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The 2021-updated guidelines of the Spanish Society of Neonatology Guidelines have moved the zone of parental discretion to 23 + 0-23 + 6 weeks. The objective of this study was to describe the changes in perinatal management at this gestational age along the last decade and to determine if a more active perinatal management has contributed to improved outcomes.Retrospective analysis of prospectively collected data from the 23-week infants included in the Spanish SEN 1500 neonatal network during the period 2010-2019. The main study outcomes were survival at discharge and survival without major morbidity of actively managed infants. Two periods were compared: 2010-2014 (Period 1) and 2015-2019 (Period 2). NICUs were classified into low activity NICUs (less than 50 admissions of very low birth weight infants per year) and high activity NICUs (50 or more admissions).A total of 381 infants were included, 182 in Period 1 and 199 in Period 2. In Period 2 an increase in the use of intrapartum magnesium sulfate (21.5% vs 39.9%,A change to a more active intention to treat infants born at 23 weeks is taking place in Spain. But the survival rate of the actively-managed infants has remained stable around 25-30% during the study period. A multidisciplinary effort is needed to improve outcomes in this population.

PDF file - 85K, Supplementary Figure S2: Specificity of TRAILR2-induced cell killing. TRAIL-sensitive cell lines were incubated with 100pM G6T8 and different concentrations of soluble TRAILR2-Fc fusion protein.

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PDF file - 98K, Supplementary Figure S4: Caspase activation by G6T8.

PDF file - 59K, Supplementary Figure S3: G6T8 is specific for human TRAILR2 and does not cross-react with other TRAIL receptors.

Activation of TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) can induce apoptosis in a variety of human cancer cell lines and xenografts, while lacking toxicity in normal cells. The natural ligand and agonistic antibodies show antitumor activity in preclinical models of cancer, and this had led to significant excitement in the clinical potential of these agents. Unfortunately, this optimism has been tempered by trial data that, thus far, are not showing clear signs of efficacy in cancer patients. The reasons for discrepant preclinical and clinical observations are not understood, but one possibility is that the current TRAILR2 agonists lack sufficient potency to achieve a meaningful response in patients. Toward addressing that possibility, we have developed multivalent forms of a new binding scaffold (Tn3) that are superagonists of TRAILR2 and can induce apoptosis in tumor cell lines at subpicomolar concentrations. The monomer Tn3 unit was a fibronectin type III domain engineered for high-affinity TRAILR2 binding. Multivalent presentation of this basic unit induced cell death in TRAILR2-expressing cell lines. Optimization of binding affinity, molecular format, and valency contributed to cumulative enhancements of agonistic activity. An optimized multivalent agonist consisting of 8 tandem Tn3 repeats was highly potent in triggering cell death in TRAIL-sensitive cell lines and was 1 to 2 orders of magnitude more potent than TRAIL. Enhanced potency was also observed in vivo in a tumor xenograft setting. The TRAILR2 superagonists described here have the potential for superior clinical activity in settings insensitive to the current therapeutic agonists that target this pathway. Mol Cancer Ther; 12(7); 1235–44. ©2013 AACR.

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection and hospitalization in infants. Nirsevimab is a monoclonal antibody to the RSV fusion protein that has an extended half-life. The efficacy and safety of nirsevimab in healthy late-preterm and term infants are uncertain. METHODS: We randomly assigned, in a 2:1 ratio, infants who had been born at a gestational age of at least 35 weeks to receive a single intramuscular injection of nirsevimab or placebo before the start of an RSV season. The primary efficacy end point was medically attended RSV-associated lower respiratory tract infection through 150 days after the injection. The secondary efficacy end point was hospitalization for RSV-associated lower respiratory tract infection through 150 days after the injection. RESULTS: A total of 1490 infants underwent randomization: 994 were assigned to the nirsevimab group and 496 to the placebo group. Medically attended RSV-associated lower respiratory tract infection occurred in 12 infants (1.2%) in the nirsevimab group and in 25 infants (5.0%) in the placebo group; these findings correspond to an efficacy of 74.5% (95% confidence interval [CI], 49.6 to 87.1; P

Background: Transplacentally transferred antibodies induced by maternal pertussis vaccination interfere with infant immune responses to pertussis primary vaccination. We evaluated whether this interference remains in toddlers after booster vaccination. Methods: In a prior phase IV, observer-blind, placebo-controlled, randomized study (NCT02377349), pregnant women in Australia, Canada and Europe received intramuscular tetanus-reduced-antigen-content diphtheria-three-component acellular pertussis vaccine (Tdap group) or placebo (control group) at 270/7–366/7 weeks’ gestation, with crossover immunization postpartum. Their infants were primed (study NCT02422264) and boosted (at 11–18 months; current study NCT02853929) with diphtheria-tetanus-three-component acellular pertussis-hepatitis B virus-inactivated poliovirus/Haemophilus influenzae type b vaccine (DTaP-HepB-IPV/Hib) and 13-valent pneumococcal conjugate vaccine. Immunogenicity before and after booster vaccination, and reactogenicity and safety of the booster were evaluated descriptively. Results: 263 (Tdap group) and 277 (control group) toddlers received a DTaP-HepB-IPV/Hib booster. Pre-booster vaccination, observed geometric mean concentrations (GMCs) for the three pertussis antigens and diphtheria were 1.4–1.5-fold higher in controls than in the Tdap group. No differences were observed for the other DTaP-HepB-IPV/Hib antigens. One month post-booster vaccination, booster response rates for pertussis antigens were ≥ 92.1% and seroprotection rates for the other DTaP-HepB-IPV/Hib antigens were ≥ 99.2% in both groups (primary objective). Higher post-booster GMCs were observed in controls versus the Tdap group for anti-filamentous hemagglutinin (1.2-fold), anti-pertussis toxoid (1.5-fold) and anti-diphtheria (1.4-fold). GMCs for the other DTaP-HepB-IPV/Hib antigens were similar between groups. Serious adverse events were reported for three toddlers (controls, not vaccination-related). One death occurred pre-booster (Tdap group, not vaccination-related). Conclusions: As a consequence of interference of maternal pertussis antibodies with infant immune responses to pertussis primary vaccination, pertussis antibody concentrations were still lower in toddlers from Tdap-vaccinated mothers before DTaP-HepB-IPV/Hib booster vaccination. After the booster, antibody concentrations were lower for filamentous hemagglutinin and pertussis toxoid but not for pertactin. The clinical significance of this interference requires further evaluation. Clinical Trial Registration. ClinicalTrials.gov: NCT02853929. publishedVersion

Group 1 innate lymphoid cells (ILCs), which comprise both natural killer (NK) cells and ILC1s, are important innate effectors that can also positively and negatively influence adaptive immune responses. The latter function is generally ascribed to the ability of NK cells to recognize and kill activated T cells. Here, we used multiphoton intravital microscopy in mouse models of hepatitis B to study the intrahepatic behavior of group 1 ILCs and their cross-talk with hepatitis B virus (HBV)–specific CD8+T cells. We found that hepatocellular antigen recognition by effector CD8+T cells triggered a prominent increase in the number of hepatic NK cells and ILC1s. Group 1 ILCs colocalized and engaged in prolonged interactions with effector CD8+T cells undergoing hepatocellular antigen recognition; however, they did not induce T cell apoptosis. Rather, group 1 ILCs constrained CD8+T cell proliferation by controlling local interleukin-2 (IL-2) availability. Accordingly, group 1 ILC depletion, or genetic removal of their IL-2 receptor a chain, considerably increased the number of intrahepatic HBV-specific effector CD8+T cells and the attendant immunopathology. Together, these results reveal a role for group 1 ILCs in controlling T cell–mediated liver immunopathology by limiting local IL-2 concentration and have implications for the treatment of chronic HBV infection.

Background Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection (LRTI) in infants. Nirsevimab is a single-dose monoclonal antibody with extended half-life that was shown to protect preterm infants 29 to < 35 weeks gestation against RSV LRTI. However, most medically attended (MA) cases occur in otherwise healthy, term infants for whom there is currently no effective RSV prevention strategy. We report the primary analysis of efficacy and safety, along with the impact of nirsevimab in late preterm and term infants (≥ 35 weeks gestation) in the phase 3 MELODY study (NCT03979313). Methods Infants were randomized 2:1 to receive one intramuscular injection of nirsevimab (50 mg if < 5 kg; 100 mg if ≥ 5 kg at dosing) or placebo entering their first RSV season. The primary endpoint was the incidence of MA RSV LRTI over 150 days postdose. Cases met predefined clinical criteria of disease severity and were confirmed by real-time reverse-transcriptase PCR. Safety was evaluated through 360 days postdose. Enrollment started on 23 July 2019 and was suspended following the declaration of the COVID-19 pandemic by the WHO on 11 March 2020. Results Overall, 1490 infants were randomized and included in the intent-to-treat population; 1465 (98%) completed the 150-day efficacy follow-up, and 1367 (92%) completed the 360-day safety follow-up. The incidence of MA RSV LRTI was 1.2% (n=12/994) in the nirsevimab group and 5.0% (n=25/496) in the placebo group, giving nirsevimab an efficacy of 74.5% (95% confidence interval [CI]: 49.6, 87.1; p< 0.0001). Nirsevimab averted 93.6 (95% CI 63.0, 124.0) MA LRTIs per 1000 infants dosed. Nirsevimab was well tolerated, with similar rates of adverse events (87.4% nirsevimab; 86.8% placebo) and serious adverse events (6.8% nirsevimab; 7.3% placebo) between groups. Conclusion In this phase 3 study, a single dose of nirsevimab protected late preterm and term infants against MA RSV LRTI over an RSV season with a favorable safety profile. Approximately 11 infants need to be immunized to prevent 1 case of LRTI; nirsevimab has the potential to be an important intervention to reduce the burden of RSV LRTI in healthy infants. Disclosures Laura Hammitt, MD, MedImmune (Grant/Research Support, Scientific Research Study Investigator, Research Grant or Support)Merck & Co., Inc. (Grant/Research Support, Scientific Research Study Investigator, Research Grant or Support)Novavax (Grant/Research Support, Scientific Research Study Investigator, Research Grant or Support)Pfizer (Grant/Research Support, Scientific Research Study Investigator, Research Grant or Support) Laura Hammitt, MD, MedImmune (Individual(s) Involved: Self): Grant/Research Support, Research grant to my institution; Merck (Individual(s) Involved: Self): Grant/Research Support, Research grant to my institution; Pfizer (Individual(s) Involved: Self): Grant/Research Support, Research grant to my institution Ron Dagan, MD, Medimmune/AstraZeneca (Grant/Research Support, Scientific Research Study Investigator, Research Grant or Support)MSD (Consultant, Grant/Research Support, Scientific Research Study Investigator, Advisor or Review Panel member, Research Grant or Support, Speaker’s Bureau)Pfizer (Consultant, Grant/Research Support, Scientific Research Study Investigator, Advisor or Review Panel member, Research Grant or Support, Speaker’s Bureau) Yuan Yuan, PhD, AstraZeneca (Employee, Shareholder) Shabhir A. Mahdi, PhD, BMGF (Research Grant or Support)EDCTP (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melody (Research Grant or Support)Minervax (Research Grant or Support)Novavax (Research Grant or Support)SAMRC (Research Grant or Support) William J. Muller, MD, PhD, Ansun (Scientific Research Study Investigator)Astellas (Scientific Research Study Investigator)AstraZeneca (Scientific Research Study Investigator)Genentech (Scientific Research Study Investigator)Gilead (Scientific Research Study Investigator)Janssen (Scientific Research Study Investigator)Karius (Scientific Research Study Investigator)Melinta (Scientific Research Study Investigator)Merck (Scientific Research Study Investigator)Nabriva (Scientific Research Study Investigator)Seqirus (Scientific Research Study Investigator)Tetraphase (Scientific Research Study Investigator) William J. Muller, MD, PhD, Ansun (Individual(s) Involved: Self): Grant/Research Support; Astellas (Individual(s) Involved: Self): Research Grant or Support; AstraZeneca (Individual(s) Involved: Self): Grant/Research Support; BD (Individual(s) Involved: Self): Research Grant or Support; Eli Lilly (Individual(s) Involved: Self): Grant/Research Support; Gilead (Individual(s) Involved: Self): Grant/Research Support; Karius, Inc. (Individual(s) Involved: Self): Grant/Research Support, Scientific Research Study Investigator; Melinta (Individual(s) Involved: Self): Grant/Research Support; Merck (Individual(s) Involved: Self): Grant/Research Support; Moderna (Individual(s) Involved: Self): Grant/Research Support; Nabriva (Individual(s) Involved: Self): Grant/Research Support; Seqirus (Individual(s) Involved: Self): Consultant; Tetraphase (Individual(s) Involved: Self): Grant/Research Support Heather J. Zar, PhD, AstraZeneca (Grant/Research Support)Novavax (Grant/Research Support)Pfizer (Grant/Research Support, Advisor or Review Panel member) Dennis Brooks, MD, AstraZeneca (Employee) Amy Grenham, MSc, AstraZeneca (Employee, Shareholder) Ulrika Wählby Hamrén, PhD, AstraZeneca R&D (Employee, Shareholder) Vaishali S. Mankad, MD, AstraZeneca (Employee) Therese Takas, BSc, AstraZeneca (Employee, Other Financial or Material Support, Own stock in AstraZeneca) Jon Heinrichs, PhD, AstraZeneca (Shareholder)Bristol Myers Squibb (Shareholder)J&J (Shareholder)Merck (Shareholder)Organon (Shareholder)Procter & Gamble (Shareholder)Sanofi (Shareholder)Sanofi Pasteur (Employee) Amanda Leach, MRCPCH, AstraZeneca (Employee, Shareholder) M. Pamela Griffin, MD, AstraZeneca (Employee) Tonya L. Villafana, PhD, AstraZeneca (Employee)

BackgroundThe ligand for the receptor tyrosine kinase FMS-like tyrosine kinase 3 (FLT3) plays an importantrole in hematopoiesis. FLT3 signaling is required for the differentiation andexpansion of dendritic cells. In the context of cancer immunity, the conventional dendritic cellsubtype 1 (cDC1) are required for the generation of tumor-specific T cell responses in mousepreclinical models. In human tumors cDC1 are often underrepresented in thetumor microenvironment, supporting the hypothesis that therapeutically increasing their number via FLT3 pathway stimulation has the potential to promote T cell-mediated anti-tumor efficacy.MethodsGS-3583 is a fusion protein composed of the extracellular domain (ECD) of human FLT3 ligand(FLT3L) combined with a modified fragment crystallizable (Fc) region of human IgG4. GS-3583was designed to induce cDC1 expansion and subsequently promote tumor-reactive T cell priming, activation and recruitment into the tumor microenvironment. To evaluate the therapeutic efficacy of FLT3 stimulation in vivo, a mouse surrogate mGS-3583was designed using the ECD of mouse FLT3L fused to an engineered mouse IgG2a Fc withattenuated binding to mouse FcgRs.Results mGS-3583 bound to recombinant mouse FLT3 with an estimated affinity of 15 nM, and to mouse FLT3-expressing cells with an EC50 of 0.15 nM. In vivo, mGS-3583 showed single agent dose-dependent tumor growth inhibition (TGI) in tumors that correlated with peripheral and intratumoral cDC1 expansion. In tumors with no initial immune infiltration, mGS-3583 led to an influx of T cells into the tumors. In addition to single agent efficacy, mGS-3583 combined effectively with programmed cell death protein (ligand)-1 (PD(L)-1) pathway blockade.ConclusionsIn vivo expansion of dendritic cells can convert uninflamed (cold) tumors to immunologically active (hot) tumors and initiate productive anti-tumor immune responses. These findings support the development GS-3583 as a promising candidate for cancer immunotherapy.

BackgroundThe ligand for the receptor tyrosine kinase FMS-like tyrosine kinase 3 (FLT3) plays an importantrole in hematopoiesis. FLT3 signaling is required for the differentiation andexpansion of dendritic cells. In the context of cancer immunity, the conventional dendritic cellsubtype 1 (cDC1) are required for the generation of tumor-specific T cell responses in mousepreclinical models. In human tumors cDC1 are often underrepresented in thetumor microenvironment, supporting the hypothesis that therapeutically increasing their number via FLT3 pathway stimulation has the potential to promote T cell-mediated anti-tumor activity.MethodsGS-3583 is a fusion protein composed of the extracellular domain of human FLT3 ligand(FLT3L) combined with a modified fragment crystallizable (Fc) region of human IgG4. GS-3583was designed to induce cDC1 expansion and subsequently promote tumor-reactive T cell priming, activation and recruitment into the tumor microenvironment. The pharmacokinetics (PK) and pharmacodynamics (PD) of GS-3583 has been characterized in a 4-week repeat dose GLP study in cynomolgus monkeys at doses ranging from 0.3 to 10mg/kg GS-3583 was given as an intravenous injection.ResultsImmunophenotyping analysis of peripheral blood cells from GS-3583 treated monkeys demonstrated a non-dose-dependent expansion of cDC1 and cDC2 populations. The peak expansion for cDC1 and cDC2 occurred at Day 8 to Day 15. At peak, there was a 160-fold relative increase in cDC1 and 150-fold increase in cDC2 at the highest dose tested. There were dose-dependent increases in the exposure (AUC and Cmax) of GS-3583. GS-3583 was well-tolerated with no mortality or adverse clinical signs.ConclusionsThe administration of GS-3583 leads to increases in cDC1 and cDC2 populations. It was well tolerated at the maximal dose tested with no adverse clinical signs. Further clinical development of GS-3583 is warranted.

This study explores the structural and functional changes associated with a low-temperature thermal transition of 2 engineered bacterial uricase mutants. Uricase has a noncovalent homotetrameric structure, with 4 active sites located at the interface of subunits. Using differential scanning calorimetry, a low-temperature transition was identified at 42°C for mutant A and at 33°C for mutant B. This transition was stabilized by the uricase inhibitor, oxonic acid, suggesting a strong structural relationship to the active site. For mutant B, there was a reversible loss of enzymatic activity above the low-temperature transition. Spectroscopic measurements demonstrated that there was also a reversible loss of secondary and tertiary structures and an increase in surface hydrophobicity. However, the hydrophobic core environment and the tetrameric structure were not altered over the low-temperature transition suggesting that the changes occurred primarily at the surface of the enzyme. The protein became aggregation-prone at temperatures approaching the cluster of higher-temperature melting transitions at 84°C, indicating these transitions represent a global unfolding of the protein. Our findings shed light on the structural changes that affect the uricase mechanism of action and provide new insights into how enzyme therapeutic development may be approached.

The analysis and accurate quantitation of bioconjugations proves challenging in the case of oligomeric proteins, especially when the size of the molecule or the nature of the conjugate do not allow the analysis of the intact protein under native conditions. In this case, analytical methods are frequently applied that result in a dissociation of non-covalently linked subunits. This limits the analysis to a description of individual subunits, thereby obscuring the accurate characterization of the overall functionalization. This situation is frequently encountered in the biopharmaceutically important case of protein PEGylation, as the biophysical properties of the PEG polymer generally make analysis and accurate quantitation for a protein with multiple conjugation sites challenging under native conditions. In this work we present a statistical measure for deriving the overall functionalization of an oligomeric protein from the data obtained from readily accessible assays that cause non-covalently associated subunits to dissociate. This approach is broadly applicable for the characterization and optimization of bioconjugation reactions for multimeric biomolecules. It should also be highly valuable for the accurate description of composition and manufacturing consistency of conjugated biotherapeutics in regulatory filings.LAY ABSTRACT: Conjugated proteins are an important class of biopharmaceuticals. For these molecules, successful drug development requires accurate methods for the quantitative characterization of protein conjugation. This task is particularly challenging in the case of proteins consisting of several, non-covalently linked subunits, especially when the size of the protein or nature of the conjugate do not allow for analysis of the intact oligomeric molecule. Many of the analytical methods used to characterize these conjugates, such as reverse phase high-performance liquid chromatography, cause the individual subunits to dissociate, making it difficult to fully understand quality attributes at the native oligomeric level. We present a method to accurately quantify the overall conjugation of an oligomeric protein in these cases when readily available assays describe only individual subunits. This should be highly valuable for process optimization and to correctly characterize the conjugated biopharmaceutical in interactions with regulatory agencies.

Background Pertussis immunization during pregnancy results in high pertussis antibody concentrations in young infants but may interfere with infant immune responses to post-natal immunization. Methods This phase IV, multi-country, open-label study assessed the immunogenicity and safety of infant primary vaccination with DTaP-HepB-IPV/Hib and 13-valent pneumococcal conjugate vaccine (PCV13). Enrolled infants (6–14 weeks old) were born to mothers who were randomized to receive reduced-antigen-content diphtheria-tetanus-three-component acellular pertussis vaccine (Tdap group) or placebo (control group) during pregnancy (270/7–366/7 weeks’ gestation) with crossover immunization postpartum. All infants received 2 or 3 DTaP-HepB-IPV/Hib and PCV13 doses according to national schedules. Immunogenicity was assessed in infants pre- and 1 month post-primary vaccination. The primary objective was to assess seroprotection/vaccine response rates for DTaP-HepB-IPV/Hib antigens 1 month post-primary vaccination. Results 601 infants (Tdap group: 296; control group: 305) were vaccinated. One month post-priming, seroprotection rates were 100% (diphtheria; tetanus), ≥98.5% (hepatitis B), ≥95.9% (polio) and ≥94.5% (Hib) in both groups. Vaccine response rates for pertussis antigens were significantly lower in infants whose mothers received pregnancy Tdap (37.5–77.1%) versus placebo (90.0–99.2%). Solicited and unsolicited adverse event rates were similar between groups. Serious adverse events occurred in 2.4% (Tdap group) and 5.6% (control group) of infants, none were vaccination-related. Conclusions Pertussis antibodies transferred during pregnancy may decrease the risk of pertussis infection in the first months of life but interfere with the infant’s ability to produce pertussis antibodies, the clinical significance of which remains unknown. Safety and reactogenicity results were consistent with previous experience. Clinical Trial Registration: ClinicalTrials.gov: NCT02422264.

The CD40/CD40L axis plays a central role in the generation of humoral immune responses and is an attractive target for treating autoimmune diseases in the clinic. Here, we report the generation and clinical results of a CD40L binding protein, VIB4920, which lacks an Fc domain, therefore avoiding platelet-related safety issues observed with earlier monoclonal antibody therapeutics that targeted CD40L. VIB4920 blocked downstream CD40 signaling events, resulting in inhibition of human B cell activation and plasma cell differentiation, and did not induce platelet aggregation in preclinical studies. In a phase 1 study in healthy volunteers, VIB4920 suppressed antigen-specific IgG in a dose-dependent fashion after priming and boosting with the T-dependent antigen, KLH. Furthermore, VIB4920 significantly reduced circulating Ki67+ dividing B cells, class-switched memory B cells, and a plasma cell gene signature after immunization. In a phase 1b proof-of-concept study in patients with rheumatoid arthritis, VIB4920 significantly decreased disease activity, achieving low disease activity or clinical remission in more than 50% of patients in the two higher-dose groups. Dose-dependent decreases in rheumatoid factor autoantibodies and Vectra DA biomarker score provide additional evidence that VIB4920 effectively blocked the CD40/CD40L pathway. VIB4920 demonstrated a good overall safety profile in both clinical studies. Together, these data demonstrate the potential of VIB4920 to significantly affect autoimmune disease and humoral immune activation and to support further evaluation of this molecule in inflammatory conditions.

Background: A quadrivalent split-virion inactivated influenza vaccine (VaxigripTetre (TM), Sanofi Pasteur; IIV4) containing two A strains (H1N1 and H3N2) and B strains from both lineages (Victoria and Yamagata) was approved in Europe in 2016 for individuals aged >= 3 years. This study examined the efficacy and safety of IIV4 in children aged 6-35 months. Methods: This was a phase III randomised controlled trial conducted in Latin America, Asia, Africa, and Europe during the Northern Hemisphere 2014/2015 and 2015/2016 and Southern Hemisphere 2014 and 2015 influenza seasons. Healthy children aged 6-35 months not previously vaccinated against influenza were randomised to receive two full doses 28 days apart of IIV4, placebo, the licensed trivalent splitvirion inactivated vaccine (IIV3), an investigational IIV3 containing a B strain from the alternate lineage. The primary objective was to demonstrate efficacy against influenza illness caused by any strain or vaccine-similar strains. Results: The study enrolled 5806 participants. Efficacy, assessed in 4980 participants completing the study according to protocol, was demonstrated for IIV4. Vaccine efficacy was 50.98% (97% CI, 37.36-61.86%) against influenza caused by any A or B type and 68.40% (97% CI, 47.07-81.92%) against influenza caused by vaccine-like strains. Safety profiles were similar for IIV4, placebo, and the IIV3s, although injection-site reactions were slightly more frequent for IIV4 than placebo. Conclusions: IIV4 was safe and effective for protecting children aged 6-35 months against influenza illness caused by vaccine-similar or any circulating strains. (C) 2018 The Authors. Published by Elsevier Ltd.

The influence of niobium Nb as a bulk constituent of phase-pure MoVTe(Sb)NbO mixed oxides, with an M1 structure has been studied. Sb and Te containing M1 phases with various amounts of Nb were prepared and characterized using several techniques such as V K-edge and Sb or Te L1-edge Xanes spectroscopy. The mechanism through which the presence of niobium affects the cationic and valence distributions in the M1 phases has been determined. Various M1 phases were tested as catalysts for the oxidation of propane to acrylic acid, or the oxidative dehydrogenation of ethane, in order to study the influence niobium on the catalyst’s properties or stability. It is shown that Nb has no influence on the catalyst’s activity, and that it has a positive effect on its selectivity to acrylic acid for propane oxidation, and to ethylene for ethane oxidation. This effect is associated with a decrease in the surface acidity of the M1 phase, which promotes unselective oxidation of propene and with a decrease in the catalyst’s oxidation power, which inhibits the over-oxidation of acrylic acid or ethylene. Both of these effects are related to the increase in Mo5+ content of the phases, which appears to be a key parameter in the selectivity of the M1 phase. Although a possible influence of Nb on the crystallinity or texture of the catalysts was not observed, in the case of the preparation techniques used in the present study, an increase in stability was observed over long periods of time on stream in the case of Nb- containing phases.

4-1BBL is a member of the tumor necrosis factor (TNF) superfamily and is the ligand for the TNFR superfamily receptor, 4-1BB. 4-1BB plays an immunomodulatory role in T cells and NK cells, and agonists of this receptor have garnered strong attention as potential immunotherapy agents. Broadly speaking, the structural features of TNF superfamily members, their receptors, and ligand-receptor complexes are similar. However, a published crystal structure of human 4-1BBL suggests that it may be unique in this regard, exhibiting a three-bladed propeller-like trimer assembly that is distinctly different from that observed in other family members. This unusual structure also suggests that the human 4-1BB/4-1BBL complex may be structurally unique within the TNF/TNFR superfamily, but to date no structural data have been reported. Here we report the crystal structure of the human 4-1BB/4-1BBL complex at 2.4-A resolution. In this structure, 4-1BBL does not adopt the unusual trimer assembly previously reported, but instead forms a canonical bell-shaped trimer typical of other TNF superfamily members. The structure of 4-1BB is also largely canonical as is the 4-1BB/4-1BBL complex. Mutational data support the 4-1BBL structure reported here as being biologically relevant, suggesting that the previously reported structure is not. Together, the data presented here offer insight into structure/function relationships in the 4-1BB/4-1BBL system and improve our structural understanding of the TNF/TNFR superfamily more broadly.

Tn3 proteins are a novel class of binding molecules based on the third fibronectin type III domain of human tenascin C. Target-specific Tn3 proteins are selected from combinatorial libraries in which three surface-exposed loops have been diversified. Here, the cocrystallization of two different Tn3 proteins in complex with CD40L, a therapeutic target for immunological disease, is reported. These crystal structures are the first to be reported of Tn3 proteins and will help to reveal how these engineered molecules achieve specific recognition of a cognate target.

Activation of TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) can induce apoptosis in a variety of human cancer cell lines and xenografts, while lacking toxicity in normal cells. The natural ligand and agonistic antibodies show antitumor activity in preclinical models of cancer, and this had led to significant excitement in the clinical potential of these agents. Unfortunately, this optimism has been tempered by trial data that, thus far, are not showing clear signs of efficacy in cancer patients. The reasons for discrepant preclinical and clinical observations are not understood, but one possibility is that the current TRAILR2 agonists lack sufficient potency to achieve a meaningful response in patients. Toward addressing that possibility, we have developed multivalent forms of a new binding scaffold (Tn3) that are superagonists of TRAILR2 and can induce apoptosis in tumor cell lines at subpicomolar concentrations. The monomer Tn3 unit was a fibronectin type III domain engineered for high-affinity TRAILR2 binding. Multivalent presentation of this basic unit induced cell death in TRAILR2-expressing cell lines. Optimization of binding affinity, molecular format, and valency contributed to cumulative enhancements of agonistic activity. An optimized multivalent agonist consisting of 8 tandem Tn3 repeats was highly potent in triggering cell death in TRAIL-sensitive cell lines and was 1 to 2 orders of magnitude more potent than TRAIL. Enhanced potency was also observed in vivo in a tumor xenograft setting. The TRAILR2 superagonists described here have the potential for superior clinical activity in settings insensitive to the current therapeutic agonists that target this pathway. Mol Cancer Ther; 12(7); 1235–44. ©2013 AACR.

Fusion of proteins to the Fc region of IgG is widely used to express cellular receptors and other extracellular proteins, but cleavage of the fusion partner is sometimes required for downstream applications. Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) is a protease with exquisite specificity for human IgG, and it can also cleave Fc-fusion proteins at a single site in the N-terminal region of the CH2 domain. However, the site of IdeS cleavage results in the disulfide-linked hinge region partitioning with the released protein, complicating downstream usage of the cleaved product. To tailor the Fc fragment for release of partner proteins by IdeS treatment, we investigated the effect of deleting regions of IgG-derived sequence that are upstream of the cleavage site. Elimination of the IgG-derived hinge sequence along with several residues of the CH2 domain had negligible effects on expression and purity of the fusion protein, while retaining efficient processing by IdeS. An optimal Fc fragment comprising residues 235-447 of the human IgG1 heavy chain sufficed for efficient production of fusion proteins and minimized the amount of residual Ig-derived sequence on the cleavage product following IdeS treatment. Pairing of this truncated Fc fragment with IdeS cleavage enables highly specific cleavage of Fc-fusion proteins, thus eliminating the need to engineer extraneous cleavage sequences. This system should be helpful for producing Fc-fusion proteins requiring downstream cleavage, particularly those that are sensitive to internal miscleavage if treated with alternative proteases.

Receptor clustering is important for signaling among the therapeutically relevant TNFR superfamily of receptors. In nature, this clustering is driven by trimeric ligands often presented in large numbers as cell surface proteins. Molecules capable of driving similar levels of clustering could make good agonists and hold therapeutic value. However, recapitulating such extensive clustering using typical biotherapeutic formats, such as antibodies, is difficult. Consequently, generating effective agonists of TNFR superfamily receptors is challenging. Toward addressing this challenge we have used lipid- and polyion complex-based micelles as platforms for presenting receptor-binding biologics in a multivalent format that facilitates receptor clustering and imparts strong agonist activity. We show that receptor-binding scFvs or small antibody mimetics that have no agonist activity on their own can be transformed into potent agonists through multivalent presentation on a micelle surface and that the activity of already active multivalent agonists can be enhanced. Using this strategy, we generated potent agonists against two different TNFR superfamily receptors and mouse tumor model studies demonstrate that these micellar agonists have therapeutic efficacy in vivo. Due to its ease of implementation and applicability independent of agonist molecular format, we anticipate that this strategy could be useful for developing agonists to a variety of receptors that rely on clustering to signal.

The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3-5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material.

IL-11 and IL-11 receptor (R)alpha are induced by Th2 cytokines. However, the role(s) of endogenous IL-11 in antigen-induced Th2 inflammation has not been fully defined. We hypothesized that IL-11, signaling via IL-11Ralpha, plays an important role in aeroallergen-induced Th2 inflammation and mucus metaplasia. To test this hypothesis, we compared the responses induced by the aeroallergen ovalbumin (OVA) in wild-type (WT) and IL-11Ralpha-null mutant mice. We also generated and defined the effects of an antagonistic IL-11 mutein on pulmonary Th2 responses. Increased levels of IgE, eosinophilic tissue and bronchoalveolar lavage (BAL) inflammation, IL-13 production, and increased mucus production and secretion were noted in OVA-sensitized and -challenged WT mice. These responses were at least partially IL-11 dependent because each was decreased in mice with null mutations of IL-11Ralpha. Importantly, the administration of the IL-11 mutein to OVA-sensitized mice before aerosol antigen challenge also caused a significant decrease in OVA-induced inflammation, mucus responses, and IL-13 production. Intraperitoneal administration of the mutein to lung-specific IL-13-overexpressing transgenic mice also reduced BAL inflammation and airway mucus elaboration. These studies demonstrate that endogenous IL-11R signaling plays an important role in antigen-induced sensitization, eosinophilic inflammation, and airway mucus production. They also demonstrate that Th2 and IL-13 responses can be regulated by interventions that manipulate IL-11 signaling in the murine lung.

There is limited knowledge on the influence of the pore size on surface phenomena (adsorption, dissolution, precipitation, etc.) at the oxide/water interface and a better understanding of the space confinement in nanoscale pores should have practical implications in different areas, such as transport of contaminants in the environment or heterogeneous catalyst preparation, to name a few. To investigate the modifications of the oxide adsorption properties at the oxide/water interface in a confined environment, the surface acidobasic and ion adsorption properties of six different aluminas (5 porous commercial aluminas with pore diameters ranging from 25 to 200 A and 1 non-porous alumina) were determined by means of acid-base titration and Ni(II) adsorption. It is shown that the confinement has a moderate impact on the alumina adsorption capacity because all materials have similar surface charging behaviours and ion saturation coverages. However, a confined geometry has a much larger impact on the ion adsorption constants, which decrease drastically when the average pore diameter decreases below 200 A. These results are discussed in terms of nanoscale pore space confinement.

The suppressors of cytokine signaling (SOCS) family of proteins act as intracellular inhibitors of several cytokine signal transduction pathways. Their expression is induced by cytokine activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and they act as a negative feedback loop by subsequently inhibiting the JAK/STAT pathway either by direct interaction with activated JAKs or with the receptors. These interactions are mediated at least in part by the SH2 domain of SOCS proteins but these proteins also contain a highly conserved C-terminal homology domain termed the SOCS box. Here we show that the SOCS box mediates interactions with elongins B and C, which in turn may couple SOCS proteins and their substrates to the proteasomal protein degradation pathway. Analogous to the family of F-box-containing proteins, it appears that the SOCS proteins may act as adaptor molecules that target activated cell signaling proteins to the protein degradation pathway.

The overexpression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations, retroviral insertion during gene therapy, or in transgenic mice models, leads to the onset of T-cell leukemias. LMO2 comprises two protein-binding LIM domains that allow LMO2 to interact with multiple protein partners, including LIM domain-binding protein 1 (Ldb1, also known as CLIM2 and NLI), an essential cofactor for LMO proteins. Sequestration of Ldb1 by LMO2 in T-cells may prevent it binding other key partners, such as LMO4. Here, we show using protein engineering and enzyme-linked immunosorbent assay (ELISA) methodologies that LMO2 binds Ldb1 with a twofold lower affinity than does LMO4. Thus, excess LMO2 rather than an intrinsically higher binding affinity would lead to sequestration of Ldb1. Both LIM domains of LMO2 are required for high-affinity binding to Ldb1 (K(D) = 2.0 x 10(-8) M). However, the first LIM domain of LMO2 is primarily responsible for binding to Ldb1 (K(D) = 2.3 x 10(-7) M), whereas the second LIM domain increases binding by an order of magnitude. We used mutagenesis in combination with yeast two-hybrid analysis, and phage display selection to identify LMO2-binding "hot spots" within Ldb1 that locate to the LIM1-binding region. The delineation of this region reveals some specific differences when compared to the equivalent LMO4:Ldb1 interaction that hold promise for the development of reagents to specifically bind LMO2 in the treatment of leukemia.

SOCS3 is essential for regulating the extent, duration, and specificity of cellular responses to cytokines such as G-CSF and IL-6. Here we describe the solution structure of SOCS3, the first structure determined for any SOCS protein, in complex with a phosphotyrosine-containing peptide from the IL-6 receptor signaling subunit gp130. The structure of the complex shows that seven peptide residues form a predominantly hydrophobic binding motif. Regions outside the SOCS3 SH2 domain are important for ligand binding, in particular, a single 15 residue alpha helix immediately N-terminal to the SH2 domain makes direct contacts with the phosphotyrosine binding loop and, in part, determines its geometry. The SH2 domain itself is remarkable in that it contains a 35 residue unstructured PEST motif insertion that is not required for STAT inhibition. The PEST motif increases SOCS3 turnover and affects its degradation pathway, implying that it has an important regulatory role inside the cell.

SOCS3 is a negative regulator of cytokine signalling that inhibits Janus kinase-signal transduction and activator of transcription (JAK-STAT) mediated signal tranduction by binding to phosphorylated tyrosine residues on intracellular subunits of various cytokine receptors, as well as possibly the JAK proteins. SOCS3 consists of a short N-terminal sequence followed by a kinase inhibitory region, an extended SH2 domain and a C-terminal suppressor of cytokine signalling (SOCS) box. SOCS3 and the related protein, cytokine-inducible SH2-containing protein, are unique among the SOCS family of proteins in containing a region of mostly low complexity sequence, between the SH2 domain and the C-terminal SOCS box. Using NMR, we assigned and determined the secondary structure of a murine SOCS3 construct. The SH2 domain, unusually, consists of 140 residues, including an unstructured insertion of 35 residues. This insertion fits the criteria for a PEST sequence and is not required for phosphotyrosine binding, as shown by isothermal titration calorimetry. Instead, we propose that the PEST sequence has a functional role unrelated to phosphotyrosine binding, possibly mediating efficient proteolytic degradation of the protein. The latter half of the kinase inhibitory region and the entire extended SH2 subdomain form a single α-helix. The mapping of the true SH2 domain, and the location of its C terminus more than 50 residues further downstream than predicted by sequence homology, explains a number of previously unexpected results that have shown the importance of residues close to the SOCS box for phosphotyrosine binding.

Results are reported concerning the synergetic effect observed in the oxidation of propane to acrylic acid over the orthorhombic M1 and hexagonal M2 phases present in the most active and selective MoVTe(Sb)NbO catalysts. The pure phases and phase mixtures containing either tellurium or antimony have been prepared and individually tested as catalysts. The results obtained confirm that the phase responsible for the catalytic properties of the efficient catalysts is phase M1, and that M2 is poorly active. Mechanical mixtures of the pure phases have also been prepared and tested. All of the catalysts have been characterized before and after the catalytic reaction by X-ray diffraction, X-ray photoelectron spectroscopy, and high-resolution electron microscopy with EDS analyses. Although the synergetic effect previously described (bifunctional catalysis with the oxidative dehydrogenation of propane molecules on the orthorhombic M1 phase and the subsequent oxidation of propene on the hexagonal M2 phase) was observed, another cause related to migration of tellurium from the M2 phase to the surface of the M1 phase was indicated. This migration should balance a loss of tellurium in the active phase occurring under the conditions of a catalytic test, but may also create new dehydrogenation sites for propene and/or anneal total oxidation sites. The hexagonal M2 phase would thus play a role of tellurium reservoir for the active M1 phase. This effect was not reversible and concerned only the tellurium. Antimony, which is less volatile, should not be lost by the active phase. Furthermore, it was shown not to diffuse at the surface of the phases.

The adsorption of pyridine on the surface of the M1 active phase of the MoVTe(Sb)O catalysts has been studied for the determination of the Bronsted and Lewis acid sites. The results obtained showed medium Lewis and Bronsted acidity for antimony containing phases and low Lewis and Bronsted acidity for tellurium ones. The incorporation of niobium in the M1 phases resulted in a decrease of both acidities. The samples characterized have been tested as catalysts in the oxidation of propane to acrylic acid. The results obtained allowed to propose correlations between the acidic properties and the catalytic selectivity. 2005 Elsevier B.V. All rights reserved.

Different spectroscopic techniques like XANES, ESR, XPS and Mossbauer spectroscopy have been used to determine the oxidation state of the various cations in the M1 and M2 phases of the MoVTe(Sb)NbO catalysts used for the oxidation or ammoxidation of propane. It was observed that the tellurium or antimony M 1o rM2 phases contained mainly Te IV , and Nb V. Molybdenum, vanadium and antimony were shown to be present as Mo VI and Mo V ,V V and V IV and Sb V and Sb III . The M1 phase which is responsible for the high efficiency of catalysts, corresponds to the total stoichiometry (AO)2� x(A2O)xM20O56 with A = Sb or Te, M = Mo, V and Nb and 0 � x � 1. It has a structure with hexagonal channels occupied by the A cations and oxides. This channel may contain an excess of oxygen which constitutes a reservoir for the catalysts and which likely plays a role in the reoxidation of the catalytic sites. The balance of the charges introduced by these oxides anions was achieved by the partial oxidation of Sb III to Sb V in M1(Sb) and by the oxidation of Mo V to Mo VI in M1(Te), Te remaining always Te IV as shown from Mossbauer spectroscopy data. The characterization of the solids after catalytic test showed very few changes in the solids structures and compositions. The study allowed concluding that the lone pair elements does not have only a role as constituents of the surface catalytic sites but also as bulk components to store oxygen in the hexagonal channels and contributes to its rapid diffusion to the surface. # 2004 Elsevier B.V. All rights reserved. reported as (Te2O)2M20O56 and (SbO)2M20O56 with M = Mo, V, Nb. The V/Mo and Nb/Mo ratios can vary, but are most frequently close to 0.3 and 0.1, respectively (4,5). The phase containing Te has an oxide over- stoichiometry while the one containing Sb, an oxide sub- stoichiometry, they may better be reported with the general composition (AO)2� x(A2O)xM20O56 with A = Te or Sb and 0 � x � 1. The first structure model for the M1 phases has been proposed in 2002 based on an isomorphism with the phase Cs0.7(Nb,W)5O14 (4). The structure of the M1 phase has been described as a corner sharing MO6 (M = Mo, V, Nb) octahedra network with tellurium or antimony cations and oxygen anions occupying sites in hexagonal channels formed by the octahedra. Besides hexagonal channels, pentagonal and heptagonal channels are present and oriented in the same direction. The heptagonal channels have been

Leukemia inhibitory factor (LIF)-induced cell signaling occurs following sequential binding to the LIF receptor alpha-chain (LIFR), then to the gp130 co-receptor used by all members of the interleukin-6 family of cytokines. By monovalently displaying human LIF on the surface of M13 phage and randomizing clusters of residues in regions predicted to be important for human LIFR binding, we have identified mutations, which lead to significant increases in affinity for binding to LIFR. Six libraries were constructed in which regions of 4-6 amino acids were randomized then panned against LIFR. Mutations identified in three distinct clusters, residues 53-57, 102-103, and 150-155, gave rise to proteins with significantly increased affinity for binding to both human and mouse LIFR. Combining the mutations for each of these regions further increased the affinity, such that the best mutants bound to human LIFR with1000-fold higher affinity than wild-type human LIF. NMR analysis indicated that the mutations did not alter the overall structure of the molecule relative to the native protein, although some local changes occurred in the vicinity of the substituted residues. Despite increases in LIFR binding affinity, these mutants did not show any increase in activity as agonists of LIF-induced proliferation of Ba/F3 cells expressing human LIFR and gp130 compared with wild-type LIF. Incorporation of two additional mutations (Q29A and G124R), which were found to abrogate cell signaling, led to the generation of highly potent antagonists of both human and murine LIF-induced bioactivity.

Several phases reported as minor or major phases in the active MoVTeNbO catalysts have been prepared and investigated for the oxidation of propane into acrylic acid. Activity and selectivity of pure phases and mixtures of phases obtained either directly from synthesis or by co-grinding have been compared. The results obtained confirmed that the orthorhombic M1 phase is the most active and selective phase and is responsible for the major part of the efficiency of the best catalysts. However, they also clearly demonstrated that a synergism due to a cooperation between phases occurs, similar to that previously proposed between the M1 [(Te2O)M20O56] and M2 [(TeO)M3O9] phases for the ammoxidation of propane. The origin of this phase cooperation is discussed.

Suppressor of cytokine signaling (SOCS)-2 is a member of a family of intracellular proteins implicated in the negative regulation of cytokine signaling. The generation of SOCS-2-deficient mice, which grow to one and a half times the size of their wild-type littermates, suggests that SOCS-2 may attenuate growth hormone (GH) signaling. In vitro studies indicate that, while SOCS-2 can inhibit GH action at low concentrations, at higher concentrations it may potentiate signaling. To determine whether a similar enhancement of signaling is observed in vivo or alternatively whether increased SOCS-2 levels repress growth in vivo, we generated and analyzed transgenic mice that overexpress SOCS-2 from a human ubiquitin C promoter. These mice are not growth-deficient and are, in fact, significantly larger than wild-type mice. The overexpressed SOCS-2 was found to bind to endogenous GH receptors in a number of mouse organs, while phosphopeptide binding studies with recombinant SOCS-2 defined phosphorylated tyrosine 595 on the GH receptor as the site of interaction. Together, the data implicate SOCS-2 as having dual effects on GH signaling in vivo.

Suppressor of cytokine signaling-3 (SOCS-3) and the protein tyrosine phosphatase SHP-2 both regulate signaling by cytokines of the interleukin-6 family, and this is dependent upon recruitment to tyrosine 757 in the shared cytokine receptor subunit gp130. To better explore the overlap in ligand binding specificities exhibited by these two signaling regulators, we have mapped the phosphopeptide binding preferences of the SH2 domains from SOCS-3 and SHP-2. Degenerate phosphopeptide libraries were screened against recombinantly produced SH2 domains to determine the sequences of optimal phosphopeptide ligands. We found that the consensus ligand binding motif for SOCS-3 was pY-(S/A/V/Y/F)-hydrophobic-(V/I/L)-hydrophobic-(H/V/I/Y), while the consensus motif for SHP-2 was pY-(S/T/A/V/I)-X-(V/I/L)-X-(W/F). We validated these data through the design of phosphopeptide ligands based on the consensus motifs and found that these bound to SOCS-3 and SHP-2 with high affinity. Finally, we have compared the affinity of SOCS-3 for binding to phosphopeptides representing putative docking sites in the gp130, leptin and erythropoietin receptors. While SOCS-3 binds with much higher affinity to a gp130 phosphopeptide than to phosphopeptides derived from the other receptors, multiple SOCS-3 binding sites are predicted to exist in the leptin and erythropoietin receptors which may compensate for weaker binding to individual sites.

Non-antibody scaffolds are increasingly used to generate novel binding proteins for both research and therapeutic applications. Our group has developed the tenth fibronectin type III domain of human tenascin-C (TNfn3) as one such scaffold. As a scaffold, TNfn3 must tolerate extensive mutation to introduce novel binding sites. However, TNfn3's marginal stability (T(m) ∼ 59°C, ΔG(unfolding) = 5.7 kcal/mol) stands as a potential obstacle to this process. To address this issue, we sought to engineer highly stable TNfn3 variants. We used two parallel strategies. Using insights gained from structural analysis of other FN3 family members, we (1) rationally designed stabilizing point mutations or (2) introduced novel stabilizing disulfide bonds. Both strategies yielded highly stable TNfn3 variants with T(m) values as high as 83°C and ΔG(unfolding) values as high as 9.4 kcal/mol. Notably, only three or four mutations were required to achieve this level of stability with either approach. These results validate our rational design strategies and illustrate that substantial stability increases can be achieved with minimal mutation. One TNfn3 variant reported here has now been successfully used as a scaffold to develop two promising therapeutic molecules. We anticipate that other variants described will exhibit similar utility.

Highly structured, peptide antagonists of the interaction between insulin-like growth factor 1 (IGF-I) and IGF binding protein 1 (IGFBP-1) have recently been discovered by phage display of naïve peptide libraries [Lowman, H. B., et al. (1998) Biochemistry 37, 8870--8878]. We now report a detailed analysis of the features of this turn-helix peptide motif that are necessary for IGFBP-1 binding and structural integrity. Further rounds of phage randomization indicate the importance of residues contributing to a hydrophobic patch on one face of the helix. Alanine-scanning substitutions confirm that the hydrophobic residues are necessary for binding. However, structural analysis by NMR spectroscopy indicates that some of these analogues are less well folded. Structured, high-affinity analogues that lack the disulfide bond were prepared by introducing a covalent constraint between side chains at positions i and i + 7 or i + 8 within the helix. Analogues based on this scaffold demonstrate that a helical conformation is present in the bound state, and that hydrophobic side chains in this helix, and residues immediately preceding it, interact with IGFBP-1. By comparison of alanine scanning data for IGF-I and the turn-helix peptide, we propose a model for common surface features of these molecules that recognize IGFBP-1.