Jan Tavernier

EVIDENCE FOR TUMOUR NECROSIS FACTOR/CACHECTIN PRODUCTION IN CANCER

Labile tumour-necrosis-factor-like (TNF) activity was detected by means of an enzyme-linked immunosorbent assay in 50% of 226 freshly obtained serum samples from cancer patients with active disease. In contrast, only 3% of 32 samples from normal subjects and 18% of 39 samples from cancer patients with no clinically evident disease were positive for this factor, with low levels of activity. Greater proportions of serum samples from patients with ovarian or oat-cell carcinoma were positive (69% and 63%) than those from patients with lymphoma (26%). RNA preparations from peripheral-blood mononuclear cells and solid tumours were probed with TNF complementary DNA; evidence of TNF messenger RNA was found in 8 of 11 samples of peripheral-blood mononuclear cells from cancer patients, but only 1 of 8 normal subjects, and in 2 of 6 colorectal tumours. As yet the inducing stimulus and the clinical significance of TNF production in cancer are not understood.

Molecular cloning of human interleukin 2 cDNA and its expression in E. coli.

A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes. Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA. A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal sequence of 20 amino acids. A mature polypeptide starting with either Met-Ala-Pro or Met-Pro was expressed in E. coli under control of the E. coli trp promoter or using a combination of the phage lambda PL promoter and a ribosome binding site derived from phage Mu. The bacterial IL2 polypeptide had a molecular weight of 15,000 daltons and accounted for more than 10% of the total E. coli proteins in fully induced cells; it was biologically active in the T-cell specific DNA synthesis assay, even after recovery from a SDS-containing polyacrylamide gel.

Dissociation of TNF-alpha cytotoxic and proinflammatory activities by p55 receptor- and p75 receptor-selective TNF-alpha mutants.

Human tumour necrosis factor alpha (TNF‐alpha) is a pleiotropic cytokine capable of killing mammalian tumour cells in vitro and in vivo, and of enhancing the proinflammatory activity of leucocytes and endothelium, the latter effects limiting its usage as an antitumour agent in humans. Using TNF‐alpha mutants with a selective capacity to bind to the TNF p55 receptor (TNFR55) or to the p75 receptor (TNFR75) we show here that these two major activities of TNF‐alpha can be dissociated. The TNFR55‐selective mutants (R32W, E146K and R32W‐S86T) which bind poorly to TNFR75 displayed similar potency to wild‐type TNF in causing cytotoxicity of a human laryngeal carcinoma‐derived cell line (HEp‐2) and cytostasis in a human leukaemic cell line (U937). However, these TNFR55‐selective mutants exhibited lower proinflammatory activity than wild‐type TNF. Specifically, TNF‐alphas priming of human neutrophils for superoxide production and antibody‐dependent cell‐mediated cytotoxicity, platelet‐activating factor synthesis and adhesion to endothelium were reduced by up to 170‐fold. Activation of human endothelial cell functions represented by human umbilical venular endothelial cell (HUVEC) adhesiveness for neutrophils, E‐selectin expression, neutrophil transmigration and IL‐8 secretion were also reduced by up to 280‐fold. On the other hand, D143F, a TNFR75‐selective mutant tested either alone or in combination with TNFR55‐selective mutants, did not stimulate these activities despite being able to cause cytokine production in TNFR75‐transfected PC60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-5 and its receptor: a drug target for eosinophilia associated with chronic allergic disease.

A characteristic feature of chronic allergic diseases such as asthma is the increase in eosinophil numbers in the inflamed tissue. In light of its specificity for the development of eosinophils, interleukin‐5 (IL‐5) is considered the most important cytokine involved in the regulation of eosinophilia. Hence, an antagonist for IL‐5 activity is a new target for drug discovery programs. We have examined the opportunity for both a random and a rational approach for the identification of such an antagonist. The elucidation of the structure of IL‐5 and the initial structure/function analysis of the ligand/receptor complex constitute a first step towards the design of antagonistic compounds. The identification of a small compound by random screening able to inhibit the IL‐5/IL‐5 receptor interaction indicated an important domain in the receptor. We examine here protein‐based IL‐5 antagonists, such as IL‐5‐muteins, soluble IL‐5 receptor constructs, and monoclonal antibodies, for their potential as IL‐5/IL‐5 receptor antagonists, and the use of a murine model of eosinophil airway inflammation for their evaluation. J. Leukoc. Biol. 57: 813–819; 1995.

Detailed analysis of the IL-5-IL-5R alpha interaction: characterization of crucial residues on the ligand and the receptor.

The receptor for interleukin‐5 (IL‐5) is composed of two different subunits. The IL‐5 receptor alpha (IL‐5R alpha) is required for ligand‐specific binding while association with the beta‐chain results in increased binding affinity. Murine IL‐5 (mIL‐5) has similar activity on human and murine cells, whereas human IL‐5 (hIL‐5) has marginal activity on murine cells. We found that the combined substitution of K84 and N108 on hIL‐5 by their respective murine counterpart yields a molecule which is as potent as mIL‐5 for growth stimulation of a murine cell line. Since the unidirectional species specificity is due only to the interaction with the IL‐5R alpha subunit, we have used chimeric IL‐5R alpha molecules to define regions of hIL‐5R alpha involved in species‐specific hIL‐5 ligand binding. We found that this property is largely determined by the NH2‐terminal module of hIL‐5R alpha, and detailed analysis defined D56 and to a lesser extent E58 as important for binding. Moreover, two additional residues, D55 and Y57, were identified by alanine scanning mutagenesis within the same region. Based on the observed homology between the NH2‐terminal module and the membrane proximal (WSXWS‐containing) module of hIL‐5R alpha we located this stretch of four amino acid residues (D55, D56, Y57 and E58) in the loop region that connects the C and D beta‐strands on the proposed tertiary structure of the NH2‐terminal module.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide sequence of the chromosomal gene for human fibroblast (β1) interferon and of the flanking regions

The nucleotide sequence of the human fibroblast (beta 1) interferon chromosomal gene and its flanking regions was determined. These results confirm the absence of intervening sequences in the gene. The presence of some sequences in the upstream flanking region homologous to similar features for other eukaryotic genes was revealed: these include not only the TATAAAT sequence and the consensus sequence (reported by Benoist et al., 1980) but also two additional motifs, one of which is so far present only in inducible genes. Furthermore, a striking similarity between the upstream flanking regions of the human beta 1 and alpha 1 interferon genes is observed.

Cloning and structure of a mouse interleukin-2 chromosomal gene

Using non-stringent hybridization with a human interleukin-2 cDNA probe, we have isolated recombinant phages from a mouse genomic DNA library cloned in the EMBL3 phage. The sequence and organization of the mouse interleukin-2 (IL-2) gene was determined. By comparison with the human IL-2 sequence, three introns can be identified with lengths of 99, ±2 400, and ±1 900 base pairs, respectively. The mouse IL-2 gene codes for a polypeptide of 169 amino acids and contains a putative signal peptide of 20 amino acids. The homology to the human interleukin-2 is 72% at the nucleotide level in the coding part and 65% at the amino acid level. An extraordinary sequence, consisting of 12 consective CAG codons coding for glutamine, is found in the first exon.

The presence of homologous regions between interferon sequences

The amino acid sequences derived from cloned interferon genes are compared. Human leukocyte and fibroblast interferons (IFN-α, IFN-β; both type I interferons) are known to be related (T. Taniguchi et al., Nature 285, 1980). Comparison of human and murine α-and β-type interferons suggests the presence of two major homology blocks within type I interferon sequences. Also, although immune interferon (IFN-γ; type II) shows obvious differences in its biological properties and genomic organization, some sequence homology with type I interferons is apparent (W. F. DeGrado et al., Nature 300, 1982;L.B. Epstein, Nature 295, 1982). This is especially true for the region 41–104 of the IFN-γ polypeptide which shows homology to both IFN-α and IFN-β. This region is almost completely confined to the third exon of the human IFN-γ gene. When superposed on a tentative 3-dimensional model of type I interferons (M.J.E. Sternberg & F.E. Cohen, Int. J. Biol. Macromol.4, 1982), a correlation with a domain encompassing two α-helical structures is found. This may have implications for the evolution of the interferon gene family.

Tumour Necrosis Factor and Interleukin-6: Structure and Mechanism of Action of the Molecular, Cellular and in Vivo Level

Tumour necrosis factor (TNF) can be induced in experimental animals by injection of Bacillus Calmette-Guerin followed, after one or two weeks, by treatment with lipopolysaccharide (LPS); serum taken a few hours later contains a high concentration of TNF (Carswell et al., 1975). Isolated macrophages, e.g. obtained from placenta, can be activated with interferon-γ (LFN-γ) and 24 h later induced to produce TNF by treatment with LPS. Also monocytic cell lines, such as the human U-937 line or the murine PU-518 line, can be induced under proper conditions to produce TNF (Mannel et al., 1980; Fransen et al., 1985; Marmenout et al., 1985). We have cloned and expressed to a high specific activity in E. coli both the human TNF (hTNF) gene (Marmenout et al., 1985) and the murine TNF (mTNF) gene (Fransen et al., 1985). Also the sequence of the rabbit TNF gene has been reported (Ito et al., 1986). TNF obtained from various species is highly homologous (about 80%). The subunit of the mature hTNF is a 157 amino acids long polypeptide (156 amino acids for mTNF). The native protein is a nearly spherical, trimeric molecule, containing 45% β-structure and little or no α-helix (Wingfield et al., 1987). TNF, as its name implies, was originally recognized as a substance causing necrosis of tumours in experimental animals; this was usually demonstrated by means of a methyl-cholanthrene-induced sarcoma, and it may be noted that obtaining effective tumour regression requires a rather strict adherence to a defined treatment protocol. Remarkably (and almost by coincidence as it later turned out), TNF is also selectively toxic to some transformed cell lines. But in the presence of concomitant treatment with interferon (IFN), many more transformed and malignant cell lines become sensitive to the cytotoxic action of TNF (Williamson et al., 1983; Fransen et al., 1986b).