Phase I study of TNFalpha AutoVaccIne in patients with metastatic cancer.

We evaluated the safety and immunogencity of a novel vaccine directed against autologous TNFalpha in a Phase I fixed dose escalation trial. The vaccine consisted of two recombinant TNFalpha proteins, with specific peptides replaced by foreign immunodominant T cell epitopes from tetanus toxoid. The main objectives were to establish a safe dose and evaluate the vaccines ability to raise neutralising TNFalpha antibodies. Secondary objectives were improvements in body weight and tumour response. Thirty-three patients were vaccinated with three doses (20, 100, or 400 mug) of TNFalpha vaccine at 2-weekly intervals adjuvanted with aluminium hydroxide. Anti-TNFalpha antibody titres were measured by both a RIA, using soluble native TNFalpha as the antigen, and by an ELISA using immobilized partly denatured TNFalpha. Eleven patients (33%) had mild grade1/2 injection site reactions at the higher doses. In 10 of 20 patients, serum antibodies recognize denatured TNFalpha in the ELISA, whereas, antibody titres against native TNFalpha in the RIA were undetectable. This suggests that the production process had partly denatured the vaccine preventing the formation of cross-reacting antibodies to native TNFalpha. In conclusion, TNFalpha vaccine was able to elicit vaccine specific antibodies. However, since the antibodies were only able to cross-react with partly denatured TNFalpha, evaluation of safety and tumour responses to the TNFalpha vaccine was compromised.

Lipocalins and cancer.

Lipocalins are mainly extracellular carriers of lipophilic molecules, though exceptions with properties like prostaglandin synthesis and protease inhibition are observed for specific lipocalins. The interest concerning lipocalins in cancer has so far been focussed to the variations in concentration and the modification of lipocalin expression in distinct cancer forms. In addition, lipocalins have been assigned a role in cell regulation. The influence of the extracellular lipocalins on intracellular cell regulation events is not fully understood, but several of the lipocalin ligands are also well-known agents in cell differentiation and proliferation. Lipophilic ligands can, after lipocalin-mediated transport to the cell surface, penetrate the cell membrane and interact with proteins in the cytosol and/or the nucleus. The signaling routes of the lipocalin ligands, retinoids and fatty acids are presented and discussed. Tumor growth in tissue is restricted by extracellular protease/protease inhibitor interactions. Several lipocalins also have protease inhibitory properties and possess the ability to interact with tumor specific proteases, revealing another pathway for lipocalins to interact with cancer cells.

Ligand preference inferred from the structure of neutrophil gelatinase associated lipocalin.

Neutrophil gelatinase associated lipocalin (NGAL), a constituent of neutrophil granules, is a member of the lipocalin family of binding proteins. NGAL can also be highly induced in epithelial cells in both inflammatory and neoplastic colorectal disease. NGAL is proposed to mediate inflammatory responses by sequestering neutrophil chemoattractants, particularly N-formylated tripeptides and possibly leukotriene B(4) and platelet activating factor. The crystal structures of NGAL display a typical lipocalin fold, albeit with an unusually large and atypically polar binding site, or calyx. The fold of NGAL is most similar to the epididymal retinoic acid-binding protein, another lipocalin, though the overall architecture of the calyces are very different. The crystal structures also reveal either sulfate ions or an adventitiously copurified fatty acid bound in the binding site. Neither ligand is displaced by added N-formylated tripeptides. The size, shape, and character of the NGAL calyx, as well as the low relative affinity for N-formylated tripeptides, suggest that neither the copurified fatty acid nor any of the proposed ligands are likely to be the preferred ligand of this protein. Comparisons between the crystal structures and the recently reported solution structure of NGAL reveal significant differences, in terms of both the details of the structure and the overall flexibility of the fold.

Interactions between neutrophil gelatinase-associated lipocalin and natural lipophilic ligands.

Neutrophils are activated by both paracrine molecules, e.g. platelet activating factor (PAF) and leukotriene B4 (LTB4), and the bacterial hydrophobic peptide N-formyl-Met-Leu-Phe (fMLP). Several mechanisms are involved in regulation of the activation, including receptor endocytosis and ligand breakdown. The interactions between the specific granule protein neutrophil gelatinase-associated lipocalin (NGAL), expressed in human neutrophils, and fMLP, PAF and LTB4, were investigated by weak affinity chromatography. NGAL was immobilised to a silica matrix and packed in a micro-column and the retention times of retarded ligands were measured and used to calculate the strength of the interactions. The association constants for fMLP were K(ass) = 0.85 x 10(3) M(-1) at 20 degrees C and 0.77 x 10(3) M(-1) at 37 degrees C, for LTB4 were K(ass) = 4.37 x 10(3) M(-1) at 20 degrees C and 3.27 x 10(3) M(-1) at 37 degrees C and for PAF were K(ass) = 25.4 x 10(3) M(-1) at 20 degrees C and 10.5 x 10(3) M(-1) at 37 degrees C. Other methods of detecting the interactions such as gel filtration, immunoprecipitation, photoactivated ligands and fluorescence quenching proved to be insufficient. The results demonstrate the superiority of weak affinity chromatography as a method of studying the interactions of the specific granule protein NGAL.

Glycosylation of natural human neutrophil gelatinase B and neutrophil gelatinase B-associated lipocalin.

Gelatinase B is a matrix metalloproteinase (MMP-9) involved in tissue remodeling, development, cancer, and inflammation. Neutrophils produce three major forms of (pro)gelatinase B: 92 kDa monomers, homodimers, and complexes of gelatinase B covalently bound to neutrophil gelatinase B-associated lipocalin (NGAL). In contrast to the case for other proteinases, little information about the glycosylation of any natural human MMP is available. Here, both gelatinase B and NGAL were purified from human peripheral blood neutrophils, and the entire contents of the released N- and O-glycan pools were analyzed simultaneously using recently developed high-performance liquid chromatography-based technology. The results are discussed within the context of the domain structure of gelatinase B and a molecular model of NGAL based on data from this study and the three-dimensional nuclear magnetic resonance (NMR) structure of the protein. More than 95% of the N-linked glycans attached to both gelatinase B and NGAL were partially sialylated, core-fucosylated biantennary structures with and without outer arm fucose. The O-linked glycans, which were estimated to comprise approximately 85% of the total sugars on gelatinase B, mainly consisted of type 2 cores with Galbeta1,4GlcNAc (lactosamine) extensions, with or without sialic acid or outer arm fucose. This paper also contains the first report of O-linked glycans attached to NGAL. Although both proteins were isolated from neutrophils and contained O-linked glycans mainly with type 2 cores, the glycans attached to individual serine/threonine residue(s) in NGAL were significantly smaller than those on gelatinase B. In contrast to NGAL, gelatinase B contains a region rich in Ser, Thr, and Pro typical of O-glycosylated mucin-like domains.

The human antibacterial cathelicidin, hCAP-18, is bound to lipoproteins in plasma.

Cathelicidins are a family of antibacterial and lipopolysaccharide-binding proteins. hCAP-18, the only human cathelicidin, is a major protein of the specific granules of human neutrophils. The plasma level of hCAP-18 is >20-fold higher than that of other specific granule proteins relative to their levels within circulating neutrophils. The aim of this study was to elucidate the background for this high plasma level of hCAP-18. Plasma was subjected to molecular sieve chromatography, and hCAP-18 was found in distinct high molecular mass fractions that coeluted with apolipoproteins A-I and B, respectively. The association of hCAP-18 with lipoproteins was validated by the cofractionation of hCAP-18 with lipoproteins using two different methods for isolation of lipoproteins from plasma. Furthermore, the level of hCAP-18 in delipidated plasma was <1% of that in normal plasma. Immunoprecipitation of very low, low, and high density lipoprotein particles with anti-apolipoprotein antibodies resulted in coprecipitation of hCAP-18. The binding of hCAP-18 to lipoproteins was mediated by the antibacterial C-terminal part of the protein. The binding of hCAP-18 to lipoproteins suggests that lipoproteins may play an important role as a reservoir of this antimicrobial protein.

Expression, purification, crystallization and crystallographic characterization of dimeric and monomeric human neutrophil gelatinase associated lipocalin (NGAL).

Crystals of the monomeric and dimeric forms of human neutrophil gelatinase associated lipocalin have been grown in hanging-drop vapor-diffusion trials using PEG as a precipitating agent with recombinant protein expressed in a baculovirus-based system. Crystals of monomeric NGAL belong to the cubic space group P432 with lattice constants a = b = c = 126.6 A; crystals of dimeric NGAL belong to the tetragonal space group P41212 (or its enantiomorph P43212) with lattice constants a = b = 54.14 and c = 121.56 A. Isomorphous crystals of the NGAL dimer can be grown in the presence of ligand: the tripeptide N-formyl-Met-Leu-Phe.

A new intracellular serine protease inhibitor expressed in the rat pituitary gland complexes with granzyme B.

We have cloned a novel serpin (raPIT5a) from a rat pituitary cDNA library which is structurally related to members of the ovalbumin subfamily of serine protease inhibitors. This new cDNA encodes a 374-amino acid protein, designated raPIT5a. raPIT5a was expressed in specific cells in the intermediate and anterior lobes of the pituitary. Recombinant raPIT5a was not secreted suggesting raPIT5a functions to inhibit intracellular proteases. Recombinant raPIT5a formed an SDS-stable complex with human granzyme B, a serine protease which induces apoptosis by activating members of the caspase enzyme family. These data suggest raPIT5a may have a role in regulating granzyme B or related enzymes and apoptosis in the pituitary gland.

Expression of rat alpha 1-microglobulin-bikunin in baculovirus-transformed insect cells.

cDNA encoding rat alpha 1-microglobulin-bikunin was ligated into the transfer vector pVL 1392 and recombined with a wild-type baculovirus. The resulting alpha 1-microglobulin-bikunin-encoding baculovirus was used to infect Trichoplusia ni (Hi-5) insect cells. The infected cells secreted alpha 1-microglobulin with maximal concentrations of 15 mg/liter 5 days after infection. The secreted proteins migrated upon SDS-PAGE as two major protein bands, 40 and 26 kDa, corresponding to alpha 1-microglobulin-bikunin and free alpha 1-microglobulin. The results suggested that the cells secreted mostly alpha 1-microglobulin-bikunin, which subsequently was cleaved in the medium, yielding free alpha 1-microglobulin. Both forms were isolated by monoclonal anti-alpha 1-microglobulin affinity chromatography, and alpha 1-microglobulin-bikunin separated from free alpha 1-microglobulin by gel chromatography. The yields of purified alpha 1-microglobulin-bikunin and free alpha 1-microglobulin were approximately 1 and 5 mg, respectively, per liter medium. Insect cell alpha 1-microglobulin displayed a size, shape, and charge heterogeneity similar to alpha 1-microglobulin isolated from rat urine. A panel of monoclonal antibodies raised against urinary alpha 1-microglobulin from several different species bound to rat urinary alpha 1-microglobulin and insect cell secreted alpha 1-microglobulin-bikunin and free alpha 1-microglobulin with approximately the same strength, indicating that the three proteins are folded in similar ways. The results of glycosidase treatments and lectin blotting indicate the absence of neuraminic acid but the presence of one N-linked oligosaccharide and an unspecified number of O-linked oligosaccharides in alpha 1-microglobulin-bikunin and free alpha 1-microglobulin.

Formation of the alpha 1-microglobulin chromophore in mammalian and insect cells: a novel post-translational mechanism?

alpha 1-Microglobulin is an immunosuppressive plasma protein synthesized by the liver. The isolated protein is yellow-brown, but the hypothetical chromophore has not yet been identified. In this work, it is shown that a human liver cell line, HepG2, grown in a completely synthetic and serum-free medium, secretes alpha 1-microglobulin which is also yellow-brown, suggesting a de novo synthesis of the chromophore by the cells. alpha 1-Microglobulin isolated from the culture medium of insect cells transfected with the gene for rat alpha 1-microglobulin is also yellow-brown, suggesting that the gene carries information about the chromophore. Reduction and alkylation or removal of N- or O-linked carbohydrates by glycosidase treatment did not reduce the colour intensity of the protein. An internal dodecapeptide (amino acid positions 70-81 in human alpha 1-microglobulin) was also yellow-brown. The latter results indicate that the chromophore is linked to the polypeptide. In conclusion, the results suggest that the alpha 1-microglobulin gene carries information activating a post-translational protein modification mechanism which is present in mammalian and insect cells.

Processing and secretion of rat alpha 1-microglobulin-bikunin expressed in eukaryotic cell lines.

The precursor protein alpha 1-microglobulin-bikunin was cleaved to the same degree whether expressed in CHO cells or in mutated CHO cells, RPE.40 cells, suggested to lack a functional form of the intracellular protease furin. Thus, alpha 1-microglobulin-bikunin probably is not cleaved in vivo by furin. However, simultaneous overexpression of the precursor and furin in COS, CHO and RPE.40 cells increased the cleavage, suggesting that compartmentalisation and concentrations of protease and precursor are important for the cleavage, besides the in vitro specificity. Expression of alpha 1-microglobulin and bikunin alone gave different protein patterns of SDS-PAGE as compared to expression of the precursor and subsequent cleavage, suggesting that the precursor protein is important for the post-translational handling of alpha 1-microglobulin and bikunin.

Cleavage of the alpha 1-microglobulin-bikunin precursor is localized to the Golgi apparatus of rat liver cells.

alpha 1-Microglobulin, a plasma protein with immunoregulatory properties, and bikunin, the light chain of the proteinase inhibitors inter-alpha-inhibitor and pre-alpha-inhibitor, are translated as a precursor protein from the same mRNA. The cosynthesis of alpha 1-microglobulin and bikunin is unique compared to other proproteins such as procomplement components and prohormones, since alpha 1-microglobulin and bikunin have no known functional connection. Different forms of intracellular rat liver alpha 1-microglobulin were isolated and characterized by amino acid sequence analysis, lectin binding and glycosidase treatment. Their subcellular distribution was studied by Nycodenz and sucrose gradient centrifugation, pulse-chase experiments, and electrophoresis with subsequent immunoblotting, using pro-C3 and prohaptoglobin as reference proteins. Two alpha 1-microglobulin-bikunin precursors (40 and 42 kDa), containing one and two N-linked oligosaccharides, respectively, were detected in the endoplasmic reticulum. After transport to the Golgi apparatus, the precursors were cleaved, probably C-terminal to the sequence Arg-Ala-Arg-Arg immediately preceding the bikunin part, yielding free sialylated 28 kDa alpha 1-microglobulin, representing the mature protein. The cleavage was almost complete in phosphatidylinositol 4-kinase-enriched membranes, previously identified as a post-Golgi compartment. A fourth intracellular form of alpha 1-microglobulin, 26 kDa, lacked sialic acid. None of the intracellular forms carried the yellow-brown chromophore associated with alpha 1-microglobulin when purified from serum and urine, suggesting that this chromophore becomes linked to the protein after its secretion from the liver cells.

Improved removal of anti-A and anti-B antibodies from plasma using blood-group-active haptens.

Highly efficient anti-blood group A and B immunoadsorbents for extracorporeal treatment were developed by immobilizing A and B trisaccharide and A tetrasaccharide haptens on Sepharose 4FF and Fractogel TSK using three different methods. The adsorption of the IgG and IgM anti-A antibodies was essentially the same regardless of the A hapten used or the method of oligosaccharide coupling. The adsorption of IgM anti-A was found to be more sensitive than IgG anti-A to changes in column flow rate. The binding of both the IgM and IgG antibodies was slightly lower at 37 degrees C than at 22 degrees C. An anti-A/anti-B adsorbent column potentially suitable for treatment of patients was prepared. A column switching system resulted in a more efficient adsorption of antibodies. Hapten leakage from the column was very low. No nonspecific adsorption of plasma proteins to the column (other than traces of albumin) could be detected.

Rat alpha 1-microglobulin: co-expression in liver with the light chain of inter-alpha-trypsin inhibitor.

A 1162 bp rat liver cDNA clone encoding the immunoregulatory plasma protein alpha 1-microglobulin was isolated and sequenced. The open reading frame encoded a 349 amino acid polyprotein, including alpha 1-microglobulin, 182 amino acids, and bikunin, the light chain of the plasma protein inter-alpha-trypsin inhibitor, 145 amino acids. The alpha 1-microglobulin/bikunin mRNA was found only in the liver when different tissues were examined. Free alpha 1-microglobulin and a polyprotein, containing both alpha 1-microglobulin and inter-alpha-trypsin inhibitor epitopes, were found in the microsomal fraction from rat liver homogenates.

The development of a C1q anti-C1q immunoadsorbent for removal of immune complexes from plasma.

An immunoadsorbent based on immobilized C1q has been developed to remove possibly pathogenic immune complexes from plasma deriving from patients suffering from systemic lupus erythematosus or rheumatoid arthritis. Traditional immobilization procedures based on, e.g., cyanogen bromide activation could not be used to produce an efficient adsorbent. However, by using antibodies directed towards C1q as handles for the immobilization of C1q it was possible to make an adsorbent that efficiently bound immune complexes in plasma. The capacity of the C1q anti-C1q adsorbent to bind artificial immune complexes such as aggregated human globulins or immune complexes from various plasma samples was evaluated. Both batch and column experiments were conducted. The typical capacity in batch was about 1 mg immune complexes/ml gel when incubated with patient plasma samples with high titers of immune complexes. Special attention has to be paid to leakage of undesirable components from the adsorbent. It was found that leakage of C1q occurred but it was not more than after covalent immobilization procedures such as cyanogen bromide.