Method of identifying agonist and antagonists for tumor necrosis related receptors TR1 and TR2

The present invention relates to tumor necrosis factor receptor (TNF-R) related polypeptides and their ligands, hereinafter referred to as TR1, TR2, TL2 and TL4. The invention relates to methods to identify agonists and antagonists of TR1, TR2, TL2 and TL4.

EXAMPLES The expression and dtermination of receptor ligand pairings for TL2, TL4, TR1 and TR2 are described below. TL2 is also known as TRAIL (Wiley et al.Immunity 3: 673-682 (1995)) or Apo-2L (Pitti et al., J. Biol. Chem. 271:12687-12690 (1996)). TR1 is also known as osteoprotegerin Simonet et al., Cell 89:309-319 (1997). TR2 is also known as HVEM (Montgomery et al., Cell 87:427-436 (1996)). Expression TR1 and TR2 were expressed as fusion proteins in which the extracellular domain of either receptor was fused at its amino terminus with the hinge-CH2-CH3 region of human IgG1. The junction between the two protein domains was engineered to include the amino acid sequence for proteolytic cleavage by Factor Xa. When expressed in this form in mammalian cells, the TR fusion proteins (TR1Fc and TR2 Fc respectively) were secreted as dimeric proteins, and were purified by protein A sepharose. The non-fused soluble receptor was generated from the TR2 or TR1Fc fusion by incubation with bovine Factor Xa and was purified away from the Fc portion by repassage over protein A sepharose and pooling of the flow through. TL2 and TL4 are both type II membrane proteins in which it is the C-terninus which is extracellular. These were expressed as secreted fusion proteins by engineering an expression DNA construct in which the DNA encoding a substantial part of the carboxyterminal region, which includes all of the residues homologous to mature TNF, was fused to an amino terminal epitope tag sequence, and an amino terminal hydrophobic signal sequence for secretion, detection and purification. When transfected into mammalian cells, these DNA constructs resulted in the secretion of soluble, epitope tagged fusion proteins (sTL2, sTL4 respectively). Specific details of the construction of each expression vector are given below. TR2 The putative transmembrane domain of translated TR2 sequence was determined by hydrophobicity using the method of Goldman et al (1) for identifying nonpolar transbilayer helices. The region upstream of this transmembrane domain, encoding the putative leader peptide and extracellular domain, was chosen for the production of an Fc fusion protein. Primers were designed to PCR the corresponding coding region from the TR2 cDNA with the addition of a Bg1II site, a Factor Xa protease cleavage site and an Asp718I site at the 3′ end. PCR with this primer pair (forward 35-mer 5′ cag gaa ttc gca gcc atg gag cct cct gga gac tg 3′ (SEQ ID NO: 6), and reverse primer 53-mer 5′ cca tac cca ggt acc cct tcc ctc gat aga tct tgc ctt cgt cac cag cca gc 3′ (SEQ ID NO: 7)) resulted in one band of the expected size. This was cloned into COSFclink to give the TR2Fclink plasmid. The PCR product was digested with EcoRI and Asp718I and ligated into the COSFclink plasmid (2, 3) to produce TR2Fclink. This vector encodes amino acids 1-192 of TR2, followed by the amino acids RSIEGRGT for Factor Xa cleavage, followed by residues 226-458 (end) of human IgG1. The IgG1 region also has a mutation of Cys230 to Ala (2). COS cells were transiently transfected with TR2Fclink and the resulting supernatant was immunoprecipitated with protein A agarose. Western blot analysis of the immunoprecipitate using goat anti-human Fc antibodies revealed a strong band consistent with the expected size for glycosylated TR2Fc (greater than 47.5 kD). CHO cells were transfected with TR2Fclink to produce stable cell lines. Five lines were chosen by dot blot analysis for expansion and were adapted to shake flasks. The line with the highest level of TR2Fc protein expression was chosen by Western blot analysis. TR1 The sequence of TR1 did not show any transmembrane region by hydrophobicity plot (Goldman et al., see TR2 above). The entire coding region of TR1 minus the terminator codon was therefore used to produce an Fc fusion construct. The TR2 insert in TR2Fclink was replaced with TR1 as follows. The 3′ end of TR1 was amplified from a TR1 cDNA using the following primers: 5′ cgc ccc ttg ccc tga cca cta 3′ (SEQ ID NO: 8) (upstream of HindIII site) and 5′ gcc att tca gat ctt aag cag ctt att ttt act ga 3′ (SEQ ID NO: 9) (replaces stop codon with Bg1II site). The PCR products were cloned into pCR2 (Invitrogen; pCR2TR1) and sequenced. TR2Fclink was digested with EcoRI Bg1II and calf intestinal phosphatase, then ligated with the EcoRI /HindIII fragment of TR1 cDNA and HindIII/Bg1II fragment of pCR2TR1 to form TR1Fclink. Confirmation of TR1Fc expression in transiently transfected COS cells was determined and stable cell lines established as for TR2Fc. TL2 The soluble form of TL2 was identical to that previously published (Wiley et al., Immunity 3:673-682 (1995)). Residues 95-281 of the full length TL2 (also known as TRAIL, Apo-2L) were fused to the tPA (tissue plasminogen activator) signal sequence and the FLAG epitope. The resulting DNA construct was transfected into COS and CHO cells, and TL2 was secreted into the supernatant. The protein (sTL2) was purified by passage over an affinity column containing the M2 anti-FLAG epitope antibody available commercially. TL4 An expression vector was constructed which contained the tPA (tissue plasminogen activator) signal sequence, an 11 amino acid sequence derived from HIV-1 gp 120 glycoprotein, six histidines, the enterokinase proteolytic sequence SDDDDK followed by residues 85-240 of the coding region of TL4. This construct was transfected into COS and CHO cells and resulted in the secretion of a soluble form of TL4 (sTL4). The protein was purified by passage over a NiNTA column (available commercially) which binds to the polyhistidine sequence at the amino terminus of the fusion protein. Cleavage of the fusion protein with enterokinase yielded mature TL4. Binding studies Surface plasmon resonance. Protein A was immobilized on to a research grade carboxymethyldextran chip (CM5) using amin coupling procedures described previously (4). Flow cell 1 was activated with NHS/EDC for 5 min. Protein A was injected a a concentration of 1 ug/ml in NaOAc buffer (10 mM, pH 5.0) until 1000RUs of protein were coupled. Remaining activated groups were blocked with a 7 min injection of 1M ethanolamine. A control surface was created by repeating the coupling procedure in a flow cell2 without incorporating protein A. In a BIAcore 2000 biosensor (BIAcore Inc. Uppsala, Sweden) TR1Fc or TR2Fc were then injected at a flow rate of 100 ul/min followed by injection of TL2 or TL4, and the binding to receptor monitored by chances in surface plasmon resonance relative to the control chip. In these experiments, TR1Fc bound to TL2 but not TL4 and TR2Fc bound to TL4 but not TL2. Receptor precipitation. 4 We examined the ability of TR1Fc and TR2Fc to precipitate TL2 or TL4 in solution followed by detection of the ligand in a western blot using antibodies against the fused epitope tag the ligands or the ligand itself. In a typical experiment, 2 ug of TR1Fc or TR2Fc receptor was incubated with 250 ng of purified TL2 or TL4 respectively in binding buffer (25 mM HEPES pH 7.2, 0.1% BSA, 0.01% TWEEN in RPMI 1640). After binding for four hours, receptor complexes were captured on protein A sepharose, centrifuged, washed with binding buffer, electrophoresed on 15% SDS PAGE and transferred for western blotting. TL4 was detected by antibodies to its epitope tag (a 1:5000 dilution of a mixture of murine monoclonal antibodies to the p 120 peptide epitope and the poyHis tail of both antibodies) and demonstrated to bind to TR2 but not TR1Fc or other TNFR related Fc fusion proteins. TL2 ws detected by a 1:5000 dilution of a rabbit polyclonal antiserum raised to TL2 expressed and purified from E. coli , and was found to bind to TR1Fc but not to TR2Fc or other TNFR related Fc fusion proteins. Specificity of binding was further confirmed by the ability of the soluble cleaved TR1 or TR2 to compete with the binding of TR1Fc to TL2 and TR2Fc to TL4 respectively. The references cited in this EXAMPLES Section are as follows: 1. Engelman-DM; Steitz-TA; Goldman-A. Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. Annu-Rev-Biophys-Biophys-Chem. 1986; 15: 321-53. 2. Johanson-K; Appelbaum-E; Doyle-M; Hensley-P; Zhao-B; Abdel-Meguid-SS; Young-P; Cook-R; Carr-S; Matico-R; et-al. Binding interactions of human interleukin 5 with its receptor alpha subunit. Large scale production, structural, and functional studies of Drosophila-expressed recombinant proteins. J-Biol-Chem. Apr. 21, 1995; 270(16): 9459-71. 3. Kumar-S; Minnich-MD; Young-PR. ST2/T1 protein functionally binds to two secreted proteins from Balb/c 3T3 and human umbilical vein endothelial cells but does not bind interleukin 1. J-Biol-Chem. Nov. 17, 1995; 270(46): 27905-13. 4. Johnsson, B., Lofas, S. And Lindquist, G. (1991). Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Anal. Biochem. 198:268-277.