Bispecific antibody target pair discovery by high-throughput phenotypic screening using in vitro combinatorial Fab libraries.

Bispecific antibodies can uniquely influence cellular responses, but selecting target combinations for optimal functional activity remains challenging. Here we describe a high-throughput, combinatorial, phenotypic screening approach using a new bispecific antibody target discovery format, allowing screening of hundreds of target combinations. Simple in vitro mixing of Fab-fusion proteins from a diverse library enables the generation of thousands of screen-ready bispecific antibodies for high-throughput, biologically relevant assays. We identified an obligate bispecific co-targeting CD79a/b and CD22 as a potent inhibitor of human B cell activation from a short-term flow cytometry signaling assay. A long-term, high-content imaging assay identified anti-integrin bispecific inhibitors of human cell matrix accumulation targeting integrins ß1 and ß6 or αV and ß1. In all cases, functional activity was conserved from the bispecific screening format to a therapeutically relevant format. We also introduce a broader type of mechanistic screen whereby functional modulation of different cell subsets in peripheral blood mononuclear cells was evaluated simultaneously. We identified bispecific antibodies capable of activating different T cell subsets of potential interest for applications in oncology or infectious disease, as well as bispecifics abrogating T cell activity of potential interest to autoimmune or inflammatory disease. The bispecific target pair discovery technology described herein offers access to new target biology and unique bispecific therapeutic opportunities in diverse disease indications.

Further delineation of the oculoauricular syndrome phenotype: A new family with a novel truncating HMX1 mutation.

Biallelic HMX1 mutations cause a very rare autosomal recessive genetic disorder termed as oculoauricular syndrome (OAS) because it is characterized only by the combination of eye and ear anomalies. We identified a new family bringing to three the total families reported with this disorder. Our proband presented with anteriorly protruded ears and malformed ear pinnae in association with microphthalmia, congenital cataract, microcornea, and iris and optic disc colobomata. Additionally, he had high and broad forehead with asymmetry giving a recognizable facial gestalt. Further, short left mandibular ramus and bifid cingulum in the boy and short right mandibular ramus in his father were observed. Mutation analysis revealed a novel homozygous nonsense mutation c.487G>T in the second exon of the HMX1 that predicted to introduce a premature stop codon at position 163 (p.E163*). Parents showed the heterozygous state of the detected mutation. Investigations in a process as complex as craniofacial development suggest that there are still additional, as yet unidentified, genes that play in orchestrate to determine the final phenotype.

Formyl-peptide receptor is not involved in the protection afforded by annexin 1 in murine acute myocardial infarct.

Recent interest in the annexin 1 field has come from the notion that specific G-protein-coupled receptors, members of the formyl-peptide receptor (FPR) family, appear to mediate the anti-inflammatory actions of this endogenous mediator. Administration of the annexin 1 N-terminal derived peptide Ac2-26 to mice after 25 min ischemia significantly attenuated the extent of acute myocardial injury as assessed 60 min postreperfusion. Evident at the dose of 1 mg/kg (approximately 9 nmol per animal), peptide Ac2-26 cardioprotection was intact in FPR null mice. Similarly, peptide Ac2-26 inhibition of specific markers of heart injury (specifically myeloperoxidase activity, CXC chemokine KC contents, and endogenous annexin 1 protein expression) was virtually identical in heart samples collected from wild-type and FPR null mice. Mouse myocardium expressed the mRNA for FPR and the structurally related lipoxin A4 receptor, termed ALX; thus, comparable equimolar doses of two ALX agonists (W peptide and a stable lipoxin A4 analog) exerted cardioprotection in wild-type and FPR null mice to an equal extent. Curiously, marked (>95%) blood neutropenia produced by an anti-mouse neutrophil serum did not modify the extent of acute heart injury, whereas it prevented the protection afforded by peptide Ac2-26. Thus, this study sheds light on the receptor mechanism(s) mediating annexin 1-induced cardioprotection and shows a pivotal role for ALX and circulating neutrophil, whereas it excludes any functional involvement of mouse FPR. These mechanistic data can help in developing novel therapeutics for acute cardioprotection.

Antiflammin-2 activates the human formyl-peptide receptor like 1.

The anti-inflammatory actions of the nonapeptide antiflammin-2, identified by homology with uteroglobin and annexin-A1 sequences, have been described in some detail, yet its mechanisms of action remain elusive. Since recent data indicate an involvement of the formyl peptide receptor (FPR)-like 1 (or FPRL-1) in the effects of annexin-A1, we have tested here the effect of antiflammin-2 with respect to this receptor family. Using HEK-293 cells expressing either human FPR and FPRL-1, and an annexin-A1 peptide as tracer ([125I-Tyr]-Ac2-26), we found that antiflammin-2 competed for binding only at FPRL-1, and not FPR, with an approximate EC50 of 1 mM. In line with data produced for the full-length protein, genuine receptor activation by antiflammin-2 was confirmed by rapid phosphorylation of extracellular-regulated kinase 1 and 2. Finally, study of the neutrophil interaction with activated endothelium under flow demonstrated an inhibitory effect of antiflammin-2, thus providing functional support to a role for the antiflammin-2/FPRL-1 anti-inflammatory axis.

Low-dose bacille Calmette-Guérin for non-muscle-invasive bladder cancer: Results of a prospective study.

OBJECTIVE: To evaluate the efficacy and safety of low-dose (45 mg) intravesical bacille Calmette-Guérin (BCG) therapy in the treatment of patients with non-muscle-invasive bladder cancer (NMIBC), as intravesical BCG is the most acceptable adjuvant therapy for NMI transitional cell carcinoma of the bladder. However, in the standard regimen, undesirable effects are the main cause of treatment discontinuation. PATIENTS AND METHODS: The present study included 37 men with primary NIMBC. All patients underwent complete TURB and 2 weeks later, a 6-week course of 45 mg BCG diluted in 50 mL isotonic saline was instilled into the bladder and retained for 2 h. Patients were evaluated for BCG efficacy (recurrence with or without progression) and safety by documentation of minor and/or major side-effects. RESULTS: There were no major or severe side-effects and no treatment discontinuations. Local adverse effects occurred in 20 patients, while systemic effects, in the form of fever, occurred in six patients (16.2%). There was recurrence in 14 patients (37.8%) after 18-34 months, with disease progression (muscle invasion) in four (10.8%) after 6-18 months. The recurrence index was 0.39/100 patients/month and the mean (range) tumour-free period was 30.97 (7-36) months. CONCLUSION: Low-dose BCG intravesical therapy is an effective adjuvant treatment in NMIBC. However, this needs to be validated in future studies and in comparison with other proposed doses and/or regimens.

LAPAROSCOPIC MANAGEMENT OF DISTAL URETERIC STONE IN BILHARZIAL URETER: RESULTS OF A SINGLE CENTER PROSPECTIVE STUDY.

No doubt, Bilharzial ureters are complicated by distal stricture due to precipitation of Bilharzial ova in distal ureter. These cases are associated with poorly functioning and grossly hydronephroic kidneys that hinder endoscopic manipulation of the coexistent distal, high burden, long standing impacted stones. Thus, laparoscopic uretrolithotomy was performed in 51 bilharzial patients with distal ureteric stones 4 trocars were used. The ureter was opened directly over the stone and the stone was extracted. A double-J stent was inserted into the ureter which was closed by 4-0 polyglactin running suture. The results showed that among 51 cases 33 males and 18 females; the mean age was 40.13 years. the mean stone size was 2.73 cm. Conversion to open surgery was in only one case; the mean operative time 92.05 (range 75-120 minutes); postoperative pain score ranged from 20 to 60, the mean number of PO analgesic request was 1.72 (range 1-3); it was once in 21, twice in 23 and thrice in 7 cases. Hospital stay ranged from 2 to 5 with a mean of 2.74 days; total duration of follow up ranged from 7 to 12 with a mean of 9.68. Stone recurrence reported in 4 cases; ureteric stricture reported in 2 cases. Stone free rate was reported to be 100%.

Laparoscopic management of distal ureteric stones in a bilharzial ureter: Results of a single-centre prospective study.

OBJECTIVE: To determine the efficacy and safety of the laparoscopic management of an impacted distal ureteric stone in a bilharzial ureter, as bilharzial ureters are complicated by distal stricture caused by the precipitation of bilharzial ova in the distal ureter. These cases are associated with poorly functioning and grossly hydronephrotic kidneys that hinder the endoscopic manipulation of the coexistent distal high burden of, and long-standing, impacted stones. PATIENTS AND METHODS: We used laparoscopic ureterolithotomy, with four trocars, to manage 51 bilharzial patients (33 men and 18 women; mean age 40.13 years) with distal ureteric stones. The ureter was opened directly over the stone and the stone was extracted. A JJ stent was inserted into the ureter, which was then closed with a 4-0 polyglactin running suture. RESULTS: The mean stone size was 2.73 cm. Conversion to open surgery was required in only one patient. The mean operative duration was 92 min, the postoperative pain score was 20-60, the mean (range) number of analgesic requests after surgery was 1.72 (1-3), comprising once in 21 patients, twice in 23 and thrice in seven. The mean hospital stay was 2.74 days, and the total duration of follow-up was 7-12 months. The stone recurred in four patients and a ureteric stricture was reported in two. All patients were rendered stone-free. CONCLUSION: Laparoscopy is a safe and effective minimally invasive procedure for distal ureteric stones in a bilharzial ureter with hydronephrosis.

Single-step renal dilatation in percutaneous nephrolithotomy: A prospective randomised study.

OBJECTIVE: To perform an economical single-step renal dilatation (RD) during percutaneous nephrolithotomy (PCNL), using directly a 30-F Amplatz dilator over the central Alken dilator, in a trial to reduce the operative duration and radiation exposure during RD while avoiding an exchange of dilators that might increase the risk of blood loss. PATIENTS AND METHODS: In a prospective randomised study including 49 patients divided into two groups, the first had RD before PCNL using the standard metallic telescopic dilators (Alken), and the second had RD using the 30-F Amplatz dilator over the central Alken dilator. The operative duration, with X-ray exposure, was calculated. The procedure outcome in terms of complications, stone-free rates and hospital stay was evaluated statistically. RESULTS: The tract was dilated correctly in all cases. The operative duration and X-ray exposure was shorter in patients undergoing single-step RD (P < 0.05). There were perioperative complications, according to the Clavien grading system, in 17 (34%) patients but there was no statistically significant difference between the groups. The stone-free rates were comparable in both groups. CONCLUSION: A single-step RD during PCNL is feasible, with a shorter operative duration and X-ray exposure. The outcomes were comparable with those of a standard metallic telescopic RD.

Uneven modulation of the annexin 1 system in osteoblast-like cells by dexamethasone.

We tested whether glucocorticoids modulated osteoblast expression of the annexin 1 system, including the ligand and two G-coupled receptors termed formyl-peptide receptor (FPR) and FPR-like-1 (FPRL-1). In Saos-2 cells, rapid up-regulation of FPR mRNA upon cell incubation with dexamethasone (0.01-1 microM) was observed, with significant changes as early as 2h and a more marked response at 24h; annexin 1 and FPRL-1 mRNA changes were more subtle. At the protein level, dexamethasone provoked a rapid externalization of annexin 1 (maximal at 2h) followed by delayed time-dependent changes in the cell cytosol. Saos-2 cell surface expression of FPR or FPRL-1 could not be detected, even when dexamethasone was added with the bone modelling cytokines interleukin-6 or interleukin-1. The uneven modulation of the annexin 1 system (mediator and its putative receptors) in osteoblasts might lead to a better understanding of how these complex biochemical pathways become operative in bone.

Annexin 1 and its bioactive peptide inhibit neutrophil-endothelium interactions under flow: indication of distinct receptor involvement.

We have tested the effects of annexin 1 (ANXA1) and its N-terminal peptide Ac2-26 on polymorphonuclear leukocyte (PMN) recruitment under flow. Differential effects of the full-length protein and its peptide were observed; ANXA1 inhibited firm adhesion of human PMNs, while Ac2-26 significantly attenuated capture and rolling without effect on firm adhesion. Analysis of the effects of ANXA1 and Ac2-26 on PMN adhesion molecule expression supported the flow chamber results, with Ac2-26 but not ANXA1 causing l-selectin and PSGL-1 shedding. ANXA1 and its peptide act via the FPR family of receptors. This was corroborated using HEK-293 cells transfected with FPR or FPRL-1/ALX (the 2 members of this family expressed by human PMNs). While Ac2-26 bound both FPR and FPRL-1/ALX, ANXA1 bound FPRL-1/ALX only. ANXA1 and Ac2-26 acted as genuine agonists; Ac2-26 binding led to ERK activation in both FPR- and FPRL-1/ALX-transfected cells, while ANXA1 caused ERK activation only in cells transfected with FPRL-1/ALX. Finally, blockade of FPRL-1/ALX with a neutralizing monoclonal antibody was found to abrogate the effects of ANXA1 in the flow chamber but was without effect on Ac2-26-mediated inhibition of rolling. These findings demonstrate for the first time distinct mechanisms of action for ANXA1 and its N-terminal peptide Ac2-26.

An overview of the effects of annexin 1 on cells involved in the inflammatory process.

The concept of anti-inflammation is currently evolving with the definition of several endogenous inhibitory circuits that are important in the control of the host inflammatory response. Here we focus on one of these pathways, the annexin 1 (ANXA1) system. Originally identified as a 37 kDa glucocorticoid-inducible protein, ANXA1 has emerged over the last decade as an important endogenous modulator of inflammation. We review the pharmacological effects of ANXA1 on cell types involved in inflammation, from blood-borne leukocytes to resident cells. This review reveals that there is scope for more research, since most of the studies have so far focused on the effects of the protein and its peptido-mimetics on neutrophil recruitment and activation. However, many other cells central to inflammation, e.g. endothelial cells or mast cells, also express ANXA1: it is foreseen that a better definition of the role(s) of the endogenous protein in these cells will open the way to further pharmacological studies. We propose that a more systematic analysis of ANXA1 physio-pharmacology in cells involved in the host inflammatory reaction could aid in the design of novel anti-inflammatory therapeutics based on this endogenous mediator.

Leukocyte antiadhesive actions of annexin 1: ALXR- and FPR-related anti-inflammatory mechanisms.

Recent investigations conducted with human neutrophils have indicated an involvement for the receptor for formylated peptides, termed FPR, and its analog FPRL1 (or ALXR because it is the receptor for the endogenous ligand lipoxin A(4)) in the in vitro inhibitory actions of the glucocorticoid-regulated protein annexin 1 and its peptidomimetics. To translate these findings in in vivo settings, we have used an ischemia/reperfusion (I/R) procedure to promote leukocyte-endothelium interactions in the mouse mesenteric microcirculation. In naive mice, the annexin 1 mimetic peptide Ac2-26 (20 to 100 microg administered intravenously prior to reperfusion) abolished I/R-induced cell adhesion and emigration, but not cell rolling. In FPR-deficient mice, peptide Ac2-26 retained significant inhibitory actions (about 50% of the effects in naive mice), and these were blocked by an FPR antagonist, termed butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe, or Boc2. In vitro, neutrophils taken from these animals could be activated at high concentrations of formyl-Met-Leu-Phe (30 microM; fMLP), and this effect was blocked by cell incubation with peptide Ac2-26 (66 microM) or Boc2 (100 microM). FPR-deficient neutrophils expressed ALXR mRNA and protein. Both ALXR agonists, lipoxin A(4) and peptide Ac2-26, provoked detachment of adherent leukocytes in naive as well as in FPR-deficient mice, whereas the CXC chemokine KC or fMLP were inactive. The present findings demonstrate that endogenous regulatory autocoids such as lipoxin A(4) and annexin 1-derived peptides function to disengage adherent cells during cell-cell interactions.

An overview of the effects of annexin 1 on cells involved in the inflammatory process

The concept of anti-inflammation is currently evolving with the definition of several endogenous inhibitory circuits that are important in the control of the host inflammatory response. Here we focus on one of these pathways, the annexin 1 (ANXA1) system. Originally identified as a 37 kDa glucocorticoid-inducible protein, ANXA1 has emerged over the last decade as an important endogenous modulator of inflammation. We review the pharmacological effects of ANXA1 on cell types involved in inflammation, from blood-borne leukocytes to resident cells. This review reveals that there is scope for more research, since most of the studies have so far focused on the effects of the protein and its peptido-mimetics on neutrophil recruitment and activation. However, many other cells central to inflammation, e.g. endothelial cells or mast cells, also express ANXA1: it is foreseen that a better definition of the role(s) of the endogenous protein in these cells will open the way to further pharmacological studies. We propose that a more systematic analysis of ANXA1 physio-pharmacology in cells involved in the host inflammatory reaction could aid in the design of novel anti-inflammatory therapeutics based on this endogenous mediator.