Extracellular domain-IgG fusion proteins for three human natriuretic peptide receptors. Hormone pharmacology and application to solid phase screening of synthetic peptide antisera.

The natriuretic peptide receptors (NPRs) are a family of three cell surface glycoproteins, each with a single transmembrane domain. Two of these receptors, designated NPR-A and NPR-B, are membrane guanylyl cyclases that synthesize cGMP in response to hormone stimulation. The third receptor, NPR-C, has been reported to function in the metabolic clearance of ligand and in guanylyl cyclase-independent signal transduction. We engineered three chimeric proteins consisting of the natriuretic peptide receptor extracellular domains fused to the Fc portion of human IgG-gamma 1. These molecules provide material for detailed studies of the human receptor's extracellular domain structure and interaction with the three human natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and type-C natriuretic peptide (CNP). The homodimeric fusion proteins, designated A-IgG, B-IgG, and C-IgG, were secreted from Chinese hamster ovary cells and purified by protein-A affinity chromatography. We present here the primary characterization of these fusion proteins as represented by the intrinsic hormone affinities measured by saturation binding and competition assays. The dissociation constant of 125I-ANP for A-IgG was 1.6 pM and for C-IgG, 1.2 pM. The dissociation constant of 125I-Y0-CNP (CNP with addition of tyrosine at the amino terminus) for B-IgG was 23 pM. The rank order of potency in competitive binding for A-IgG was ANP greater than BNP much greater than CNP, whereas for B-IgG the ranking was CNP much greater than ANP greater than BNP. For C-IgG, we observed ANP greater than CNP greater than or equal to BNP. These data demonstrate that the receptor-IgG fusion proteins discriminate among the natriuretic peptides in the same manner as the native receptors and provide a basis for future structural studies with these molecules. The purified fusion proteins have a variety of potential applications, one of which we illustrate by a solid phase screening assay in which rabbit sera from a series of synthetic-peptide immunizations were titered for receptor reactivity and selectivity.     

Natriuretic peptides in cardiovascular diseases

The natriuretic peptide family comprises atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), dendroaspis natriuretic peptide (DNP), and urodilatin. The activities of natriuretic peptides and endothelins are strictly associated with each other. ANP and BNP inhibit endothelin-1 (ET-1) production. ET-1 stimulates natriuretic peptide synthesis. All natriuretic peptides are synthesized from polypeptide precursors. Changes in natriuretic peptides and endothelin release were observed in many cardiovascular diseases: e.g. chronic heart failure, left ventricular dysfunction and coronary artery disease.     

Guanylyl cyclase / atrial natriuretic peptide receptor-A: role in the pathophysiology of cardiovascular regulation

Atrial natriuretic factor (ANF), also known as atrial natriuretic peptide (ANP), is an endogenous and potent hypotensive hormone that elicits natriuretic, diuretic, vasorelaxant, and anti-proliferative effects, which are important in the control of blood pressure and cardiovascular events. One principal locus involved in the regulatory action of ANP and brain natriuretic peptide (BNP) is guanylyl cyclase / natriuretic peptide receptor-A (GC-A/NPRA). Studies on ANP, BNP, and their receptor, GC-A/NPRA, have greatly increased our knowledge of the control of hypertension and cardiovascular disorders. Cellular, biochemical, and molecular studies have helped to delineate the receptor function and signaling mechanisms of NPRA. Gene-targeted and transgenic mouse models have advanced our understanding of the importance of ANP, BNP, and GC-A/NPRA in disease states at the molecular level. Importantly, ANP and BNP are used as critical markers of cardiac events; however, their therapeutic potentials for the diagnosis and treatment of hypertension, heart failure, and stroke have just begun to be realized. We are now just at the initial stage of molecular therapeutics and pharmacogenomic advancement of the natriuretic peptides. More investigations should be undertaken and ongoing ones be extended in this important field.     

利钠肽家族基因与心血管疾病研究新进展

利钠肽家族是一组由心肌细胞分泌的激素,主要包括A型、B型和C型利钠肽,具有相似的基因结构和生理学效应,可对心血管系统产生血压调节、抗心肌肥厚、抗心肌纤维化和抗心肌弛缓等保护作用。利钠肽受体A、B和C亦介导多种生理活性,调节心血管稳态。利钠肽受体A选择性结合A型、B型利钠肽。利钠肽受体B结合C型利钠肽。利钠肽受体C结合各型利钠肽,通过受体介导的内化和退化作用清除血液循环中利钠肽。对利钠肽家族及其受体基因单核甘酸多态性及功能研究显示,其与多种心血管疾病(房颤、高血压、心力衰竭等)的易感性相关。利钠肽家族及其受体基因缺失的转基因小鼠表现为心肌肥厚、心肌纤维化,与高血压、心肌病及心力衰竭的发生发展相关。各种导致心肌肥厚和缺血性损伤的刺激均参与利钠肽及其受体基因的表达调控。临床将脑钠肽作为左室功能障碍和心力衰竭失代偿的一个预测指标。静脉注射重组脑钠肽已经成为治疗急性心力衰竭的有效手段。深入了解利钠肽家族基因变异及其信号调控有助于探索心血管疾病的病理生理机制,为临床诊疗开辟新思路。     

利钠肽家族基因与心血管疾病研究新进展

利钠肽家族是一组由心肌细胞分泌的激素, 主要包括A型、B型和C型利钠肽, 具有相似的基因结构和生理学效应, 可对心血管系统产生血压调节、抗心肌肥厚、抗心肌纤维化和抗心肌弛缓等保护作用。利钠肽受体A、B和C亦介导多种生理活性, 调节心血管稳态。利钠肽受体A选择性结合A型、B型利钠肽。利钠肽受体B结合C型利钠肽。利钠肽受体C结合各型利钠肽, 通过受体介导的内化和退化作用清除血液循环中利钠肽。对利钠肽家族及其受体基因单核甘酸多态性及功能研究显示, 其与多种心血管疾病(房颤、高血压、心力衰竭等)的易感性相关。利钠肽家族及其受体基因缺失的转基因小鼠表现为心肌肥厚、心肌纤维化, 与高血压、心肌病及心力衰竭的发生发展相关。各种导致心肌肥厚和缺血性损伤的刺激均参与利钠肽及其受体基因的表达调控。临床将脑钠肽作为左室功能障碍和心力衰竭失代偿的一个预测指标。静脉注射重组脑钠肽已经成为治疗急性心力衰竭的有效手段。深入了解利钠肽家族基因变异及其信号调控有助于探索心血管疾病的病理生理机制, 为临床诊疗开辟新思路。     

Natriuretic peptides cause relaxation of human esophageal mucosal muscle

Natriuretic peptides have been demonstrated to cause relaxation of the human gallbladder muscle through interaction with natriuretic peptide receptor-B (NPR-B/NPR2). Effects of natriuretic peptides in the human esophageal muscle were unknown. To investigate the effects of natriuretic peptides in the human esophagus, we measured relaxation of muscularis mucosae strips isolated from the human esophagus caused by C-type natriuretic peptide (CNP), brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP) and des[Gln(18), Ser(19), Gly(20), Leu(21), Gly(22)]ANP(4-23) amide (cANP(4-23)), a selective natriuretic peptide receptor-C (NPR-C) agonist. In endothelin-1 or carbachol-contracted mucosal muscle strips, CNP caused moderate, sustained and concentration-dependent relaxation. BNP caused a very mild relaxation whereas ANP and cANP(4-23) did not cause any relaxation. CNP was much more potent than BNP and ANP in causing relaxation. These suggest the existence of NPR-B mediating relaxation. The CNP-induced relaxation was not affected by tetrodotoxin or atropine in endothelin-1-contracted esophageal strips and not by tetrodotoxin in carbachol-contracted strips, indicating a direct effect of CNP on the human esophageal muscularis mucosae. Taken together, these results demonstrate that natriuretic peptides cause relaxation of the muscularis mucosae of the human esophagus and suggest that the relaxation is through interaction with NPR-B. Natriuretic peptides may play an important role in the control of human esophageal motility.     

C-type natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain

Two types of natriuretic peptide, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), very similar to each other in structure and in pharmacological effect, are known to be present in mammalian heart and brain. In our present survey for unidentified peptides in porcine brain extracts, we found a new peptide of 22 amino acid residues, eliciting a potent relaxant activity on chick rectum. The amino acid sequence determined for the peptide shows remarkable similarity to those of ANP and BNP, especially in the 17-residue sequences flanked by two cysteine residues. The peptide shows a pharmacological spectrum similar to ANP and BNP. Thus, the peptide was designated "C-type natriuretic peptide (CNP)", the third member to join the natriuretic peptide family. In contrast to ANP and BNP, CNP terminates in the second cysteine residue, lacking a further C-terminal extension.     

Dendroaspis natriuretic peptide binds to the natriuretic peptide clearance receptor

Dendroaspis natriuretic peptide (DNP) is a newly-described natriuretic peptide which lowers blood pressure via vasodilation. The natriuretic peptide clearance receptor (NPR-C) removes natriuretic peptides from the circulation, but whether DNP interacts with human NPR-C directly is unknown. The purpose of this study was to test the hypothesis that DNP binds to NPR-C. ANP, BNP, CNP, and the NPR-C ligands AP-811 and cANP(4-23) displaced [(125)I]-ANP from NPR-C with pM-to-nM K(i) values. DNP displaced [(125)I]-ANP from NPR-C with nM potency, which represents the first direct demonstration of binding of DNP to human NPR-C. DNP showed high pM affinity for the GC-A receptor and no affinity for GC-B (K(i)>1000 nM). DNP was nearly 10-fold more potent than ANP at stimulating cGMP production in GC-A expressing cells. Blockade of NPR-C might represent a novel therapeutic approach in augmenting the known beneficial actions of DNP in cardiovascular diseases such as hypertension and heart failure.     

Dendroaspis natriuretic peptide is the most potent natriuretic peptide to cause relaxation of lower esophageal sphincter

Atrial natriuretic peptide (ANP) causes relaxation in the opossum lower esophageal sphincter. The effects of dendroaspis natriuretic peptide (DNP) and other natriuretic peptides in the lower esophageal sphincter were not known. We measured the relaxation of transverse strips from the guinea pig lower esophageal sphincter caused by DNP, ANP, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and a natriuretic peptide receptor-C agonist des[Gln(18), Ser(19), Gly(20), Leu(21), Gly(22)]ANP(4-23) amide (cANF(4-23)) in vitro. In resting strips of the guinea pig lower esophageal sphincter DNP and BNP caused marked relaxations. Furthermore, in both sarafotoxin S6c and carbachol-contracted lower esophageal sphincter strips, DNP caused marked and BNP caused moderate, concentration-dependent relaxations. ANP as well as CNP caused mild relaxations. In contrast, cANF(4-23) did not cause relaxation. The relative potencies for natriuretic peptides to cause relaxation were DNP>BNP>ANP>=CNP in both sarafotoxin S6c and carbachol-contracted lower esophageal sphincter strips. The DNP and BNP-induced relaxations were not affected by tetrodotoxin or atropine, suggesting that the natriuretic peptide-induced response was not neutrally mediated. In conclusion, these results demonstrate that natriuretic peptides cause the relaxation of the guinea pig lower esophageal sphincter. DNP is the most potent natriuretic peptide to cause lower esophageal sphincter relaxation, which might be mediated by natriuretic peptide receptor-A or a novel DNP-selective natriuretic peptide receptor.     

Apparent B-type natriuretic peptide selectivity in the kidney due to differential processing

Two natriuretic peptides, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), are found principally in the heart. In preliminary experiments with mouse kidney cells or slices, we found mouse BNP1-45 much more potent than ANP1-28 in causing elevations of cGMP (>50-fold). The guanylyl cyclase-A (GC-A) receptor has been suggested to represent the primary means by which both peptides signal. In cultured cells overexpressing GC-A, BNP and ANP were almost equivalent in potency, suggesting that a receptor unique for BNP exists in the kidney. However, in mice lacking the GC-A gene, neither BNP nor ANP significantly elevated cGMP in kidney slices. Phosphoramidon, a neutral endopeptidase inhibitor, shifted the apparent potency of ANP to values equivalent to that of BNP, suggesting these kidney cell/slices rapidly degrade ANP but not BNP. Mass spectroscopic analysis confirmed that ANP is rapidly cleaved at the first cysteine of the disulfide ring, whereas BNP is particularly stable to such cleavage. Other tissues (heart, aorta) failed to significantly degrade ANP or BNP, and therefore the kidney-specific degradation of ANP provides a mechanism for preferential regulation of kidney function by BNP independent of peripheral ANP concentration.     

Natriuretic peptides: a new lipolytic pathway in human adipocytes.

Atrial natriuretic peptide (ANP) receptors have been described on rodent adipocytes and expression of their mRNA is found in human adipose tissue. However, no biological effects associated with the stimulation of these receptors have been reported in this tissue. A putative lipolytic effect of natriuretic peptides was investigated in human adipose tissue. On isolated fat cells, ANP and brain natriuretic peptide (BNP) stimulated lipolysis as much as isoproterenol, a nonselective beta-adrenergic receptor agonist, whereas C-type natriuretic peptide (CNP) had the lowest lipolytic effect. In situ microdialysis experiments confirmed the potent lipolytic effect of ANP in abdominal s.c. adipose tissue of healthy subjects. A high level of ANP binding sites was identified in human adipocytes. The potency order defined in lipolysis (ANP > BNP > CNP) and the ANP-induced cGMP production sustained the presence of type A natriuretic peptide receptor in human fat cells. Activation or inhibition of cGMP-inhibited phosphodiesterase (PDE-3B) (using insulin and OPC 3911, respectively) did not modify ANP-induced lipolysis whereas the isoproterenol effect was decreased or increased. Moreover, inhibition of adenylyl cyclase activity (using a mixture of alpha(2)-adrenergic and adenosine A1 agonists receptors) did not change ANP- but suppressed isoproterenol-induced lipolysis. The noninvolvement of the PDE-3B was finally confirmed by measuring its activity under ANP stimulation. Thus, we demonstrate that natriuretic peptides are a new pathway controlling human adipose tissue lipolysis operating via a cGMP-dependent pathway that does not involve PDE-3B inhibition and cAMP production.     

Engineering a Monomeric Fc Domain Modality by N-Glycosylation for the Half-life Extension of Biotherapeutics

Human IgG is a bivalent molecule that has two identical Fab domains connected by a dimeric Fc domain. For therapeutic purposes, however, the bivalency of IgG and Fc fusion proteins could cause undesired properties. We therefore engineered the conversion of the natural dimeric Fc domain to a highly soluble monomer by introducing two Asn-linked glycans onto the hydrophobic C_H3-C_H3 dimer interface. The monomeric Fc (monoFc) maintained the binding affinity for neonatal Fc receptor (FcRn) in a pH-dependent manner. We solved the crystal structure of monoFc, which explains how the carbohydrates can stabilize the protein surface and provides the rationale for molecular recognition between monoFc and FcRn. The monoFc prolonged the in vivo half-life of an antibody Fab domain, and a tandem repeat of the monoFc further prolonged the half-life. This monoFc modality can be used to improve the pharmacokinetics of monomeric therapeutic proteins with an option to modulate the degree of half-life extension.     

Monomeric IgG1 Fc molecules displaying unique Fc receptor interactions that are exploitable to treat inflammation-mediated diseases

The IgG1 Fc is a dimeric protein that mediates important antibody effector functions by interacting with Fcγ receptors (FcγRs) and the neonatal Fc receptor (FcRn). Here, we report the discovery of a monomeric IgG1 Fc (mFc) that bound to FcγRI with very high affinity, but not to FcγRIIIa, in contrast to wild-type (dimeric) Fc. The binding of mFc to FcRn was the same as that of dimeric Fc. To test whether the high-affinity binding to FcγRI can be used for targeting of toxins, a fusion protein of mFc with a 38 kDa Pseudomonas exotoxin A fragment (PE38), was generated. This fusion protein killed FcγRI-positive macrophage-like U937 cells but not FcγRI-negative cells, and mFc or PE38 alone had no killing activity. The lack of binding to FcγRIIIa resulted in the absence of Fc-mediated cytotoxicity of a scFv-mFc fusion protein targeting mesothelin. The pharmacokinetics of mFc in mice was very similar to that of dimeric Fc. The mFc's unique FcγRs binding pattern and related functionality, combined with its small size, monovalency and the preservation of FcRn binding which results in relatively long half-life in vivo, suggests that mFc has great potential as a component of therapeutics targeting inflammation mediated by activated macrophages overexpressing FcγRI and related diseases, including cancer.     

Natriuretic peptides cause relaxation of human and guinea-pig gallbladder muscle through interaction with natriuretic peptide receptor-B

Atrial natriuretic peptide (ANP) binding sites have been demonstrated in the guinea-pig gallbladder muscle with unclear function. To investigate effects of natriuretic peptides in the gallbladder, we measured relaxation of isolated human and guinea-pig gallbladder strips caused by natriuretic peptides, including C-type natriuretic peptide (CNP), brain natriuretic peptide (BNP) and ANP, as well as des[Gln18, Ser19, Gly20, Leu21, Gly22]ANP(4-23) amide (cANP(4-23)), a selective natriuretic peptide receptor-C (NPR-C) agonist. Results in the human gallbladder were similar to those in the guinea-pig gallbladder. CNP, BNP, ANP and cANP(4-23) alone did not cause contraction or relaxation in resting gallbladder strips. However, in carbachol or endothelin-1-contracted strips, CNP caused moderate, sustained and concentration-dependent relaxation. The relaxation was not affected by tetrodotoxin or atropine in endothelin-1-contracted gallbladder strips and not by tetrodotoxin in carbachol-contracted strips. These indicate a direct effect of CNP on the gallbladder muscle. The relative potencies for natriuretic peptides to cause relaxation were CNP>BNP> or = ANP. cANP(4-23) did not cause relaxation. These indicate the existence of the natriuretic peptide receptor-B (NPR-B) mediating the relaxation. Taken together, these results demonstrate that natriuretic peptides cause relaxation of human and guinea-pig gallbladder muscle through interaction with the natriuretic peptide receptor-B.     

X-ray Crystal Structures of Monomeric and Dimeric Peptide Inhibitors in Complex with the Human Neonatal Fc Receptor,FcRn

The neonatal Fc receptor, FcRn, is responsible for the long half-life of IgG molecules in vivo and is a potential therapeutic target for the treatment of autoimmune diseases. A family of peptides comprising the consensus motif GHFGGXY, where X is preferably a hydrophobic amino acid, was shown previously to inhibit the human IgG:human FcRn protein-protein interaction (Mezo, A. R., McDonnell, K. A., Tan Hehir, C. A., Low, S. C., Palombella, V. J., Stattel, J. M., Kamphaus, G. D., Fraley, C., Zhang, Y., Dumont, J. A., and Bitonti, A. J. (2008) Proc. Natl. Acad. Sci. U.S.A., 105, 2337–2342). Herein, the x-ray crystal structure of a representative monomeric peptide in complex with human FcRn was solved to 2.6 Å resolution. The structure shows that the peptide binds to human FcRn at the same general binding site as does the Fc domain of IgG. The data correlate well with structure-activity relationship data relating to how the peptide family binds to human FcRn. In addition, the x-ray crystal structure of a representative dimeric peptide in complex with human FcRn shows how the bivalent ligand can bridge two FcRn molecules, which may be relevant to the mechanism by which the dimeric peptides inhibit FcRn and increase IgG catabolism in vivo. Modeling of the peptide:FcRn structure as compared with available structural data on Fc and FcRn suggest that the His-6 and Phe-7 (peptide) partially mimic the interaction of His-310 and Ile-253 (Fc) in binding to FcRn, but using a different backbone topology.     

Natriuretic peptide receptors in cultured rat diencephalon

To characterize the type of cell expressing natriuretic peptide receptors in the brain and the nature of these receptors, we conducted studies in primary cultured glial and neuronal cells derived from fetal rat diencephalon. The glial predominant cultures (95% of total cells and glial fibrillary acidic protein positive) expressed nearly a 10-fold greater specific binding of the natriuretic peptides to cell surface receptors compared with the neuron-predominant cultures. Scatchard analysis of binding studies with 125I-atrial natriuretic peptide (ANP) and 125I-brain natriuretic peptide (BNP) revealed a single class of receptors with dissimilar affinities (0.25 +/- 0.09 and 0.74 +/- 0.07 nM, respectively, n = 3 experiments p less than 0.01) but similar numbers of binding sites for both peptides (93 and 88 fmol/mg of protein, respectively). Cross-linking of 125I-ANP and BNP to cultured glia followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography identified distinct bands at either approximate Mr 130,000, or 102,000 and 66,000, corresponding to two high molecular weight (B) receptors and one low molecular weight (C) receptor described in other tissues. Different subtypes of astrocytes appeared to express different B receptors. Binding and cross-linking of radiolabeled ANP or BNP were competitively inhibited equally by unlabeled ANP or BNP, indicating that ANP and BNP probably bind the same receptors. The glial cultures functionally expressed a receptor(s) with guanylate cyclase activity; BNP was less potent than ANP in stimulating cGMP at lower concentrations. These results indicate that both high and low molecular weight natriuretic peptide receptors are expressed in astrocyte-predominant cultures from the fetal diencephalon and suggest that glia participate in several actions of ANP which are probably mediated through this area of the brain.     

The effect of galanin, CGRP and ANP on spontaneous smooth muscle activity of rat uterus

In the present study the effect of the newly isolated peptides galanin, calcitonin gene-related peptide (CGRP) and atrial natriuretic peptide (ANP) was examined on spontaneous uterine smooth muscle activity on the rat in vitro. Galanin showed a slight and insignificant stimulatory effect on the amplitude, while both CGRP and ANP were found to be potent inhibitors of the uterine smooth muscle contractions. In connection with the recent demonstration of galanin and CGRP nerves in the genital tract, these pharmacological findings suggest that the peptides may participate in the control of nervous control of uterine muscular activity, although the exact physiological roles remain to be clarified.     

Acute volume expansion as a physiological stimulus for the release of atrial natriuretic peptides in the rat

The concentration of immunoreactive atrial natriuretic peptide(s) (ANP) was measured in normovolemic conscious rats and 15 min after 10% and 20% blood volume expansion. A 20% blood volume expansion caused a 2-fold increase in plasma ANP. While plasma ANP increased linearly, atrial levels of ANP remained unaltered. The increase in plasma ANP parallelled increases of central blood volume and central venous pressure. It is concluded that acute blood volume expansion is a major physiological stimulus for the release of atrial natriuretic peptides into the circulation.     

Paradoxical antagonism of PACAP receptor signaling by VIP in Xenopus oocytes via the type-C natriuretic peptide receptor

Atrial natriuretic peptide (ANP) and the closely-related peptides BNP and CNP are highly conserved cardiovascular hormones. They bind to single transmembrane-spanning receptors, triggering receptor-intrinsic guanylyl cyclase activity. The "truncated" type-C natriuretic peptide receptor (NPR-C) has long been called a clearance receptor because it lacks the intracellular guanylyl cyclase domain, though data suggest it might negatively couple to adenylyl cyclase via G(i). Here we report the molecular cloning and characterization of the Xenopus laevis type-C natriuretic peptide receptor (XNPR-C). Analysis confirms the presence of a short intracellular C-terminus, as well as a high similarity to fish and mammalian NPR-C. Injection of XNPR-C mRNA into Xenopus oocytes resulted in expression of high affinity [(125)I]ANP binding sites that were competitively and completely displaced by natriuretic analogs and the unrelated neuropeptide vasoactive intestinal peptide (VIP). Measurement of cAMP levels in mRNA-injected oocytes revealed that XNPR-C is negatively coupled to adenylyl cyclase in a pertussis toxin-sensitive manner. When XNPR-C was co-expressed with PAC(1) receptors for pituitary adenylyl cyclase-activating polypeptide (PACAP), VIP and natriuretic peptides counteracted the cAMP induction by PACAP. These results suggest that VIP and natriuretic peptides can potentially modulate the action of PACAP in cells where these receptors are co-expressed.     

Development of a novel, nonimmunogenic, soluble human TNF receptor type I (sTNFR-I) construct in the baboon.

Pharmacokinetics and immunogenicity of six different recombinant human soluble p55 tumor necrosis factor (TNF) receptor I (sTNFR-I) constructs were evaluated in juvenile baboons. The constructs included either an sTNFR-I IgG1 immunoadhesin (p55 sTNFR-I Fc) or five different sTNFR-I constructs covalently linked to polyethylene glycol. The constructs were administered intravenously three times, and pharmacokinetics and immunogenicity were examined over 63 days. All of the constructs were immunogenic, with the exception of a 2.6-domain monomeric sTNFR-I. To evaluate whether the nonimmunogenic 2.6-domain monomeric construct could protect baboons against TNF-alpha-induced mortality, baboons were pretreated with 1, 5, or 10 mg/kg body wt and were compared with baboons receiving either placebo or 1 mg/kg body wt of the dimeric 4.0-domain sTNFR-I construct (n = 3 each) before lethal Escherichia coli bacteremia. The monomeric construct protected baboons and neutralized TNF bioactivity, although greater quantities were required compared with the dimeric 4.0-domain sTNFR-I construct. We conclude that E. coli-recombinant-derived human sTNFR-I constructs can be generated with minimal immunogenicity on repeated administration and still protect against the consequences of exaggerated TNF-alpha production.