Markers for the Development of Preeclampsia

Angiogenic biomarkers, including soluble fms-like tyrosine kinase 1 (sFlt1), are thought to be predictors of preeclampsia onset; however, improvement is needed before a widespread diagnostic test can be utilized. Here we describe the development and use of diagnostic monoclonal antibodies specific to the two main splice variants of sFlt1, sFlt1-1 and sFlt1-14. These antibodies were selected for their sensitivity and specificity to their respective sFlt1 isoform in a capture ELISA format. Data from this pilot study suggest that sFlt1-1 may be more predictive of preeclampsia than total sFlt1. It may be possible to improve current diagnostic platforms if more specific antibodies are utilized

Identification and Characterization of Broadly Neutralizing Human Monoclonal Antibodies Directed against the E2 Envelope Glycoprotein of Hepatitis C Virus▿

Nearly all livers transplanted into hepatitis C virus (HCV)-positive patients become infected with HCV, and 10 to 25% of reinfected livers develop cirrhosis within 5 years. Neutralizing monoclonal antibody could be an effective therapy for the prevention of infection in a transplant setting. To pursue this treatment modality, we developed human monoclonal antibodies (HuMAbs) directed against the HCV E2 envelope glycoprotein and assessed the capacity of these HuMAbs to neutralize a broad panel of HCV genotypes. HuMAb antibodies were generated by immunizing transgenic mice containing human antibody genes (HuMAb mice; Medarex Inc.) with soluble E2 envelope glycoprotein derived from a genotype 1a virus (H77). Two HuMAbs, HCV1 and 95-2, were selected for further study based on initial cross-reactivity with soluble E2 glycoproteins derived from genotypes 1a and 1b, as well as neutralization of lentivirus pseudotyped with HCV 1a and 1b envelope glycoproteins. Additionally, HuMAbs HCV1 and 95-2 potently neutralized pseudoviruses from all genotypes tested (1a, 1b, 2b, 3a, and 4a). Epitope mapping with mammalian and bacterially expressed proteins, as well as synthetic peptides, revealed that HuMAbs HCV1 and 95-2 recognize a highly conserved linear epitope spanning amino acids 412 to 423 of the E2 glycoprotein. The capacity to recognize and neutralize a broad range of genotypes, the highly conserved E2 epitope, and the fully human nature of the antibodies make HuMAbs HCV1 and 95-2 excellent candidates for treatment of HCV-positive individuals undergoing liver transplantation

Identification of human monoclonal antibodies specific for human SOD1 recognizing distinct epitopes and forms of SOD1.

Mutations in the gene encoding human SOD1 (hSOD1) can cause amyotrophic lateral sclerosis (ALS) yet the mechanism by which mutant SOD1 can induce ALS is not fully understood. There is currently no cure for ALS or treatment that significantly reduces symptoms or progression. To develop tools to understand the protein conformations present in mutant SOD1-induced ALS and as possible immunotherapy, we isolated and characterized eleven unique human monoclonal antibodies specific for hSOD1. Among these, five recognized distinct linear epitopes on hSOD1 that were not available in the properly-folded protein but were available on forms of protein with some degree of misfolding. The other six antibodies recognized conformation-dependent epitopes that were present in the properly-folded protein with two different recognition profiles: three could bind hSOD1 dimer or monomer and the other three were specific for hSOD1 dimer only. Antibodies with the capacity to bind hSOD1 monomer were able to prevent increased hydrophobicity when mutant hSOD1 was exposed to increased temperature and EDTA, suggesting that the antibodies stabilized the native structure of hSOD1. Two antibodies were tested in a G93A mutant hSOD1 transgenic mouse model of ALS but did not yield a statistically significant increase in overall survival. It may be that the two antibodies selected for testing in the mouse model were not effective for therapy or that the model and/or route of administration were not optimal to produce a therapeutic effect. Therefore, additional testing will be required to determine therapeutic potential for SOD1 mutant ALS and potentially some subset of sporadic ALS

Antibody delivery to hSOD1-G93A transgenic mice.

<p>(A) HuMabs 37_L-63 (red) and 120_c (green) and an irrelevant isotype-matched HuMab (IR Mab, blue) were delivered to the lumbar intrathecal space of hSOD1-G93A transgenic mice via osmotic pump from mouse age 65 to 115 (days). Complete two limb paralysis was used as an endpoint of disease and the day of disease endpoint was used to calculate the percent survival each day. For each group, the mean days to reach the endpoint (mean survival) and P value from the Mantel-Cox test (log-rank) were calculated using JMP and are listed below the graph. (B) HuMab 37_L-63 (red) and an irrelevant isotype-matched HuMab (IR Mab, blue) were delivered by intraperitoneal injection to hSOD1-G93A transgenic mice. Antibody dosing was initiated at mouse age of 65 days and continued once per week for the duration of the mouse survival. Endpoint and statistics were calculated as above.</p

HuMab epitope location in predicted hSOD1 structure.

<p>(A) The crystal structure of one monomer of a hSOD1 dimer (2C9V) is indicated in ribbon model from Rasmol. The bound zinc is indicated as a yellow sphere. The two cysteines involved in the intramolecular disulfide bond (C57 and C146) are displayed as wireframe in light blue. The phenylalanine at position 50 and glycine at position 51, two residues mutated at the dimer interface to generate apo-hSOD1-monomer, are displayed as wireframe in dark grey. HuMab epitopes are indicated as follows: 16_L-40 amino acids 40–47 in yellow, 3_L-42 amino acids 42–49 in red, the overlap of these two epitopes is orange, 37_L-63 amino acids 63–71 in dark blue, 11_L-80 amino acids 80–88 in purple, and 33_L-112 amino acids 112–121 in green. (B) The same orientation molecule in (A) is displayed as a space-filling model. (C) The ribbon model from (A) is rotated 90 degrees on the x-axis to view from the bottom of the molecule in (A). (D) The same orientation molecule in (C) is displayed as a space-filling model.</p

HuMabs recognize different regions of the hSOD1 protein.

<p>(A) Full-length hSOD1 (amino acids 1–153) and various portions of the protein were expressed in bacteria fused to the carboxy-terminus (C-terminus) of thioredoxin (Trx) and containing a C-terminal 6-histidine tag used for purification (Trx-hSOD1-WT-his). Each protein is represented in the figure as a hashed line with the beginning and ending amino acid number listed below the line. The proteins were coated on ELISA plates and binding of HuMabs (listed at the top right) detected with goat-anti-human antibody conjugated to alkaline phosphatase followed by PNPP substrate addition. ELISA results are listed to the right of the schematic; positive recognition is indicated by a plus sign while signals equivalent to background are indicated by a minus sign. HuMabs recognizing only full-length hSOD1 were designated conformation dependent and are noted below with a C. HuMabs with an epitope that mapped to a linear sequence of amino acids are noted below with an L. (B) Minimal linear epitopes were determined with amino-terminal biotin-labeled overlapping peptides coated on streptavidin ELISA plates. Binding of HuMabs was assessed as described in A. The epitopes are noted as a grey box for each linear-epitope HuMab with the amino acids (aa) bound indicated. To distinguish these epitopes throughout the rest of the manuscript each linear-epitope HuMab has a subscripted L for linear followed by the initial amino acid of the epitope.</p

Increased hydrophobicity of hSOD1 can be inhibited by a subset of hSOD1 specific HuMabs.

<p>(A) hSOD1-G85R, hSOD1-G93A, various hSOD1-specific HuMabs, and an irrelevant isotype-matched antibody (IR Mab) were separately incubated with 5 mM EDTA at room temperature (RT) or 45°C for 4 hrs. Hydrophobicity was measured with ANS dye fluorescence using a Victor 3 plate reader at 390 nm excitation and 460 nm emission and is reported as the fold change in relative fluorescence units from RT to 45°C. hSOD1-G85R (B) and hSOD1-G93A (C) were mixed with irrelevant antibody or various HuMabs with 5 mM EDTA and incubated at RT or 45°C for 4 hrs. Hydrophobicity was measured as indicated above. The mean of replicates is noted with a thick black bar with the standard deviation indicated. Samples with insufficient material for replicates are indicated with the single data point as a thick grey bar. Statistical significant differences from irrelevant antibody were determined using a Student’s t-test with P<0.01 (***), <0.05 (**), and <0.10 (*) indicated above the black bar for each sample.</p