Dual reactivity disulfide bridging reagents; enabling new approaches to antibody fragment bioconjugation

Disulfide bridging, also known as disulfide stapling, is a powerful strategy for the construction of site-selective protein bioconjugates. Here we describe the first examples of a new class of such reagents, containing a ‘stable-labile’ design. These dual-reactive reagents are designed to form a stable bond to one cysteine and a labile bond to the second; resulting in a robust attachment to the protein with one end of the bridge, whilst the other end serves as a reactive handle for subsequent bioconjugation. By incorporating thioesters into these bridges, we demonstrate that they are primed for native chemical ligation (NCL) with N-terminal cysteines; offering an alternative to the requirement for C-terminal thioesters for use in such ligations. Alternatively, the use of hydrazine as the ligating nucleophile enables a separate cargo to be attached to each cysteine residue, which are exploited to insert variably cleavable linkers. These methodologies are demonstrated on an antibody fragment, and serve to expand the scope of disulfide bridging strategies whilst offering a convenient route to the construction of multifunctional antibody fragment conjugates

A Systematic Review of Comparative Efficacy of Treatments and Controls for Depression

Background: Although previous meta-analyses have examined effects of antidepressants, psychotherapy, and alternative therapies for depression, the efficacy of these treatments alone and in combination has not been systematically compared. We hypothesized that the differences between approved depression treatments and controls would be small. Methods and Findings: The authors first reviewed data from Food and Drug Administration Summary Basis of Approval reports of 62 pivotal antidepressant trials consisting of data from 13,802 depressed patients. This was followed by a systematic review of data from 115 published trials evaluating efficacy of psychotherapies and alternative therapies for depression. The published depression trials consisted of 10,310 depressed patients. We assessed the percentage symptom reduction experienced by the patients based on treatment assignment. Overall, antidepressants led to greater symptom reduction compared to placebo among both unpublished FDA data and published trials (F = 38.5, df = 239, p<0.001). In the published trials we noted that the magnitude of symptom reduction with active depression treatments compared to controls was significantly larger when raters evaluating treatment effects were un-blinded compared to the trials with blinded raters (F = 2.17, df = 313, p<0.05). In the blinded trials, the combination of antidepressants and psychotherapy provided a slight advantage over antidepressants (p = 0.027) and psychotherapy (p = 0.022) alone. The magnitude of symptom reduction was greater with psychotherapies compared to placebo (p = 0.019), treatment-as-usual (p = 0.012) and waiting-list (p<0.001). Differences were not seen with psychotherapy compared to antidepressants, alternative therapies or active intervention controls. Conclusions: In conclusion, the combination of psychotherapy and antidepressants for depression may provide a slight advantage whereas antidepressants alone and psychotherapy alone are not significantly different from alternative therapies or active intervention controls. These data suggest that type of treatment offered is less important than getting depressed patients involved in an active therapeutic program. Future research should consider whether certain patient profiles might justify a specific treatment modality

Ariel - Volume 6 Number 4

Editors Mark Dembert J.D. Kanofsky Frank Chervenak John Lammie Curt Cummings Entertainment Robert Breckenridge Joe Conti Gary Kaskey Photographer Larry Glazerman Overseas Editor Mike Sinason Humorist Jim McCann Staff Ken Jaffe Bob Sklaroff Halley Faust Jim Burk

Red cell and platelet transfusions in neonates: a population based study

Objectives: Reports of neonatal transfusion practices have focused predominantly on premature neonates admitted to neonatal intensive care units (NICU), however little is known about transfusion among other neonates. This study aimed to describe the use of blood products among all neonates. Design: Linked population-based birth and hospital discharge data from New South Wales (NSW), Australia was used to determine rates of blood product transfusion in the first 28 days of life. The study included all livebirths ≥23 weeks’ gestation in NSW between 2001 and 2011. Results: Between 2001-2011, 5326 of 989,491 live born neonates received a blood product transfusion (5.4 per 1000 births). Transfusion rates were 4.8 per 1000 for red cells, 1.3 per 1000 for platelets and 0.3 per 1000 for exchange transfusion. High transfusion rates were seen in neonates with prior in-utero transfusion (631/1000), congenital anomaly requiring surgery (440/1000) or haemolytic disorder (106/1000). Among transfused infants, 7% received transfusions in a hospital without a NICU. Of those transfused, 64% were born ≤32 weeks gestation (n=3384, 255/1000 births), with 96% of these receiving red cells. 36% were born >32 weeks gestation (n= 1942, 1.98/1000 births), with 76% receiving red cells and 38% receiving platelets. Conclusions: In this population based study, high transfusion rates were seen in neonates with haemolytic disorders or requiring surgery, as well as in those born preterm. Thirty-six percent of neonates who received blood products were born >32 weeks gestation and 7% were transfused in hospitals without a NICU.NHMRC, AR

Ariel - Volume 6 Number 4 (Alternate Version)

Editors Mark Dembert J.D. Kanofsky Frank Chervenak John Lammie Curt Cummings Entertainment Robert Breckenridge Joe Conti Gary Kaskey Photographer Larry Glazerman Overseas Editor Mike Sinason Humorist Jim McCann Staff Kenn Jaffe Bob Sklaroff Halley Faust Jim Burke Jay Amsterdam Morton A. Klein Nancy Redfer

An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behavior

Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-Antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A - on the opposite face of the molecule - more liable to adopt an 'open' state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin-enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the 'open' state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation

Age of blood and adverse outcomes in a maternity population

BACKGROUND In recent times there has been debate around whether longer storage time of blood is associated with increased rates of adverse outcomes following transfusion. It is unclear whether results focused on cardiac or critically ill patients apply to a maternity population. This study investigates whether older blood is associated with increased morbidity and readmission in women undergoing obstetric transfusion. STUDY DESIGN AND METHODS Women giving birth in hospitals in New South Wales, Australia between July 2006‐December 2010 were included in the study population if they had received between 1‐4 red cell units during the birth admission. Information on women’s characteristics, transfusions and outcomes were obtained from 5 routinely collected datasets including blood collection, birth and hospitalisation data. Generalised propensity score methods were used to determine the effect of age of blood on rates of severe morbidity and readmission, independent of confounding factors. RESULTS Transfusion data were available for 2990 women, with a median age of blood transfused of 20 days (interquartile range 14,27 days). There were no differences in the age of blood transfused between women with and without severe morbidity (21 (14,28) vs 22 (15,30) days), and in women readmitted or not (22 (14,28) vs 22 (16,30) days). After considering potential confounding factors, no relationship was found between the age of blood transfused and rates of severe morbidity and readmission. CONCLUSION Among women receiving low volume transfusions during a birth admission, there was no evidence of increased rates of adverse outcomes following transfusion with older blood.NHMRC, Australian Red Cross, NSW Clinical Excellence Commission, AR

A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin

WHAT FACTORS CONTRIBUTE TO HOSPITAL VARIATION IN OBSTETRIC TRANSFUSION RATES?

Background & Objectives: To explore variation in red blood cell transfusion rates between hospitals, and the extent to which this can be explained. A secondary objective was to assess whether hospital transfusion rates are associated with maternal morbidity. Materials & Methods: Linked hospital discharge and birth data were used to identify births (N=279,145) in hospitals with at least 10 deliveries per annum between 2008-2010 in New South Wales, Australia. To investigate transfusion rates, a series of random effects multilevel logistic regression models were fitted, progressively adjusting for maternal, obstetric and hospital factors. Correlations between hospital transfusion and maternal, neonatal morbidity and readmission rates were assessed. Results: Overall, the transfusion rate was 1.4% (hospital range 0.6 to 2.9) across 89 hospitals. Adjusting for maternal casemix, reduced the variation between hospitals by 26%. Adjustment for obstetric interventions further reduced variation by 8% and a further 39% after adjustment for hospital type. At a hospital level, high transfusion rates were moderately correlated with maternal morbidity (0.56, p=0.01) and low Apgar scores (0.54, p=0.002), but not with readmission rates (0.18, p=0.28). Conclusion: Both casemix and practice differences contributed to the variation in transfusion rates between hospitals. The relationship between outcomes and transfusion rates was variable, however low transfusion rates were not associated with worse outcomes.Partnership Grant from the Australian National Health and Medical Research Council NHMRC (#1027262), the Australian Red Cross and the NSW Clinical Excellence Commission, NHMRC Senior Research Fellowship (#1021025). ARC Future Fellowship (#120100069)

WHAT FACTORS CONTRIBUTE TO HOSPITAL VARIATION IN OBSTETRIC TRANSFUSION RATES?

Background & Objectives: To explore variation in red blood cell transfusion rates between hospitals, and the extent to which this can be explained. A secondary objective was to assess whether hospital transfusion rates are associated with maternal morbidity. Materials & Methods: Linked hospital discharge and birth data were used to identify births (N=279,145) in hospitals with at least 10 deliveries per annum between 2008-2010 in New South Wales, Australia. To investigate transfusion rates, a series of random effects multilevel logistic regression models were fitted, progressively adjusting for maternal, obstetric and hospital factors. Correlations between hospital transfusion and maternal, neonatal morbidity and readmission rates were assessed. Results: Overall, the transfusion rate was 1.4% (hospital range 0.6 to 2.9) across 89 hospitals. Adjusting for maternal casemix, reduced the variation between hospitals by 26%. Adjustment for obstetric interventions further reduced variation by 8% and a further 39% after adjustment for hospital type. At a hospital level, high transfusion rates were moderately correlated with maternal morbidity (0.56, p=0.01) and low Apgar scores (0.54, p=0.002), but not with readmission rates (0.18, p=0.28). Conclusion: Both casemix and practice differences contributed to the variation in transfusion rates between hospitals. The relationship between outcomes and transfusion rates was variable, however low transfusion rates were not associated with worse outcomes.Partnership Grant from the Australian National Health and Medical Research Council NHMRC (#1027262), the Australian Red Cross and the NSW Clinical Excellence Commission, NHMRC Senior Research Fellowship (#1021025). ARC Future Fellowship (#120100069)