WO1993006489A1

WO1993006489A1 - Method to detect tumor necrosis factor (tnf) as bioactive form

Info

Publication number: WO1993006489A1
Application number: PCT/IT1992/000116
Authority: WO
Inventors: Angelo Corti
Original assignee: Tecnogen Scpa
Priority date: 1991-09-23
Filing date: 1992-09-17
Publication date: 1993-04-01
Also published as: ITRM910710A0; AU2690592A; EP0605635A1; ITRM910710A1; JPH07502113A; IT1249708B

Abstract

Disclosed is a method to detect oligomeric, biologically active TNF, which method uses reactants only capable of reacting with TNF as oligomeric form, and incapable of reacting with TNF as complexed form onto receptor, or with TNF as monomeric form. A kit for carrying out the method is disclosed as well.

Description

"METHOD TO DETECT TUMOR NECROSIS FACTOR (TNF) AS BIOACTIVE FORM"

The present invention relates to a method to determine Tumor Necrosis Factor (TNF) as bioactive form.

More particularly, the present invention relates to a method to detect oligomeric, bioactive TNF, also in the presence of monomeric TNF, or TNF as complexed form with receptors, both biologically inactive.

Alpha-TNF, also designated "cachectin", is a cytokine mainly secreted by activated macrophages.

TNF was initially identified in serum of mice infected with "Calmette Guerin" bacillus (BCG), owing to its capability of inducing the haemorrhagic necrosis of some tumors in experimental animal models.

At a later time, it was discovered that TNF is among the main inflammation mediators. In particular circumstances of acute and /or chronic production, TNF can cause pathologic conditions, such as septic shock and lipid dismetabolism (cachexia), typical of patients affected by infective chronic and neoplastic pathologies.

It was furthermore verified that TNF plays a role, together with other cytokines and lymphokines, in immunoregulation, angiogenesis, cellular differentiation and growth processes (Beutler B. and Cerami A., 1989, Ann. Rev. Immunol. 7, 625-655).

The marked cytotoxicity of TNF for some tumoral cell lines and its capability of inducing nechrosis of some tumors in vivo suggest potential pharmacological applications in anti-neoplastic therapies. Furthermore, the development of compounds capable of neutralizing the toxic effects induced by endogenic TNF under hyper- production conditions, such as, e.g., is septic shock, are equally well potentiaIly useable for therapeutical purposes.

TNF is thought to perfom its pleiotropic effects through receptors present on the membrane of target cells. Actually, two different receptor types have been purified and characterized (Smith C.A. et al., 1990, Science 248, 1019-1023; Schall T.J. et al., 1990, Cell 61, 361-370). Furthermore, soluble forms of such molecules, corresponding to their extra-cellular portions, displaying powerful TNF inhibitory activity, were purified from urines of normal subjects and, in larger amount, from urines of feverish patients affected by chronic kidney failure, or from serum of patients affected by neoplastic diseases (Nophar Y. et al., 1990 EHBO J. 9, 3269-3278).

A large number of experimental evidences suggest that the manifestation of its various biological activities by TNF, at cellular or tissutal level, is regulated by the modulation of the expression of membrane receptors and release of soluble forms.

Besides the different molecular forms of receptors, also the existence was described of different molecular forms of TNF. TridimensionaI structure analysis showed that TNF is a molecule composed by three identical sub-units of 17.5 KD, strictly associated with one another. Studies of analytical ultracentrifugation, gel electrophoresis, cross-linking, X ray dispersion and gel filtration showed that TNF is in oligomeric form also in solution (Wingfield P, Pain R.K. and Craig S., 1987, FEBS Lett. 211, 179-184; Arakawa T. and Yphatis D.A., 1987, J. Biol. Chem. 262, 7484-7435; Lam K.S., Scuderi P. and Salomon S.E., 1988, J. Biol. Response Hod. 7, 267-275; Lewit-Benteley A. et al., 1988, J. Mol. Biol. 199, 389- 392). The oligomeric structure, i.e., the bioactive form, seems however to be unstable at bioactive Cpicomolar) concentrations, with monomeric TNF being partially formed, which is characterized by poor or null capability of binding the receptor and hence of performing biological activity, in terms of cytotoxicity. It was consequently hypothesized that changes in quaternary TNF structure, consequent to changes in concentration, might perform an important role in the regulation of physio-pathological processes induced by such a molecule (Smith R.A. and Baglioni C., 1987, J. Biol. Chem. 262, 6951-6954).

In some pathologic situations, such as septic shock (Waage et al., 1987, Lancet i, 355), rejection of renal transplantations (Haury et aI., 1987, J. Exp. Med. 166, 1132), parasitic infections (Scuderi et al., 1986, Lancet ii, 1364) and various neoplasiae (Balkwill et al., 1987, Lancet ii, 1229), the plasmatic Levels of TNF display a considerable increase. The development of fast and simple analytical methods for in vitro measuring TNF levels from biological fluids is hence of considerable importance from clinic and diagnostic view points.

TNF levels in biological fluids under normal and pathologic conditions are commonly determined by means of traditional immunologic determinations or biological assays. The low correlation leveI between the results obtained from both assay types on pathologic sera suggests the existence of bioinactive antigenic forms (Balkwill et al., 1987, Lancet ii, 1229), probably constituted by monomeric TNF, or soluble-receptor- complexed TNF.

The level of TNF as bioactive form is usually determined in biological fluids by means of bioassays based on the cytotoxic effect displayed by TNF on sensible cells [e.g., L929 cells (Kramer et al., 1986, J. Immunol. Meth. 93, 210) and WEHI 164 cells (Espevik et al., 1986, J. Immunol. Heth. 95, 99)].

The presently used immunoassays make it possible total antigenic TNF to be determined -- which is potentially constituted by free oligomeric TNF, which is bioactive, and monomeric TNF, or soluble-receptor- Gomplexed THF, both biologically inactive. It was demonstrated that under pathologic conditions, TNF antigen can reach nanomolar levels (Balkwill et al., 1987, Lancet ii, 1229).

The biological determination of TNF on sensible cells shows a series of problems connected with execution difficulties: such tests require accurate specificity checks, special equipment for cellular cultures, keeping cellular lines in culture for long time periods, and a specialistic experience in cell cultivation, all the above not always being available. Furthermore, execution of assay normally requires from 3 to 4 days; it is decidedly long and laborious and does not make it possible the analysis of a large number of samples to be carried out. As a consequence, such a methodology is decidedly inadequate for routine analyses in clinic laboratories.

Furthermore, the execution of the biological assay requires that the sample is diluted in a suitable medium for cellular growth, normally containing, serum, or other factors produced by the same cells and therefore uncontrollable, which may alter the actual concentration of TNF as bioactive form in the analysed sample.

Therefore, the need results evident for having available a method to in vitro detect TNF, which discriminates the bioactive form (i.e., oligomeric, not inhibitor-complexed, TNF) from the bioinactive form

(monomeric TNF and/or soluble-receptor-complexed TNF), easy to be used also by non-specialized staff, and which does not require cell cultivations.

Therefore, an Object of the present invention is a method to detect TNF as bioactive form comprising the steps of:

- immobilizing on a substrate, a capturing reactant capable of only reacting with TNF molecules as oligomeric form, but not with TNF as complexed form with the receptor or TNF as monomeric form;

- incubating said capturing reactant with a sample;

- incubating said sample with a detector reactant; - washing;

- performing the measurement, by means of a reaction of said detector reactant.

Preferably, said detector reactant is capable of only reacting with molecules of TNF in oligomeric form, but not with TNF as complexed form with a receptor or TNF as monomeric form conjugated with a detection system; said capturing reactant has an affinity constant for oligomeric TNF higher than 10⁹ M_{- 1} and an affinity constant for receptor-complexed TNF Lower than 10⁵ M^-1.

According to a preferred form of practical embodiment, said capturing reactant and said detector reactant comprise the same reactant molecule and oligomeric TNF contains at least two binding sites for said reactant molecule.

Still according to the invention, said capturing reactant and said detector reactant comprise polyclonal or monoclonal anti-TNF antibodies, which preferably are neutralizers of TNF/receptor bond, still more preferably, monoclonal Hab-78 antibody (GB patent application No. 9028123.1; Barbanti P. et al. "2^nd Int. Congress on Trauma, Shock and Sepsis: Hechanisms and Therapeutic Approaches", 1991, Munich). According to an alternative form of practical embodiment, said capturing reactant and said detector reactant comprise TNF receptors or synthetic molecules or recombinant molecules.

According to preferred forms of practical embodiment, said substrate comprises titration microslabs or polystyrene tubes or poly-(vinyl chloride) tubes, and said detection system comprises enzymatic molecules of fluorescent compounds or chemoluminescent compounds or radioactive compounds or colloidal metals, or coloured latex particles. Said enzymatic molecules are preferably comprised within the group consisting of peroxidase, alkaline phosphatase, urease, beta-galactosidase and said colloidal metals are comprised within the group consisting of gold, silver, copper, platinum, copper hydroxide, aluminum hydroxide, vanadium oxide, manganese hydroxide.

Still according to the present invention, said measurement is carried out by causing said detector reactant to react with chromogenic or fluorogenic substrates, and reading the signal.

A further Object of the present invention is a kit to detect, from a sample, TNF as bioactive form, which kit comprises:

- a capturing reactant immobilized on a substrate, capable of only reacting with TNF molecules in oligomeric form, but not with receptor-complexed TNF or TNF in monomeric form;

- solutions to incubate said sample;

- a detector reactant capable of only reacting with TNF molecules in oligomeric form, but not with receotor- complexed TNF or TNF in monomeric form, conjugated with a detection system;

- a wash solution;

- a reference sample.

Preferably, said capturing reactant and said detector reactant comprise Mab-78 antibody, and said kit also comprises a detection system for total TNF.

The present invention is disclosed now by means of application examples thereof, in which reference is made to Figure 1, which shows the assay calibration line.

Example 1

Preparation of the solid phase for carrying out the assay for TNF in oligomeric form

The monoclonal antibody, endowed with TNF neutralizing activity, was immobilized on poly-(vinyl chloride) slabs, as follows: to each well of 96-well slabs, 50 μl of 50 mM sodium phosphate buffer pH 7.3, containing 0.15 M sodium chloride (PBS) and 10 μg/ml of monoclonal Mab-78 antibody (GB patent application No. 9028123.1; Barbanti P. et al. "2^nd Int. Congress on Trauma, Shock and Sepsis: Mechanisms and Therapeutic Approaches", 1991, Munich) were added. The slabs were then incubated at room temperature (r.t.) for 2 hours and were then blocked with a 3% calf serum albumine solution, for 2 hours at r.t.. The slabs prepared in that way can be stored some weeks at 4°C.

E x a m pl e 2

Preparation of detector reactants for carrying out the assay for TNF in oligomeric form

Monoclonal Mab-t8 antibody was biotinylated as follows: a solution containing 1 mg of Mab-78/ml, 600 μl, was mixed with 6 μ l of N-hydroxysuccinimidic ester of D-biotinyl-6-amino-caproic acid, 10 mg/ml in dimethyl-suIfoxide, and was incubated for a 4-hour time at r.t.. Then 60 μl of a 1 M lysine solution was added. The mixture was further incubated for 1 hout at r.t., and was then dialyzed overnight against PBS. The product was diluted to 3 ml with PBS, and was stored at -20ºC.

Example 3

Determination of TNF in oligomeric form The slabs sensibilized with Mab-8 antibody as disclosed in Example 1, were washed by filling the wells with PBS, and emptying them (3 times). The wells were then filled with the samples to be analysed, suitably diluted with PBS containing 1% BSA (PBS-B). The slab was incubated again overnight at 4°C and was washed with PBS containing 0.05% Tween 20 (PBS-T). At the end, to each well 50 μl of biotinylated Nab-Neu, diluted in the ratio of 1:200 with PBS-B containing 0.05% Tween 20 (PBS-BT) was added. After a 2-hour incubation at 37°C, the slabs were washed with PBS-T, were filled with 50 μl/well of a solution of streptavidine-peroxidase, available from the market (Sigma Chemical Co.), diluted in the ratio of 1:1000 with PBS-BT, and were then incubated again for 1 hour at 37°C. After being washed (8 times) with PBS-T, the slab was stained with a chromogenic solution containing ABTS [2,2'-azino-di-(3-ethyl-benzylthiazoline sulfonate) ], available from the market (from Kirkegaard and Perry Laboratories Inc.).

In Figure 1, the assay calibration line is reported, which was obtained with recombinant human TNF purchased from Esquire Chemie AG (Zurich, Switzerland). Example 4

Determination of specifity of assay for

oligomeric TNF

Oligomeric and monomeric TNF were prepared by dissociation of recombinant DNF with 0.1% Triton X-100, as described in literature [Smith R.A., Baglioni C. (1987), J. Biol. Chem. 262, 0951-09543 and were separated by HPLC gel-filtration on TSK3000SW chromatographic column (LKB-Pharmacia) (flow rate: 0.5 ml/minute). The chromatographic fractions corresponding to monomeric TNF resulted to be totally inactive at the assay for oligomeric TNF. The presence of monomeric TNF in such fractions was checked by means of a conventional assay performed with polyclonal ant-TNF antibodies as described in Example 6.

Example 5

Preparation of the solid phase for the assay for

total antigenic TNF

Polyclonal anti-TNF Mab-78 antibody was prepared by immunization of Balb/c mice and was purified according to known methods from literature (Bringman et al., 1987, Hybridoma 5, 489). Purified immunoglobulins were immobilized on poly-(vinyl chloride) slabs, as follows: to each well of 96-well slabs, 50 μl of 50 mM sodium phosphate buffer pH 7.3, containing 0.15 M sodium chloride (PBS) and 10 μg/ml of immunoglobulins were added. The slabs were then incubated 2 hours at r.t. and were then blocked with a 3% calf serum albumine solution, for 2 hours at r.t.. The slabs prepared in that way can be stored some weeks at 4°C. Example 6

Determination of total antigenic TNF The slabs sensibilized with mouse anti-TNF immunoglobulins as described in Example 5, were washed by filling the wells with PBS, and emptying them (3 times). The wells were then filled with the samples to be analysed, suitably diluted with PBS containing 1% B3A (PBS-B). The slab was incubated again, overnight, at 4°C, and was washed with PBS containing 0.05% Tween 20 (PBS-T). At the end, to each well 50 μl was added of IP 300 rabbit anti-TNF antiserum (Genzyme Corporation) diluted 1:2000 with PBS-B containing 0.05% Tween 20 (PBS-BT). After 2 hours of incubation at 37°C, the slabs were washed with PBS-T, were filled with 50 ul/well, of a solution, available from the market (Sigma Chemical Co.), of goat anti-rabbit- immunoglobuIin globulins conjugated with peroxidase, diluted 1:1000 with PBS-BT, and were incubated again, for 1 hour at 37°C. After being washed (8 times) with PBS-T, the slab was stained with a chromogenic solution containing ABTS [2,2'-azino-di-(3-ethyl-benzyIthiazoline sulfonate)], available from the market (from Kirkegaard and Perry Laboratories Inc.).

The present invention has been disclosed with specific reference to some of its preferred forms of practical embodiment, but it must be understood that variations and /or modifications aiming at reducing the necessary steps for the assay, and at increasing the sensibility thereof may be supplied by those skilled in the art without thereby departing from the scope of protection of the same invention.

Claims

C l a i m s

1. Method to detect TNF as bioactive form comprising the steps of:

- immobilizing, on a substrate, a capturing reactant capable of only reacting, with TNF molecules as oligomeric form, but not with TNF as complexed form with the receptor or TNF as monomeric form;

- incubating said capturing reactant with a sample;

- incubating said sample with a detector reactant;

- washing;

- performing the measurement, by means of a reaction of said detector reactant.

2. Method to detect TNF as bioactive form according to claim 1, characterized in that said detector reactant is capable of only reacting with molecules of TNF in oligomeric form, but not with receptor-complexed TNF or TNF in monomeric form conjugated with a detection system.

3. Method to detect TNF as bioactive form according to any of preceding claims, characterized in that said capturing reactant has an affinity constant for oligomeric TNF higher than 10⁹ M^{- 1} and an affinity constant for receptor-complexed TNF lower than 10⁵ M^-1.

4. Method to detect TNF as bioactive form according to any of preceding claims, characterized in that said capturing reactant and said detector reactant comprise the same reactant molecule and oligomeric TNF contains at least two binding sites for said reactant molecule.

5. Method to detect TNF as bioactive form according to any of preceding claims, characterized in that said capturing reactant and said detector reactant comprise polyclonal or monoclonal anti-TNF antibodies,

6. Method to detect TNF as bioactive form according to claim 5, characterized in that said anti- TNF antibodies are neutralizers of TNF/receptor bond.

7. Method to detect TNF as bioactive form according to claim 6, characterized in that said anti- TNF neutralizer antibody is Mab-78 antibody.

8. Method to detect TNF as bioactive form according to any of claims from 1 to 4, characterized in that said capturing reactant and said detector reactant comprise TNF receptors or synthetic molecules or recombinant molecules.

9. Method to detect TNF as bioactive form according to any of preceding claims, characterized in that said substrate comprises titration microslabs or polystyrene tubes or poly-(vinyl chloride) tubes and said detection system comprises enzymatic molecules of fluorescent compounds or chemo luminescent compounds or radioactive compounds or colloidal metals, or coloured latex particles.

10. Method to detect TNF as bioactive form according to claim 9, characterized in that said enzymatic molecules are comprised within the group consisting of peroxidase, alkaline phosphatase, urease, beta-galactosidase and said colloidal metals are comprised within the group consisting of gold, silver, copper, platinum, copper hydroxide, aluminum hydroxide, vanadium oxide, manganese hydroxide.

11. Method to detect TNF as bioactive form according to claim 10, characterized in that said measurement is carried out by causing said detector reactant to react with chromogenic or fluorogenic substrates, and reading the signal.

12. Kit to detect, from a sample, TNF as bioactive form, characterized in that said kit comprises:

- solutions to incubate said sample;

- a detector reactant capable of only reacting with TNF molecules in oligomeric form, but not with receptor- complexed TNF or TNF in monomeric form, conjugated with a detection system;

- a wash solution;

- a reference sample.

13. Kit to detect, from a sample, TNF as bioactive form according to claim 12, characterized in that said capturing reactant and said detector reactant comprise Mab-78 antibody, and said kit also comprises a detection system for total TNF.