IL78445A - Antibodies to subtypes of (2'-5') oligo-a-synthetase, an assay for said subtypes, and a method and kit for monitoring interferon activity - Google Patents

Description

78445/3 t3\y n 'i , ΐυπ. >Ό-Α- n tna (2'-5') ϊ?¾> οηιση- >ηπ!? α .73ΐ. ΐ οιο. >Ν m yo jnpit? PIDTVI πυ>νη , DmoNn o>:non->nn ANTIBODIES TO SUBTYPES OF (2 '-5') OLIGO-A- SYNTHETASE , AN ASSAY FOR SAID SUBTYPES, AND A METHOD AND KIT FOR MONITORING INTERFERON ACTIVITY Yeda Research and Development Co. Ltd. niJUDi i n»i7 man yi T/658 78445/3 - 1 - Many of the biological effects of interferon appear to be mediated by the induction of new mRNAs and proteins in cells exposed to IFNs (for review: Revel, 1984; Lebleu and Content, 1982; Baglioni and Nilsen, 1983). Among these IFN-induced proteins two groups appear particularly important: 1) translation regulatory enzymes (ds R A dependent protein kinase and (2 '-5') oligo A synthetase, (2 '-5') oligo A-activated nuclease, 2 'phosphodiesterase) and 2) cell surface antigens (HLA-A, B,C, B2-microglobulin, HLA-DR) . Other cellular and excreted proteins play probably important roles as well (Weil et al, 1983; Chebath et al, 1983; Wallach et al, 1983) . With the exception of the HLA genes (Malissen et al, 1982, Schamboeck et al, 1983), the structure and sometimes the function of the IFN-induced proteins is unknown and so is the mechanism by which IFN activate specifically these genes. To address these questions, several cDNAs from IFN-induced genes have recently been cloned (Chebath et al, 1983; Merlin et al, 1983; Friedman et al, 1984; Samanta et al, 1984). In particular the cDNA and gene coding for the human (2'-5') oligo A synthetase was studied, a ds RNA-activated enzyme that converts ATP into ppp(A2'pA)n oligomers (Kerr and Brown, 1978) which in turn bind to and activate the latent RNase F (Schmidt et al, 1978). The (2 '-5') oligo A synthetase is strongly induced in cells by all three types of human IFNs, and its increase is a good marker of IFN activity (Wallach et al, 1982). The enzyme is induced during differentiation of hematopoietic cells, and denotes an autocrine secretion of IFN-β (Yarden et al, 1984) . The enzyme is similarly induced late in the S phase of synchronized embryo fibroblasts (Wells and Mallucci, 1985) . The enzyme activity drops when cell growth starts (Etienne-Smekens et al, 1983; Creasey et al, 1983) and appears to be invol- ved in the antigrowth effect of IFN (Ki chi et al, (1981). Deficiency in the (2'-5') oligo A synthetase or in the (2'-5') oligo A-ac-tivated RNase F have also been correlated with partial loss of the antiviral effects of IFNs (Salzberg et al, 1983; Epstein et al, 1981), although this is probably not the only mechanism by which IFN inhibits virus growth (Lebleu and Content,, 1982). The (2·-5') oligo A nucleotides have been detected in many eukaryotic cells and even in bacteria (Laurence et al, 1984) and the synthetase is likely to be a wide-spread enzyme. The enzyme has been purified from mouse (Dougherty et al, 1980) and human cells (Yand et al, 1981 ; Revel et al, 1981); a large and a small form of the enzyme have been observed (Revel et al, 1982; St. Laurent et al, 1983) but their structures were not elucidated.

The (2'-5') oligo A synthetase, induced in cells exposed to IFNs (Hobanessian et al, 1977; Zilberstein et al, 1978) has a number of unusual properties. Its main activity is the synthesis from ATP of 5'triphosphorylated short oligo A chains (of up tc 15 A, with mainly di ers to pentamers), but in contrast to other RNA polymerases, it adds adenylate or one other nucleoide specifically to the 2' OH of adenylate in oligo A (Kerr and Brown, 1978; Samanta et al, 1980), or to other (oligo)nucleotides with a free 2ΌΗ adenylate such as NAD (Ball, 1980) or even tRNA (Ferbus et al, I98I). To be active, the enzyme has to bind to double-stranded RNA stretches of minimum 50 bp (Minks et al, 1979), and must therefore possess several binding sites: for nucleoside triphosphates, for 2'OH adenosine polynucleotides and for double stranded RNA. The enzyme binds to 2', 5' ADP-Sepharose (Johnston et al, 1980), to poly (rI)(rC)-agarose (Hovanessian et al, 1977) and to Cibacron Blue-Sepharose (Revel et al, I98D. In different cells, the (2·-5') oligo A synthetase activity is in the cytosol (Revel et al, 198D or in ribosomal salt washes (Dougherty et al, 1980), as well as in the nuclear sap (Nilsen et al, 1982b) and even in large amounts in the nuclear matrix. It is notable that cellular RNAs can replace poly (rI)(rC) for activation of the enzyme (Revel et al, 1980) and the synthetase may even have a role in Hn. RNA processing (Nilsen et al, 1982a).. Some (2 '--5'-) oligo A synthetase is bound to plasma membranes and can be incorporated in budding virions (Wallach and Revel, 1980) . These complex interactions may ensure a localized action of the (2·-5') oligo A system (Nilsen and Baglioni, 1983) and explain its multiple suggested roles in normal and virus-infected cells. The synthetase amounts to less than 0.1% of the proteins in IFN-treated cells, and its structure could not be determined directly.

It is possible to use measurements of (2 '-5') oligo A synthetase levels to determine whether cells in vitro or in vivo have been exposed to IFN and respond to it. This measurement can be used as an assay for IFN in unknown solutions, by exposing cells to said solutions and determining the increase in (2'-5') oligo A synthetase levels (Revel et al., US patent 4,302,533). The measurement can also be used to establish whether IFN is produced in increased amounts in whole organisms including man.

CLINICAL APPLICATIONS OF (2'-5') OLIGO A SYNTHETASE MEASUREMENTS It has been established that the (2'-5') oligo A synthetase level is rather constant in peripheral blood mononuclear cells (PBMC) of healthy individuals (Schattner et al, 1981b). An increase in (2'-5') oligo A synthetase is seen in PBMC of patients with acute viral infections (Schattner et al., 1981b; Schoenfeld et al., 1985), with persistent viral infections (Wallach et al., 1982), with autoimmune diseases and with a number of other syndromes suspected of infectious origin such as Jacob- reuzfeld disease (Revel et al., 1982). The basal (2'-5') oligo A synthetase level is lower in granulocytes but large increase in viral infections are seen (Schattner et al., 1984). Increase of (2' -5*) oligo A synthetase enzyme in PBMC of AIDS patients was recently reported (Read et al., 1985). In animal models, it was shown that increase in (2'-5') oligo A synthetase level is rapid and more constant than appearance of IFN in the blood (Schattner et al.1982a) . (2'-5') oligo A synthetase remains high for several weeks while the IFN peak is transient.

The (2'-5') oligo A synthetase increases during differentiation of haematopoietic cells as a result of autocrine secretion of IFN-β 78445/3 - 4 - (Yarden et al, 1984). Decreased (2 '-5') oligo A synthetase levels are seen in acute leukemias with numerous blast cells (Wallach et al. 1982; Schattner et al . 1982b).

Another important application of (2 '-5') oligo A synthetase measurements is in the monitoring of patients under IFN therapy. Besides clinical changes, it is possible to establish that the patients respond to IFN by measuring the PBMC. (2 '-5') oligo A synthetase level which increases 5-10 fold during systemic IFN-a as well as β treatment (Schattner et al. 1981a; Schoenfeld et al. 1984) . It is clear that assay of other IFN-induced activities or molecules can be used as well as the assay of the (2 '-5') oligo A synthetase enzyme, but this method has been the most widely used (Read et al., 1985; Merritt et al., 1985). In all these studies, the enzymatic assay measuring the conversion of ATP into (2 '-5') (A)n oligomers has usually been employed (Revel et al., US patent 4, 302, 533) .

The present invention provides an antibody against the 40KD, 46KD 67KD and 100KD forms of (2 '-5') oligo A synthetase selected from an antibody against all four of said forms obtainable by immunizing an animal with an antigenic peptide comprising the amino acid sequence : Glu-Lys-Tyr-Leu-Arg-Arg-Gln-Leu-Thr-Lys- Pro-Arg-Pro-Val-lie-Leu-Asp-Pro-Ala-Asp, and an antibody against one of said forms which does not cross-react with the other three forms, obtainable by immunizing an animal with an antigenic peptide comprising the amino acid sequence : Arg-Pro-Pro-Ala-Ser-Ser-Leu-Pro-Phe- Ile-Pro-Ala-Pro-Leu-His-Glu-Ala .

The antibody against all above four forms of (2 '-5') oligo A synthetase may be obtained in accordance with the invention by immunizing an animal with peptide B, said peptide B comprising the 78445/3 - 5 - amino acid residues 284-303 common to clones E16 and E18 of Figs. 8A and 8B and having the amino acid sequence: Glu-Lys-Tyr-Leu-Arg-Arg-Gln-Leu-Thr-Lys- Pro-Arg-Pro-Val-Ile-Leu-Asp-Pro-Ala-Asp, The antibody against one of the forms of (2 '-5') oligo A synthetase which does not cross-react with the other three forms may be obtained in accordance with the invention by immunizing an animal with peptide C, said peptide C comprising the amino acid residues 348-346 set fo.rth in Fig.- 8A and having the amino acid sequence: •Arg-Pro-Pro-Ala-Ser-Ser-Leu-Pro-Phe- Ile-Pro-Ala-Pro-Leu-His-Glu-Ala .

The present invention also provides an antibody against all above four forms of (2 '-5') oligo A synthetase which is conjugated with a label to form a labelled antibody. The label may be fluorescent, radioactive or may be an enzyme.

In another aspect, the present invention provides an assay for the 40KD, 46KD, 67KD and 100KD forms of (2'-5*) oligo A synthetase in cells which comprises incubating the cells with a labelled antibody and detecting cells bearing (2'-5') oligo A synthetase activity in any of said forms by means of said label. The assay may be a fluorescent immunoassay, a radioimmunoassay or an enzyme immunoassay, depending on the label employed.

The cells employed in an assay according to the invention may be e.g. mononuclear blood cells.

A kit for the detection of all form forms of (2'-5') oligo A synthetase in cells comprising an antibody against all four forms of (2'-5') oligo A synthetase conjugated with a label, is also part of the present invention.

- Sa¬ in another aspect the present invention provides a method of monitoring interferon activity in a subject in vitro, which comprises measuring the amount of (2 '-5') oligo A synthetase in a cell of the subject at predetermined time intervals, determining the differences in the amount of said synthetase in the cell of the subject within the different time intervals, by contacting the synthetase with an antibody of the present invention so as to form a complex therewith and determining the amount of complex so formed and determining therefrom the amount of synthetase in the cell of the subject and thereby the interferon activity of the subject.

The above method may further comprise the extraction of (2 '-5') oligo A synthetase from a cell which has been exposed to interferon, labelling the extracted synthetase with an identifiable marker to form a labelled synthetase, contacting the labelled synthetase with the antibody under suitable conditions so as to form a labelled synthetase-antibody complex and detecting the marker in the complex, thereby detecting the synthetase.

In the above method, the marker may be, e.g. 35s-methionine . : In another aspect, the invention provides a kit for carrying out the above method, comprising an antibody against all four forms of (2 '-5') oligo A synthetase or against one of the four forms which does not cross-react with the other three forms and may be labelled as above, materials for extracting the synthetase, materials for labelling the synthetase, and materials for detecting the marker in determining the amount of synthetase. 6 - Description of the Figures Figure 1 depicts the (2' -5 ' ) oligo A synthetase activity which is adsorbed on anti-B and anti-C IgG-Protein A-Sepharose was measured as described in Example · The scheme underneath shows the position of peptide B and peptide C in the two (2 ' -5 ' ) oligo A synthetase forms E16 and E18 sequenced by Benech et al. (1985b). The blackened area indicates the part of E18 which differs from the E16 molecule.

Figure 2 depicts electrophoresis and immunoblotting of extracts from human cells as described in Example 2. The position of the 4 forms of (2'-5') oligo A synthetase is indicted by the numbers on the right of each blot. M = C-protein molecular weight markers. IFN treatment is indicated by +.

Figure 3 depicts electrophoretic immunoblots of extracts from human SV80 cells with anti-(2'-5') oligo A synthetase peptide B. Left: crude cytoplasmic extract (S1.5), cell sap (S100) and Microsomes (P100). Right: Na deoxycholate 10% extract of microsomes (DOC-soluble) , high salt wash of microsomes (RWF) and microsomal pellet after salt extraction (Microsomes- Cl) .

Figure 4 depicts the fractionation of S100 and high-salt wash of microsomes (RWF) on DEAE-cellulose and carboxy ethyl-cellulose, followed by glycerol gradient. The (2'-5') oligo A synthetase profile and protein detected by anti-B are shown below the gradients. Electrophoretic immunoblot show fraction CM (CM-cellulose eluate from the S100 proteins non-adsorbed to DEAE-cellulose) on the left blot. On the right-hand blot fraction, DE (DEAE-cellulose eluate of RWF) and fraction GG. (heavy peak, of 80-100 kd from glycerol gradient of fraction DE). - 7 - 78445/3 Figure 5 depicts the double stranded RNA requirements of various forms of (2'-5') oligo A synthetase from SV80 cells. Fractions are labelled as in Figure 19. Enzymatic activity measured at indicated concentrations of poly (rI)(rC).

Figure 6 depicts the radio-immunoassay of (2'-5') oligo A synthetase 125 with anti-B IgGjand I-Protein A as described -in Example 3· Autoradiography is shown. Cells treated for 16 hours with 500 U/ml IFN-ft1 or left untreated.

Figure 7 depicts the use of anti-B for immunofluorescence microscopic detection of elevated (2'-5') oligo A synthetase levels in lymphocytes from blood bf - patient'' with viral disease (middle panel). Right: control with normal serum: left: blood from healthy donor with anti-B stain. (Lymphocytes do not stain, only macrophages or granulocytes give unspecific background.

Figures 8 A and B depict the nucleotide sequences of the two (2 '-5') oligo A synthetase cDNAs. The nucleotides of the 1.8 kb cDNA clone 48-1 are numbered as for the 1.6 kb cDNA clone 9-21. Amino acid numbering is given in parantheses. Translation starts at the first or second codon of the ATGATG sequence. Limits between exons are shown by vertical bars. (Glycos.) indicates a possible glycosylation site in El8. Single base variations, possibly allelic differences, were detected between clones or genomic DNA in the 1.6 kb sequence at 376 (T for C), 525 (G for A), 807 (G for C), 811 (A for G); in the 1.8 kb sequence at 1087 (G for A), 1115 (G for C).

Detailed description of the invention An enzyme having (2'-5') oligo A synthetase activity has the amino acid sequence set forth in Figure 8A. This enzyme comprises about 364 amino acids and has a molecular weight of about 41,500 daltons. Another enzyme having (2 '-5) oligo A synthetase activity comprises the sequence of amino acids 1-364 set forth in Figure 8A and the sequence of amino acids 290-400 set forth in Figure 8B. This enzyme comprises about 400:- amino acids and has a molecular weight of about 46,000 daltons. 78445/3 - 8 - Antibodies of the present invention recognize and immunoprecipitate (2'-5') oligo A synthetase.

A method of monitoring interferon activity in a subject comprises measuring the amount of (2 '-5') oligo A synthetase in a cell or body fluid of the subject at predetermined time intervals, determining the differences in the amount of said synthetase in the cell or body fluid of the subject within the different time intervals, and determining therefrom the amount of. synthetase in the cell or body fluid of the subject and thereby the interferon activity' of the subject. The amount of synthetase may be measured by contacting the synthetase with an antibody of the .present invention so as to form a complex therewith and determining the amount of complex so formed.

A: method. of monitoring interferon activity may further comprise the extraction of (2'-5') oligo A synthetase from a' cell or body fluid which has been exposed to interferon, labelling the extracted synthetase with an identifiable marker to form a labelled synthetase, contacting the labelled synthetase with an antibody of the present invention under suitable conditions so as to form a labelled synthetase-antibody complex, and detecting the marker in the complex, •55 thereby detecting the synthetase. The marker may be S-methionine.

A kit for carrying out the method of monitoring interferon activity comprises an antibody of the present invention, materials for extracting the synthetase, materials- for labelling the synthetase, and materials for detecting the marker and determining the amount of synthetase.

The invention will now be illustrated by the following non-limiting examples : EXAMPLE 1 OBTETION OF ANTIBODIES TO (2 '-5') OLIGO A SYNTHETASE Two peptide sequences were chosen from the total aminoacid sequences of E16 and E18 , to serve as antigens for the induction of antibodies against the native (2'-5') oligo A synthetase activity molecule.

Peptide B: GLU LYS TYR LEU ARG AEG GLN LEU THR LYS PRO ARG PRO VAL ILE LEU ASP PRO ALA ASP comprises aminoacids 284 to 303 common to both E18 and E16 sequences. Peptide C: ARG PRO PRO ALA SER SER LEU PRO PHE ILE PRO ALA PRO LEU HIS GLU ALA comprises the C terminus of E16 (residues 348 to 364). Both peptides were synthesized by the solid-phase peptide synthesis method of Barany and Merrifield ( 1980) . After purification on Sephadex G25 columns in 2M acetic acid, the peptides were linked to Keyhole Limpet Hemocyanin (Calbiochem) . Esterification of the NH2 -terminal arg-inine of peptide C with p-aminophenylacetic acid allowed to coval-ently link the peptide to the carrier protein through its amino-terminus (Spirer et al, 1977) . Peptide B was coupled to the carrier protein by ethylene diamine carbodiimide (Hoare and Koshland, 1967 ) .

Rabbits were injected subcutaneously with 1 mg carrier-coupled peptide (equivalent to 0.2 mg pure peptide) which was emulsified in complete Freund's adjuvant. Rabbits were boosted twice at two weeks intervals with 0.5 mg of carrier-coupled peptide in incomplete adjuvant, and were continued until maximal antibody response. The titer of antibodies in the rabbit sera were measured in enzyme-linked immunosorbent assays (Green et al, 1982) using the carrier-free peptides.

EXAMPLE 2 USE OF ANTI-(2'-5') OLIGO A SYNTHETASE ACTIVITY PEPTIDE ANTIBODIES TO DETECT THE ENZYME Extracts of IFN-treated human cell cultures The fibroblastoid cell line SV80 and the amniotic cell line Wish were grown to confluent monolayers on plastic dishes and the Daudi cell line was grown in suspension to 1.5x10^ cells/ml. Cultures were treated for 16-24 hours with rIFN-jy , 500 U/ml. The human rIFN-¾1 - 10 - was produced by genetically engineered CHO cells and purified to homogeneity by monoclonal antibody affinity chromatography (Chernajovsky et al, 1984).

Cells were washed twice with phosphate buffered saline (PBS) at U°C and lysed in the cold in Buffer A: 20mM Hepes buffer, pH 7.5, 5mM Mg acetate, 30 100 uM phenylmethyl sulfonyl fluoride (PMSF), 10% glycerol and 0.5% Nonidet P-40 (NPM0). Nuclei and unbroken cells were eliminated by centrifugation at 1,500γ for 10 min. The supernatant (S1.5) was centrifuged 10 min at 15,000γ in an Eppendorf Microfuge to obtain mitochondria and lysosomes-free supernatant (S1 ). Protein concentrations were measured by Micro- assays (Bradford, 1976).

Centrifugation of S15 for 2 hours at 100,0007 in a Beckman refrigerated ultracentrifuge was used to prepare cell sap (S100) and microsomes (P100) fractions.

EXAMPLE 3 ASSAY OF THE (2'-5') OLIGO A SYNTHETASE Aliquots of S15 containing 1-2 ug protein were incubated in 20μ1 reactions containing 25mM Hepes buffer, pH 7.5, 20mM Mg acetate, 1mM dithiothreitol, 1.5mM ATP, M Ci of32P-«-ATP, 50ug/ml poly(rl)(rC) (from PL-Biochemicals) for 2 hours at 30°C. After boiling for 5 min and Microfuge centrifugation, Bacterial Alkaline Phosphatase was added at 25 U/ml to an aliquot and the reaction incubated for 2 hours at 37°C. From 2 to 7μ1 were spotted on Whatmann 3MM paper and analyzed by electrophoresis at 3,000 V in pyridine/acetic acid pH 3-5. After autoradiography, the (A2'p)nA oligomers spots were cut out and counted.

EXAMPLE 4 ELECTROPHORETIC-TRANSFER IMMUNOBLOTS Aliquots of crude cellular fractions (30ug protein) were adjusted with Laemmli's sodium-dodecyl-sulphate-polyacrylamide gel electro-phophoresis loading buffer (Laemmli, 1970) and boiled 10 min before 14 electrophoresis on 7.5 or 10 % gels. Ainersham's C-methylated proteins were used as molecular weight standards (10 cpm). Electro-phoretic transfer onto nitrocellulose paper (Schleicher and Schull BA85) was carried out in 25 mM Tris-base, 192mM glycine and 20% methanol. The blots were preincubated in 0.09M NaCl, 0.01M Tris-HCl pH 7.5, 10% (v/v) of a 1% fat milk solution, 10% (v/v) heat-inactivated fetal calf serum and 0.05% Tween-20, either for 2 hours at 37°C or overnight at 4°C followed by 30 min at 37°C. Blots were then incubated with anti-(2'-5f) oligo A synthetase peptide B antibodies in form of rabbit IgG O.lmg/ml, for 2 hours at 37°C. Blots were washed 5 times for 10 min in 4% fetal calf serum and incubated in the 6 125 complete above preincubation mixture containing 10 cpm/ml of i-protein A (Amersham, 30 mCi/mg) for 1 hour at 37°C Blots were washed and subjected to autoradiography.

EXAMPLE 5 IMMU OPRECIPITATION OF (2'-5') OLIGO A SYNTHETASE ACTIVITY AND LABELED PROTEINS First aliquots of 5-10μ1 anti-(2'-5') oligo A synthetase peptide B rabbit serum were adsorbed on 3mg of Protein A-Sepharose (Pharmacia) equilibrated in PBS with 3% Bovine serum albumin (BSA), for 30 min at room temperature, then washed with PBS-1% BSA. For immunoprecipitation of the (2· -5') oligo A synthetase enzymatic activity, aliquots of 2ug of S15 proteins in a final volume of 20μ1 of buffer A were adsorbed on the above pelleted IgG-Protein A Sepharose for 2 hours at 4°0. The suspension was diluted 5 fold in buffer A and the supernatant transferred to another tube. The pellet was washed 3 times with 0.5 ml buffer A and was then suspended in 25ul of the enzyme reaction mixture (see above). Activity was measured also on aliquots of the non-bound supernatant.

For labeling (2'-5') oligo A synthetase, Wish cells. were grown to confluent monolayers on 3cm plastic dishes and treated for 12 hours with 500 U/ml rIFN-*i1. The medium was replaced by 0.5 ml - - methionine-free DMEM (GIBCO) containing 500μ01 of 35S-methionine (Amersham 400 mCi/mmol) and cells incubated for 2 hours, washed with PBS and homogenized in Buffer A. The S15 was used for immunoprecip- 7 itation. About 10 cpm of S15 proteins were added to the pelleted Portein A-Sepharose and mixed for 2 hours at 4°C. The Beads were washed with 1% BSA, 1% NP40, 2M KC1 in PBS and twice with PBS only. Samples were analysed by sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis.

EXAMPLE 6 USES OF ANTI-PEPTIDE ANTIBODIES TO DETECT THE (2'-5') OLIGO A SYNTHETASE ACTIVITY Immunoprecipitation of the (2'-5') oligo A synthetase activity by anti-(2'-5') oligo A synthetase activity peptide antibodies The antiserum B was raised against a peptide co mon to the E16 and E18 sequences, while antiserum C was raised against a peptide found only in E16. We used these antibodies to verify that they immuno-precipitate the (2'-5') oligo A synthetase activity specifically. The (2 '-5') oligo A synthetase activity adsorbed on the immune xgG-Protein A Sepharose and that remaining in the supernatant were compared to the same fractions obtained by using non-immune IgG.

Extracts from two cell lines which express preferentially either the 1.6 kb RNA (Wish cells) or the 1.8 kb RNA (Daudi cells) were compared. Antibodies C (El6-specific) were 20 times more efficient to adsorb the activity from Wish cells than normal serum. Substract-ing the background with normal serum, allows to evaluate what is specifically bound to anti-B and anti-C (Fig. 1 ). Anti-C retained the (2 '-5') oligo A synthetase activity from Wish cells but not from Daudi cells, in line with the absence of E16 mRNA and 40 kd protein in these cells (see Example 7). Anti-B adsorbed (2'-5') oligo A synthetase activity from both Daudi and Wish cell extracts.

The antibodies produced against peptides deduced from the cloned cDNAs, recognize, therefore, specifically different (2'-5') oligo A synthetase forms. - 13 - EXAMPLE 7 IMMUNOBLOT ANALYSIS OF THE DIFFERENT FORMS OF (2'-5') OLIGO A SYNTHETASE ACTIVITY FROM HUMAN CELLS The antibodies against peptide B were tested for their ability to bind specifically to (2·-5') oligo A synthetase activity in crude extracts of human cells separated by sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis and blotted electrophoretically onto nitrocellulose paper. In addition to being recognized by the antibodies in immunoblots, we expect genuine (2'-5') oligo A synthetase proteins present in extracts of these human cells to be induced by IFN treatment, and we therefore looked only at the induced proteins revealed by the immunoblots (Fig. 2 ).

The cell lines Daudi, Wish and SV80 were compared because of their differences in the pattern of expression of the (2 '-5') oligo A synthetase .mRNAs (see Example 6 ). Antibodies B detect as expected a 45-46 kd protein similar in size to the E18 product, in Daudi cells and no 40 kd which would correspond to E16 whose mRNA is not expressed by Daudi cells. In contrast, the 40 kd El6 protein is present in Wish cells without 46 kd E18 in line with the absence of 1.8 kb RNA in these cells. Both proteins are detected by anti-B in SV80 cells. These results demonstrate that human cells produce the 40 and 46 kd proteins and this only when they express the 1.6 and 1.8 kb RNAs respectively.

The immunoblots with anti-B also reveal that there are not only two forms of (2' -5') oligo A synthetase in human cells but probably four different forms. This can be deduced from the fact that in addition to the 40 and 46 kd proteins, anti-B clearly detected two other proteins of 100 kd and 67 kd which are induced by IFN (Fig. 2 and 3). The 100 kd was not detected in Daudi cells, showing that the large proteins detected by anti-B are also expressed in a cell-specific pattern. The fact that the anti-B was raised against a peptide derived from the sequence of genuine (2'-5') oligo A synthetase forms,- and that the two larger proteins are induced by_IFN, makes it very likely that they belong to the (2'-5') oligo A - 14 - synthetase system. To ascertain that these are (2 '-5') oligo A synthetase .forms, we have purified (2'-5') oligo A synthetase from different cellular fractions of SV80 and followed in parallel the protein detected by antibodies B.

Separation of the different active forms of (2'-5') oligo A synthetase The separation of the different protein species detected by antibodies B is shown in Fig.18 and19- Most of the MO kd protein remains in the 100,000gsupernatant (S100) of NPMO cytoplasmic extraxts from SV80 cells. It is not adsorbed on DEAE-cellulose in low salt and adsorbs to CM-cellulose from which it elutes at high salt concentration. In contrast, the 100 kd protein is almost absent from S100 and is concentrated in the microsomal pellet (P100) from which it can be solubilized by Na deoxycholate (DOC) or 0.5M Cl. This protein was adsorbed on DEAE-cellulose at low salt and elutes at high salt. The 67 kd and M5-M6 kd remain partly in S100 but are relatively concentrated per mg protein in the microsomal pellet. They appear to be less readily extractable from microsomes by DOC or KCl.

Sedimentation on glycerol gradients showed that the activity purified from S100 after CM-cellulose, parallels the sedimentation of the M0 kd protein El6. The fraction purified from P100 and eluted from DEAE-cellulose, containing the 100 and M6 kd proteins,, separated into two peaks on glycerol gradients, sedimenting as 80,000 and M5,000 Mr proteins. The (2'-5') oligo A synthetase in the heavy peak parellels the presence of the 100 kd protein.

Different enzymatic properties of the various (2 '-5') oligo A synthetase activity forms The M0 kd protein from the glycerol gradient has an optimal pH of 6.8 for its activity, and is only 25% as active at pH 7.8. Moreover, no activity of the M0 kd (2'-5') oligo A synthetase can be observed at concentrations of poly (rl)(rC) lower than 1ug/ml and the maximal activity requires 50-100ug/ml (Fig. 5). The same high ds - RNA requirement was found for_ the E16 cDNA product produced by recom- - 15 - binant DNA technology in E.coli.

The 100 kd protein after the glycerol gradient, has an optimal pH of 7.6 for its (2'-5') oligo A synthetase and is less active at acidic pH. It is maximally active already at extremely low concentrations of poly (rl)(rC) or in its absence, and its activity is even inhibited by high ds RNA concentrations. This strongly suggests that the different (2'-5') oligo A synthetase forms, because of their different cytoplasmic localizations and enzymatic properties, are used by the cells under different conditions.

Many observations suggest that the IFN-induced (2 '-5') oligo A synthetase is involved in two distinct, seemingly opposite, phases of cell growth (cell.cycling and growth inhibition) in addition to its possible role in the antiviral effect (reviewed in Revel, 1984). This may be relevant to the issue of multiple (2'-5') oligo A synthetase forms. In synchronized cell cultures we have observed that (2'-5' ) oligo A synthetase behaves as a cell-cycle protein (Mallucci et al, 1985). Thus, synchronized cultures of Mouse embryo fibroblasts exhibit a sharp rise in (2'-5') oligo A synthetase activity and (2'-5') oligo A synthetase mRNA at the end of the S-phase followed by a rapid disappearance of the RNA and enzyme activity when the cells proceed to G2. Anti-mouse IFN antibodies reduced the (2'-5') oligo A synthetase induction. In this system we also observed that the (2'-5') oligo A synthetase RNA which accumulates in S-phase is a large 4-5 kb transcript different from the 1.7 kb RNA species which accumulates in the same cells when treated with exogenous IFN. This suggests that the S-phase (2'-5') oligo A synthetase is a different form of the enzyme than that in cells growth-arrested by exogenously added IFN. Because of its large mRNA, it is likely to be like the 100 kd, a low ds RNA requiring form. Anti-B antibodies detected also the (2'-5') oligo A synthetase multiple forms in mouse cells.

These considerations illustrate the advantage of being able to assay independently the 4 forms of (2'-5') oligo A synthetase, which ..may. vary individually in various physiological conditions and - 16 - diseases.

EXAMPLE 8 USE OF ANTI-(2'-5') OLIGO A SYNTHETASE ACTIVITY PEPTIDES ANTIBODIES FOR IMMUNOASSAYS OF (2'-5') OLIGO A SYNTHETASE ACTIVITY Since the anti-B antibodies recognizes all the forms of (2'-5') oligo A synthetase, it can be used for an immunoassay of (2'-5') oligo A synthetase in unfractionated extracts of human cells either from cultures or directly obtained from patients.

An example of a solid-phase radio-immunoassay is shown in Fig. 6 ■ Wish and Daudi cells, either treated by IFN or untreated, were lysed by the NP40-containing Buffer A and ST5 prepared by microfuge cent-rifugation as described above. Aliquots containing T to 10yg of protein were directly applied to nitrocellulose paper (or to other protein-binding paper) and the sheet treated with anti-3 as for regular immunoblots (Example 7) . The autoradiography shows 125 ' that χ-Protein A binds only to the samples originating from IFN-treated cells. It is clear that this assay could- be used also in form of enzyme-linked immunoassay (ELISA) by replacing the labeled ?rotein-A by Peroxydase or£-galactosidase conjugated anti-rabbit ¾G.

The immunoassay of (2 '-5' ) -oligo A synthetase is rapid: 20 min. for- cell..extract preparation, 2 hours for anti-3 and Protein-A adsorption and washing. The assay is sensitive and very small amounts of cell extracts suffice to measure that (2'-5') oligo A synthetase level. It is specific, no signal being obtained in non-IFN treated cells.

EXAMPLE 9 IMMUNOFLUORESCENCE MICROSCOPY DETECTION OF ( '-5') OLIGO A SYNTHETASE IN CELLS Enzymatic assays- have established (see prior art) that (2'-5') oligo A- synthetase is elevated in peripheral blood mononuclear - - cells of patients with viral infections. The anti-(2,-5') oligo A synthetase peptide antibodies can be used in immunofluorescence microscopy to detect (2'-5') oligo A synthetase elevation in cells in general, and white blood cells in particular.

Blood (2 ml) was withdrawn from a healthy donor and from a patient with acute viral illness. The mononuclear blood cells were separated by Ficoll-Hypaque (Pharmacia) centrifugation , and spread on glass coverslips directly or by the use of a cytospin microfuge (micro-hematocrite) . The cells were washed in PBS, fixed for 30 min in 3% paraformaldehyde at room temperature (RT), rinsed with PBS and treated with 0.5% Triton-X100 in Hank's salts for 5 min, rinsed again with PBS and with PBS-2% gelatin. The coverslips were then incubated with antiB serum diluted 1:5 in PBS-gelatin applied as a 40 1 droplet on parafilm onto which the coverslip were deposited. After 60 min RT, the coverslips were rinsed in PBS-gelatin and FITC conjugated anti-rabbit IgG (BioYeda) diluted 1:20 was applied by the parafilm procedure. After 20 min at RT, coverslips were washed twice with PBS-gelatin, then with l^Oand mounted on microscopic slides with Miviol 4-88 (Hoechst)-Glycerol (2.5g Moviol, 6g Glycerol and 6 ml H20 to which 12 ml of 0.2M Tris.HCl pH 8.5 were added, followed by incubation at 50°C and clarification 15 min at 5,000γ). Parallel coverslips were processed using normal rabbit serum instead of anti-B. Slides were observed in a Zeiss fluorescence microscope and photographed on Polaroid film with 30 seconds exposures.

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Claims (1)

1. 78445/4 An antibody against the 40KD, 46KD, 67KD and 100KD forms of (2 '-5') oligo A synthetase selected from an antibody against all four of said forms obtainable by immunizing an animal with an antigenic peptide comprising the amino acid sequence: Glu-Lys-Tyr-Leu-Arg-Arg-Gln-Leu-Thr-Lys- Pro-Arg-Pro-Val-Ile-Leu-Asp-Pro-Ala-Asp, and an antibody against one of said forms which does not cross-react with the other three forms, obtainable by immunizing an animal with an antigenic peptide comprising the amino acid sequence: Arg-Pro-Pro-Ala-Ser-Ser-Leu-Pro-Phe- Ile-Pro-Ala-Pro-Leu-His-Glu-Ala. An antibody according to claim 1, against all four of the 40 D, 46KD, 67KD and 100KD forms of (2 '-5') oligo A synthetase. An antibody according to claim 1 against one of the 40KD, 46KD, 67 D and 100KD forms of (2 '-5') oligo A synthetase, which does not cross-react with the other three forms. An antibody according to claim 2, said antibody being obtained by immunizing an animal with peptide B, said peptide B comprising the amino acid residues 284-303 common to clones E16 and E18 of Figs. 8A and 8B and having the amino acid sequence: Glu-Lys-Tyr-Leu-Arg-Arg-Gln-Leu-Thr-Lys- Pro-Arg-Pro-Val-Ile-Leu-Asp-Pro-Ala-Asp. 27 78445/3 5. An antibody according to claim 3, said antibody being obtained by immunizing an animal with peptide C, said peptide C comprising the amino acid residues 348-346 set forth in fig. 8A and having the amino acid sequence: Arg-Pro-Pro-Ala-Ser-Ser-Leu-Pro-Phe- Ile-Pro-Ala-Pro-Leu-His-Glu-Ala. 6. An antibody according to claim 2, said antibody being conjugated with a label to form a labelled antibody. 7. An antibody according to claim 6, wherein the label is a fluorescent label. 8. An antibody according to claim 6, wherein the label is a radioactive label. 9. An antibody according to claim 6, wherein the label is an enzyme . 10. An assay for the 40 D, 46 D, 67KD and 100KD forms of (2 '-5') oligo A synthetase in cells which comprises incubating the cells with the labelled antibody of claim 6 and detecting cells bearing (2 '-5') oligo A sythetase activity in any of said forms by means of said label. 11. An assay of claim 10 in which the assay is a fluorescent immunoassay. 12. An assay of claim 10 in which the assay is a radioimmunoassay. 28 78445/5 13. An assay of claim 10 in which the assay is an enzyme immunoassay. 14. An assay of claim 10 in which the cells are mononuclear blood cells . 15. A kit for the detection of all four forms of (2 '-5') oligo A synthetase in cells comprising the antibody of claim 6. 16. A method of monitoring interferon activity in a subject in vitro, which comprises measuring the amount of (2 '-5') oligo A synthetase in a cell of the subject at predetermined time intervals, determining the differences in the amount of said synthetase in the cell of the subject within the different time intervals, by contacting the synthetase with the antibody of any of claims 2 to 9 so as to form a complex therewith and determining the amount of complex so formed and determining therefrom the amount of synthetase in the cell of the subject and thereby the interferon activity of the subject. 17. The method of claim 16 further comprising the extraction of (2 '-5') oligo A synthetase from a cell which has been exposed to interferon, labelling the extracted synthetase with an identifiable marker to form a labelled synthetase, contacting the labelled synthetase with the antibody under suitable conditions so as to form a labelled synthetase-antibody complex, and detecting the marker in the complex, thereby detecting the synthetase. 29 78445/1 18. The method of claim 17 wherein the marker is 35S-methionine . 19. A kit for carrying out the method according to claim 17, comprising the antibody of any of claims 2 to 8, materials for extracting the synthetase, materials for labelling the synthetase, and materials for detecting the marker in determining the amount of synthetase . 30