WO1999054737A1

WO1999054737A1 - Immunodiagnostic method for granzymes

Info

Publication number: WO1999054737A1
Application number: PCT/NL1998/000344
Authority: WO
Inventors: Cornelis Erik Hack; Liesbeth Spaeny-Dekking
Original assignee: Stichting Sanquin Bloedvoorziening
Priority date: 1998-04-17
Filing date: 1998-06-16
Publication date: 1999-10-28
Also published as: AU7940898A

Abstract

A method for determining a granzyme in a liquid sample by subjecting the liquid sample to an immunoassay for detecting the presence or measuring the level of said granzyme. The immunoassay uses at least one monoclonal antibody capable of specifically binding said granzyme. Useful granzyme-specific monoclonal antibodies are obtained when immunization is done with non-denatured soluble granzyme. Included is a method for determining a complex of a granzyme and an inhibitor of the granzyme. The granzyme can be human granzyme A or B, and the complex can be granzyme A/antithrombine III complex. The invention also covers suitable granzyme-specific monoclonal antibodies and immunoassay kits for determining granzyme in a liquid sample. The presence or level of granzyme, or complex of granzyme and granzyme inhibitor, may be used as marker for degranulation of cytotoxic T cells or natural killer cells and, indirectly, for activation of the immune system.

Description

IMMUNODIAGNOSTIC METHOD FOR GRANZYMES

Field of the Invention

This invention is in the field of immunology/biochemistry and describes a method to detect degranulation of cytotoxic T lymfocytes (CTL) and natural killer (NK) cells in vivo and in vitro.

Background of the Invention

Activated cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells induce apoptosis in target cells by at least two different mechanisms (B.Lowin et al . , 1994, Nature 370 : 650; D.Kagi et al . , 1994, Nature 369 : 31) . One involves the Fas/APO-1 mediated pathway, the other the granule- exocytosis pathway...

The latter pathway comprises a number of proteins, including perforin, granzymes (Granule-associated enzymes) and T cell restricted intracellular antigen (TIA-1) , stored in granules . Upon contact of the cytotoxic cell with a target cell , the cytoplasmic granules are directed to the contact area whereafter the constituents of these are released into the intercellular space. In the presence of extracellular Ca-ions, perforin molecules polymerize into transmembranic channels inserting into the membrane of the target cell (D.Masson et al . , 1985, J Biol Chem 260 : 9069; E.R.Podack, 1985, Immunol Today 6 : 21) thereby allowing the other granule constituents to enter the target cell. These in turn trigger activation of the apoptosis execution machinery in the target cell .

Granzymes A, B as well as perforin are the main effector molecules (in humans) of the granule exocytosis pathway. Therefore, the presence of these proteins or expression of their genes in NK cells or CTL has been used to assess activation of these cells in pathological processes (J.A.Kummer et al . , 1993, J Immunol Meth 163 : 77). As described therein, the presence of GrA or GrB in activated CTL or NK cells is detected by immunofluorescence or immunohistochemistry using monoclonal antibodies against human GrA or GrB. These antibodies were raised against recombinant GrA and GrB proteins produced in bacteria. However, as this method requires the availability of specimens of the tissues involved, clinical applicability of the method is limited. Moreover, the monoclonal antibodies raised against recombinant granzymes, which are obtained in insoluble form in inclusion bodies in the bacteria, are of limited use as they can only bind denatured granzyme. In vi tro studies have shown that proteases of the cytotoxic granules may be released during degranulation of cytotoxic cells (H.Takayama et al . , 1987, J Immunol 138: 566) . The release of granzymes by cells was measured by an activity assay, in which the conversion of a chromogenic substrate by active granzymes is measured.

Until now the only available assays to measure granzymes released by cells are activity assays. The most widely used assay is the so-called BLT-assay for granzyme A, which measures the conversion of the substrate N-alpha-benzyloxy- carbonyl-L-lysine thiobenzyl ester (BLT) (M.S. Pasternack et al . , 1985, Nature 314: 743). These assays, however, are not suitable to measure granzymes in biological fluids since they have a rather low sensitivity detecting at the best a few nanograms of granzyme per ml. Moreover, they do not measure granzymes complexed to proteinase inhibitors . The latter is important since plasma and interstitial fluid contain high concentrations of various proteinase inhibitors, including some that can inhibit the activity of granzymes . 3

Summary of the Invention

We here describe the production and characterization of monoclonal antibodies against native human granzymes (i.e. soluble granzymes) , and the application of these antibodies in immunoassays, in particular quantitative and sensitive Enzyme-linked immunoabsorbent assays (ELISA's), for the measurement of granzymes, in particular human granzymes A and B (GrA and GrB) .

The ELISA's for the measurement of native granzyme A (GrA) and B (GrB) were found to specifically detect these proteases at picogram concentrations.

To discriminate between zymogen (released by dying CTL or NK cells) and active granzymes we also developed a sensitive and specific ELISA for granzyme A complexed to antithrombin III (GrA-ATIII complexes) .

Upon in vitro degranulation of CTL, increased levels of GrA and GrB were detected in culture supernantant . Low levels of GrA, GrB and GrA-ATIII were present in plasma of healthy individuals, whereas significantly higher levels circulated in patients with ongoing CTL/NK cell response, in particular patients suffering from viral infections (Epstein Barr Virus, Human Immune Deficiency Virus) .

The invention provides methods for measurement of soluble granzymes, and demonstrates in vivo and in vi tro that these proteases are released during degranulation by CTL and NK cells. Therefore, the presence of soluble granzymes is a diagnostic marker for the degranulation of cytotoxic cells, which in its turn is an indication for an activated immune system. Therefore, the present invention describes a method to assess degranulation of CTL and/or NK cells in vi tro and in vivo, i.e. the measurement of extracellular granzymes using specific and sensitive immunoassays, particularly ELISA's. Brief Description of the Drawings

FIGURE 1

ELISA for soluble human granzyme B. Dilutions of purified soluble GrB (-o-) or purified GrA (-•-) as a control, were tested. Data indicate mean (± SD) of 5 experiments.

FIGURE 2

ELISA for soluble human granzyme A. Dilutions of purified soluble GrA (-•-) as well as of GrB (-o-) as a" control were tested. Data indicate mean (± SD) obtained in 5 experiments.

FIGURE 3

ELISA for human granzyme A-ATIII complex. a. Dilutions of purified soluble GrA were added to plasma anticoagulated with heparin or EDTA and incubated for 30 sec at room temperature. Data indicate mean (+ SD) 5 experiments . b. Similar experiment as in a, except that GrA was incubated with purified ATIII (0.5 micromol) in the presence or absence of heparin instead of plasma.

FIGURE 4

Specificity of the GrA mAb. Wells of ELISA plates were coated with 2 μg/ml GrA, GrB, human neutrophil elastase (HNE) , trypsin (Tryp) or Chymotrypsin (Chymotryp) and incubated with biotinylated mAb. Binding of the latter was detected with streptavidin-peroxidase. The figure shows the results obtained with mAb GA28 and GA34.

FIGURE 5

Release of soluble granzymes during degranulation. Levels of GrA (A) or GrB (B) were measured at various times after induction of degranulation of the human CTL cell line JS 136 by stimulation with anti-CD2 and PMA (open bars) . Levels of granzymes released in the medium by cells not stimulated with anti-CD2 or PMA, are represented by hatched bars.

FIGURE 6

Levels of soluble GrA (A) and GrB (B) in plasma (pi) , serum (se) or synovial fluid (sf) samples of healthy individuals (healthy) , patients with rheumatoid arthritis (RA) , or persons infected with EBV or HIV-1. The lines represent the median level for each group.

FIGURE 7

Correlation between levels of soluble GrA and soluble GrB detected in blood samples obtained from patients suffering from EBV (n=14) or HIV-1 (n=25) . (Spearman r=Q .6678) .

FIGURE 8

Course of GrA levels (-•-) in relation to IgM anti-VCA (EBV) or p24 antigen levels (-■-) in patients suffering from EBV (A,B,C,D) or HIV-1 (E) . The days after serum conversion (EBV) or days after onset of clinical symptoms (HIV-1) are given at the X-axis; left Y-axis gives GrA levels (pg/ml) , right Y-axis gives IgM anti-VCA (absorption units; panels A- D) or p24 antigen (pg/ml; panel E) .

FIGURE 9

Levels of GrA-ATIII complexes in plasma samples from normal healthy volunteers in comparison to those of GrA and GrB (A) , and in serial plasma samples from patients undergoing graft versus host disease (GVHD) or from patients with cancer treated with interleukin-2 (tumor) . Levels in healthy controls (controls) are shown for comparison. Detailed Description of the Invention

The invention will be more fully understood after a consideration of the following description of the invention. The invention provides in a first aspect a method for determining a granzyme in a liquid sample, comprising subjecting said liquid sample to an immunoassay for detecting the presence or measuring the level of said granzyme, which immunoassay uses a monoclonal antibody capable of specifically binding said granzyme. Granzymes are serine proteases stored in the cytotoxic granules of CTL and NK cells. The invention is not limited to the detection of human granzymes, but may more widely be applied in the detection of granzymes of animals in general, in particular vertebrates, especially mammals. In some species, such as mice, many different granzymes are known

(D.E. Jenne et al . , 1988, Immunol Rev 103: 53). With respect to human granzymes, so far only granzymes A and B are well characterized, but other granzymes may exist.

The method of the invention requires the availability of monoclonal antibodies which are capable of binding granzymes in the dissolved state, i.e. native or soluble granzymes. The phrase "soluble granzymes" encompasses not only granzymes which are present in solution in a free form, i.e. not bound to other substances, but also encompasses granzymes which are present in solution bound to other substances, such as proteoglycans to which granzymes are bound within the cytotoxic granules in CTL and NK cells (D. Masson et al . , 1990, Biochemistry 29: 11229), and dissolved complexes of a granzyme and an inhibitor of the granzyme . To produce such monoclonal antibodies capable of binding granzymes in solution, in a dissolved state, it is necessary that the granzyme used for immunization is in non-denatured state. Non-denatured granzymes may be obtained by isolation from a natural source, such as the cytotoxic granules in CTL and NK cells. Alternatively, they may be obtained by such a recombinant production method which yields non-denatured granzyme. For example, non-denatured granzymes may be obtained by granzyme expression in eukaryotic host cells (J.A. Kummer et al . , 1996, J Biol Chem 271: 9281) . Another possibility is to produce recombinant granzyme in bacteria in the form of a fusion protein with -a carrier protein, e.g. in Escherichia coli bacteria as a fusion protein with thioredoxin. To facilitate proper cleavage of the fusion protein, such as to liberate the mature granzyme, it is preferable^to design the fusion protein such as to contain a suitable cleavage site between the granzyme and the carrier protein, for example an enterokinase cleavage site. Alternatively, immunization may be carried out with the whole fusion protein. Monoclonal antibodies obtained by using a non-denatured granzyme may be selected for desirable properties. It has been found that some antibodies specifically bind granzymes which are not bound to proteoglycans . In some applications it may be desirable to use such antibodies which only bind dissolved granzymes which are free from proteoglycans . Other antibodies were found to bind soluble granzymes, regardless whether they are bound to proteoglycans or not .

The phrase "which immunoassay uses a monoclonal antibody capable of specifically binding said granzyme" means that at least one granzyme-specific monoclonal antibody capable of binding to soluble granzyme is used in the immunoassay. The number of granzyme-specific monoclonal antibodies used in the immunoassay depends on the immunoassay method involved (sandwich immunoassay, competitive immunoassay, etc.) and the substance to be determined (a granzyme per se, or a complex of a granzyme and an inhibitor of said granzyme) .

Certain embodiments of this invention apply a monoclonal antibody which specifically binds a (dissolved) complex of a granzyme and an inhibitor of the granzyme. As a particular example thereof, complexes of GrA and its inhibitor ATIII 8

may be mentioned. To obtain monoclonal antibodies which specifically bind such a complex, immunization may be done with the complex rather than the granzyme per se .

To determine a complex of a granzyme and an inhibitor of said granzyme, it is also possible, however, and preferred, to use a monoclonal antibody specifically binding the said granzyme in combination with a monoclonal antibody which specifically binds the inhibitor. One of these antibodies would be used to immobilize (bind to a solid support) the analyte and the other to attach a label to the"analyte.

The type of immunoassay used for determining the presence or level of a granzyme or granzyme-inhibitor complex (which are further referred to herein as the analyte) in a liquid sample is not limited to a particular methodology. The immunoassay may be of the sandwich immunoassay type, the competitive immunoassay type, or any other suitable type. Monoclonal antibody specifically binding the granzyme or granzyme-inhibitor complex may be used for immobilizing the analyte, for labeling the analyte, or both. Immobilization and labeling reactions may be carried out simultaneously, or one after the other, in either order. Any suitable label may be used, such as well known enzyme labels, fluorescent or (chemi) luminescent labels, dyes, radiolabels, biotin, etc. The label may be attached to a monoclonal antibody which binds the analyte (i.e. a monoclonal antibody specifically binding a granzyme, granzyme-inhibitor complex, or granzyme inhibitor), or to an antibody (e.g. an anti-mouse Ig antibody) which binds to the analyte-binding monoclonal antibody which serves to mediate labeling of the immobilized analyte. Preferably, however, the method is a sandwich ELISA, in which a monoclonal antibody specifically binding a granzyme is used as analyte-capturing antibody to bind analyte to a solid surface and another monoclonal antibody specifically binding the same granzyme or an inhibitor of the same granzyme is used to label, or mediate labeling of, the solid surface-bound analyte. If both monoclonal antibodies used bind the same granzyme, it is preferred that they bind to different non-overlapping epitopes. If one of the monoclonal antibodies used binds a granzyme and the other an inhibitor of the granzyme, it is also possible to use the antibody to the inhibitor for immobilizing the analyte and the granzyme antibody for labeling.

The liquid sample may be derived from any biological or body fluid, including, e.g., blood, blood plasma, lymph, urine, saliva, sputum, interstitial fluid, perspiration, nasal discharge, semen, etc. The words "derived from" are meant to cover the possibility of merely taking a portion of a body fluid, and the possibility of preparing a sample by mixing a portion of a body fluid with suitable components, such as buffers, diluents, solvents, surfactants, chelating or metal sequestering agents, stabilizers, enzyme inhibitors, etc.

According to the invention, the level of analyte in the sample may be used as a marker for the degranulation of CTL and/or NK cells and, indirectly, as a marker for activation of the immune system. Thus, the invention can be used for many purposes, e.g. to determine the effect of vaccination, to determine the presence of an infection, such as a viral infection, the occurrence of autoimmune reactions, the occurrence of cancer, etc. It may be required to carry out further analysis to determine the cause of the activation of the immune system. In many cases, the invention only allows to determine that the immune system has been activated, and does not allow to determine the cause of said activation. In another aspect, the subject invention provides a granzyme-specific monoclonal antibody capable of binding the granzyme in non-denatured state. Preferred embodiments are human GrA-specific monoclonal antibodies and human GrB- specific monoclonal antibodies, especially those which are capable of binding the granzyme when present in a soluble 10

complex with an inhibitor of said granzyme.

In yet another aspect the subject invention provides an immunoassay kit for determining a granzyme in a liquid sample, which kit comprises a monoclonal antibody capable of specifically binding said granzyme. Preferred embodiments are a sandwich immunoassay kit for determining granzyme, in which catcher antibody and labeling antibody are different granzyme-specific monoclonal antibodies; and a sandwich immunoassay kit for determining a complex of granzyme and granzyme inhibitor, in which a granzyme-specific monoclonal antibody is used as catcher antibody and a granzyme inhibitor-specific monoclonal antibody is used as labeling antibody, or vice versa. Most preferably, the immunoassay kit is a kit for a sandwich ELISA. . Yet another aspect of the subject invention is a method for diagnosing activation of the immune system in an animal, comprising preparing a liquid sample of a body fluid and subjecting said liquid sample to an immunoassay to therein detect the presence or measure the level of a granzyme, wherein said immunoassay uses a monoclonal antibody specifically binding said granzyme. In certain preferrred embodiments, said immunoassay detects the presence or measures the level of a complex of said granzyme and an inhibitor of said granzyme . Several patents/patents applications and scientific articles are referred to below that discuss various aspects of the materials and methods used to realize the invention. It is intended that all of the references be entirely incorporated by reference . To more clearly define the present invention, it will be described in two sections. The first section contains a description of the development of the various ELISAs to measure granzymes. The second section describes application of these ELISAs for in vivo and in vitro diagnostics. Both the ELISAs as well as their applications are intended to 11

come into the scope of the invention.

EXAMPLES

I. ELISA's for soluble granzymes

1.1. MATERIAL AND METHODS

Proteins Granzyme B was isolated from the human leukaemia T cell line YT-Indy by HPLC (9) . Granzyme A (GrA) was purified to homogeneity from IL-2 -activated lymphocytes (W.L. Hanna et al . , 1993. Peptides 4: 398).

Recombinant human granzyme A was also used for immunization of mice. This recombinant GrA was made as a fusion protein in Escherichia coli with thioredoxin using an appropriate thioredoxin fusion vector (E.R. LaVallie et al . , 1993, Biotechnology 11: 187-193) as can be commercially obtained from Invitrogen, Leek, The Netherlands. The fusion protein was constructed to consist of the thioredoxin sequence, a linker peptide containing an enterokinase cleavage site, the sequence of mature GrA (without pre- or pro-sequence) , and a His-tag. The recombinant protein was purified by Zinc-ion chelate chromatography using commercially available resin (Boehringer Mannheim, Ingelheim, Germany) .

Human neutrophil elastase (Elastin Products. Co. Inc., Owensville, MO); trypsin (Sigma Corp., St. Louis, MO); chymotrypsin (Sigma) ; and biotinylated goat-anti-mouse polyclonal antibodies (CLB, Amsterdam, the Netherlands) were obtained from commercial sources.

Production and purification of monoclonal antibodies (mAb) against native GrA and B Balb/b mice were immunized subcutaneously with 25 μg of 12

either native GrA or B suspended in complete Freund's adjuvant, followed by 3 to 6 subsequent injections of 25 μg of the protein suspended in incomplete Freund's adjuvant at 2 weeks-intervals. At the time the animals produced high antibody titers, fusion of the spleen and lymph node cells with mouse myeloma Sp2/0-Ag 14 cells was performed according to standard procedures (C.E.Hack et al . , 1988, J Immunol 141: 1602-1609) . Conditioned medium from the hybridomas was screened for the presence of anti-GrA or B mAb, using ratanti-mouse Ig-Sepharose suspensions together with ¹²⁵I -radio- labeled native GrA or B, similarly as described previously for anti-C3 mAb (C.E. Hack et al . , 1988, J Immunol 141: 1602) . The supernatant of antibody producing hybridomas was concentrated by ammoniumsulfate precipitation (50% w/v) followed by dialysis against phosphate buffered saline, pH 7.4 (PBS) . Thereafter, mAb were purified by protein G- affinity-chromatography (Pharmacia Fine Chemicals, Uppsala, Sweden) according to instructions of the manufacturer. Subclasses of mAb were determined by an isotyping dipstick method (Innogenetics, Antwerp, Belgium) . All mAb were of the IgGl kappa subclass .

Production and purification of a mAb against human antithrombin III (ATIII) Using a similar procedure as described for GrA and GrB, mAbs against human ATIII were prepared in Balb/b mice. One of the antibodies obtained, mAb ATIII -0 was used in the experiments. It was purified from conditioned culture medium using protein G-affinity-chromatography as described above.

Competition radioimmunoassay (RIA) for the initial characterization of the anti-granzyme mAb

Limiting amounts of Sepharose-coupled mAb were added to ¹²⁵I-radiolabeled GrA or B (2 to 4 ng) that had been preincubated for one hour at room temperature with 2 μg of a 13

second mAb. The mixture was then incubated by head over head rotation for at least 4 hours at room temperature. Next, the Sepharose beads were washed with PBS-0.05% (w/v) Tween-20 and the amount of bound ¹²⁵I-radiolabeled GrA or B was measured. A mAb was considered to bind to similar or overlapping epitopes when interaction of the second antibody with ¹²⁵I-labeled GrA or B was inhibited by more than 50%.

Biotinylation of antibodies Purified antibodies were biotinylated using" long chain biotinyl-N-hydroxysuccinimide ester solfonacid (Pierce Chemical Co., Rockford, IL) according to instructions of the manufacturer .

Granzyme B ELISA

Purified mAb GB11 was incubated at of 2 μg/ml in 0.1 M sodium carbonate/bicarbonate buffer, pH 9.6, for 16 hours at 4°C in microtiterplates (Nunc Maxisorb Immunoplate ; 100 μl/ well) . The plates were then washed with PBS-0.02% (w/v) Tween-20. An identical washing procedure was performed after each incubation step which consisted of 100 μl, except for the blocking step (150 μl) . After coating, residual binding sites were blocked by a 45 minutes-incubation with PBS 2% (v/v) cow milk. Samples and standards (purified native GrB at different concentrations) were pretreated with 40 μg/ml hyaluronidase (Sigma) for 30 minutes at room temperature in PBS-0.02% Tween-20. Samples were then appropriately diluted in High Performance ELISA buffer (CLB) and incubated for 1 hour. Next, the plates were incubated with an excess of biotinylated GB10 mAb (0.5 μg/ml) together with 1% (v/v) normal mouse serum for 1 hour. In the next step, the plates were incubated for 30 minutes with streptavidin-polymerized horseradish peroxidase (CLB) , whereafter bound peroxidase was visualized by incubation with a solution of 100 μg/ml 3 , 3 ' , 5 , 5 ' -tetramethyl-benzidine (Merck, Darmstadt , Germany) 14

and 0.003% (v/v) H₂0₂ in 0.11 M sodium acetate buffer, pH 5.5. The reaction was stopped by addition of an equal volume of 2 M H₂S0₄ to the wells. Finally, the absorbance at 450 nm was read on a Titertek Multiscan plate reader (Labsystems, Helsinki, Finland) .

Granzyme A ELISA

The GrA ELISA was essentially performed as described for the GrB except that GA28 (2 μg/ml) was the coating mAb and biotinylated GA34 (0:5 μg/ml) was used to det ct bound GrA.

ELISA for granzyme A-antithrombin III complexen.

GrA-ATIII complexen were determined with an ELISA as described for GrA antigen (see above) except that a different biotinylated was used, i.e. ATIII-0. The procedure was otherwise similar as that described above for GrA.

1.2. RESULTS

ELISA for soluble GrB

Balb/c mice were immunized with native human GrB isolated from the human leukaemia cell line YT-Indy. From the fusion experiments 3 mAb of the IgGl kappa subclass were obtained that bound ¹²⁵I-labelled GrB in solution: GB10, GB11 and GB12. Purified mAb were either coupled to Sepharose beads or biotinylated. As determined by radioimmunoassay competition experiments and sandwich ELISA experiments, mAb GB11 and GB12 recognized the same or overlapping epitopes, whereas mAb GB10 appeared to bind to a different epitope. Pilot experiments revealed that the optimal combination was GB11 as the coating antibody and biotinylated GB10 as the detecting antibody. Using this combination, a highly reproducible ELISA was developed in which soluble GrB was detected at a range of 3 ng to 4 pg per ml (Figure 1) . GrA did not cross-react in this system (Figure 1) . 15

ELISA for soluble GrA

A similar procedure as described for GrB mAb was used to raise mAb against purified human GrA. Twenty-six antibodies (GA10-GA35) , all of the IgGl kappa subclass, were obtained. After purification, coupling to Sepharose beads and biotinylation, mAb were characterized by radioimmunoassay competition experiments and sandwich ELISAs. Five groups of mAb were identified, each group recognizing one or overlapping epitopes (results not shown) . Testing combinations of mAb from the different groups ^'revealed that mAb GA28 as a catching and GA34 as a detecting antibody provided the most sensitive assay for soluble GrA. With this highly reproducible ELISA GrA was detected at a range from 16 pg to 18 ng per ml (Figure 2) . As can be seen from Figure 2 GrB did not cross-react in this system.

ELISA for GrA-ATIII complexes

The ELISA for GrA antigen does not discriminate between inactive pro-GrA and active GrA. As active GrA in vivo will be bound to inhibitors, we also developed an ELISA for GrA- antithrombin III (GrA-ATIII) complexes, since these complexes will only be formed with active GrA and not pro- GrA. By substitution of biotinylated mAb GA34 for biotinylated mAb ATIII-0 an ELISA for GrA-ATIII complexes was obtained. As is shown in Figure 3, this ELISA did not react with purified GrA (or GrB) , but only when GrA was complexed to ATIII either present in heparin-plasma or purified.

Specificity of the mAb used in the ELISA's

To ascertain that the mAb used in the ELISAs were specific for either GrA or GrB and did not react with other serine proteases that are homologous to granzymes, we did additional ELISA experiments. Wells were coated with neutrophil elastase, trypsin or chymotrypsin, and incubated 16

with the mAb GA28, GA34, GB10 and GB11. GrA mAbs only bound to GrA and not to GrB, elastase, trypsin and chymotrypsin (Figure 4) . MAb GB10 and GB11 only bound to GrB, and not to elastase, trypsin and chymotrypsin (not shown) .

Thus, these results show that we succeeded in developing sensitive and specific ELISA's for soluble granzymes, and also in developing an ELISA for GrA-ATIII complexes, which latter assay only detects active GrA (complexed with ATIII) , and not pro-GrA.

II. APPLICATIONS OF THE ELISAS

II.l. PATIENTS, MATERIAL AND METHODS

Degranulation experiments

JS-136 cells were harvested after 6 days of stimulation with JY cells (12) . Cells were washed in Iscove's supplemented with 0.02% (w/v) bovine serum albumin (BSA) and resuspended at a concentration of 1 x 10⁶ cells/ml in

Iscoves modified Dulbecco's medium (IMDM)-0.02% (w/v) BSA, containing three mAbs directed against CD2 (4B2, 6G4 and Hie 27; CLB) each at 1 μg/ml and phorbol myristic acetate (PMA) at a final concentration of 1 ng/ml . Control cells (not stimulated with anti-CD2 and PMA) were resuspended in IMDM- 0.02% BSA. Cell suspensions (100 μl/well) were put into 96- well round bottom microtiter plates (Greiner, Frickenhausen, Germany), and incubated for varying times. Aliquots of the supernatant were collected and centrifuged for 8 min at 1500 rpm to remove intact cells. Medium was stored at -20°C until testing.

Biological samples

Plasma samples of healthy volunteers were collected by venipuncture using tubes (Venoject, Terumo Europe N.V. , 17

Leuven, Belgium) containing soybean trypsin inhibitor (0.1 mg/ml) , 10 mM EDTA and 20 mM benzamidine . Plasma samples from patients with rheumatoid arthritis (RA; n=10) , obtained at the Daniel den Hoed Clinic in Rotterdam, were collected in tubes containing 10 mM EDTA and 0.05% (w/v)

Polybrene (13) . Simultaneously, synovial fluid (SF) samples were taken from involved knees in a plastic syringe and immediately transferred into siliconized Vacutainer tube (Becton Dickinson, Plymouth, England) containing 10 mM EDTA and 0.05% Polybrene. All patients fulfilled tKe revised American Rheumatism Association criteria for definite RA (F.C.Arnett et al . , 1988, Arthr Rheum 31: 315). Similar procedures were used to obtain serial plasma samples from patients with malignant melanoma or renal cell carcinoma during treatment with recombinant interleukin-2 (Daniel den Hoed Clinic) or from patients undergoing graft versus host disease following allogenic bone marrow transplantation (Academic Hospital Leiden) .

Blood from HIV-1 infected patients were collected in tubes (Venoject) containing 15 USP Heparin. Diagnoses were based on the presence of antibodies against HIV-1 p24 protein in plasma of the patients.

All blood samples were centrifuged for 10 minutes at 1300 g, whereafter supernatants were centrifuged again for 5 minutes. The final supernatants were stored in small aliquots at -70°C until use.

In addition, serum samples were obtained from 14 patients in various stages of acute EBV infection as determined by immunoblotting for EBV-Early Antigen_D (EA_r,) , viral capsid antigen (VCA) or Epstein-Barr nuclear antigen (EBNA) (J.M. Middeldorp et al . , 1988, J Virol Meth 21: 133). EBV-IgG and IgM levels were further defined by peptide ELISA (WJM Van Grunsven et al . , 1994, J. Infect . Dis . 170: 13). 18

Statistical analysis

Data are presented as median value and range unless indicated otherwise. For statistical analysis, values below the detection limit were set at 1 pg/ml. The significance of the differences between subject groups was assessed using the nonparametric Wilcoxon-Mann-Whitney test. A double-sided p value of less than 0.05 was considered to indicate a significant difference.

1.2. RESULTS

Degranulation experiments

Degranulation experiments were performed and the medium of the cells was tested for the presence of GrA and GrB. After 6 days of stimulation of the human CTL clone JS 136, degranulation was induced by resuspending the cells in BSA containing IMDM medium supplemented with anti-CD2 mAbs together with PMA. At various times of incubation, samples from the medium were taken and tested for the presence of soluble granzymes. Already after 30 minutes-incubation detectable levels of both GrA and GrB were found (Figure 5) . Soluble GrA and GrB levels continued to increase until 5 hours of incubation.

Detection of soluble granzyme A and B in plasma of healthy- individuals

Granzymes are expressed by peripheral blood mononuclear cells of healthy individuals (G. Sunder-PIassman et al . , 1990, Kidney Int 37: 1350; C.Berthou, et al . , 1995, Blood 86: 3500) . Therefore, soluble granzymes may be present in normal blood. Indeed, most plasma samples from healthy individuals contained detectable levels of soluble granzymes (Figure 6; Table 1) . 19

Table 1: Levels of soluble granzymes in healthy individuals, or patients with RA, EBV or HIV-1 infections

GrA GrB qrOUD n median ranσe E n median ranαe E

(pg/ml) (pg/ml)

norm. 54 33.5 1 - 121 _ 52 11.5 1 - 113 _

EBV^* 14 270.5 1 - 5500 0.0001 14 60.0 1 - 4000 0.393

HIV-1" 34 113.5 1 - 1683 0.0001 25 20.0 1 - 52 0.014

RA sf^* 10 5600 73 - 69554 - 10 3183.0 75 - 26538 -

RA pi^* 10 1 1 - 1139 0.454 10 20.0 1 - 1918 0.055

^*RA= rheumatoid arthritis; pl= plasma;- sf= synovial fluid; EBV= Epstein- Barr Virus; HIV-1= Human Immune Deficiency Virus, p indicates two sided p-value for the difference between the indicated group and normal individuals ( MW-test) .

The median level was 33.5 pg per ml (range 1-121) for soluble GrA, and 11.5 pg per ml (1-130) for GrB. These values were obtained from plasma samples prepared from blood collected in soybean trypsin inhibitor (100 μg/ml) , EDTA (10 mM) and benzamidine (10 mM) . Plasma samples prepared from blood collected in citrate (10.5 mM) , EDTA (10 mM) or heparin (15 USP) yielded similar results, as did serum samples obtained from the volunteers (data not shown) .

Detection of soluble grazizyme A and B in plasma and synovial fluid of RA patients

Immunohistochemical and biochemical studies have shown local production of GrA, GrB and PFN in inflamed rheumatoid joints (L.H. Young, et al. , 1992, Am J Pathol 140: 1261; P.P.Tak et al., 1994, Arthr Rheum 37: 1735; U.Mϋller- adner et al., 1995, Arthr Rheum 38: 477) . Therefore, plasma and 20

synovial fluid samples of 10 RA patients fulfilling the ARA criteria were tested for the presence of soluble granzymes . Both soluble GrA and GrB were detected in the synovial fluid samples of the RA patients. In most cases the values markedly exceeded plasma levels detected in normal volunteers (Figure 6; Table 1). In contrast, plasma levels of GrA were similar to controls while GrB tended to be somewhat higher. Since rheumatoid factors present in the plasma and SF could lead to spurious results, we performed additional specificity control studies. First ,'^Λ e did not observe absorption values significantly above background for ELISAs where the GrA coating antibody was used combined with the biotinylated GrB mAb, and vice versa . Second, rheumatoid factor levels did not correlate with either GrA or B levels. Third, addition of purified rheumatoid factors to the ELISAs did not alter the levels of granzymes detected. To ascertain whether soluble GrA or GrB were produced locally in the joints, the ratio between SF and plasma samples was calculated for each patient. This ratio exceeded 1.0 in all patients with a range from 5.66 to 69554 for soluble GrA and from 4.4 to 3775 for GrB. Thus, consistent with the finding that RA synovium contains activated CTLs, soluble GrA and GrB are produced locally in the joints of patients with rheumatoid arthritis.

Circulating levels of soluble GrA and GrB in patients with EBV or HIV-1 infections

Cytotoxic T cells and NK cells are involved in the immune response against a variety of viral infections (R.V.Blanden, 1974, Transplant Rev 19: 56; Y.F.Bukowski et al . , 1985, Eur J Immunol 161: 40; T.J.Braciale et al . , 1986, J^" Σmmuno2 137: 995) . Therefore, we tested blood samples from patients with symptomatic EBV or HIV-1 infection, for the presence of soluble GrA and GrB. Serum samples of 14 patients at various 21

stages of EBV infection (Table 2) contained elevated levels of both soluble GrA (median: 156; range: 1-5500) and soluble GrB (60; 1-4000) . Levels of GrA were significantly higher than those in the healthy control group, whereas those of GrB tended to be higher (Figure 6; Table 1) .

Table 2 : Soluble granzyme levels in patients in different stages of the Epstein-barr virus infection as defined by immunoblot analysis of EBV specific antibody responses.

sample EBV-IgG EBV-IgM phase of inf GrA pg/ml GrB pg/ml

1 + (Z) ± (EA_c-pl38) unusual IM 1 1

2 ++ (Ea_D+Z) ++ (EA_o+Z+VCA) acute IM 168 35

3 + (EA,,) ± (EA-.) early IM 243.5 1

4 ± (Z) + (EAu+Z) early IM 359.5 115

5 ± (Z) + (EAp) early IM 623 134

6 ++ (EA_D+Z) ++ (EA_D+Z) acute IM 485 140

7 +++ (EA„+Z) +++ (EA_D+Z) acute IM 864.5 210

8 + (EA_D) ++ (EA_D+Z) acute IM 296.5 85

9 +++ (EAπ+Z) ++ (EA-,) acute IM 5500 4000

10 ++ (EA_D+Z) + (EA_D+VCA) acute IM 92 1

11 + (VCA) Convalescent 68 1

12 + VCA Convalescent 77 1

13 + (EAo+Z) + (EA„+Z) acute IM 144 1

14 ++ (EA ++ (EA_D+Z) acute IM 3000 1700

IM= Infectious Mononucleosis, Z= Zebra (BZLF protein, EAπ= Early Diffuse Antigen (BMRFi+ BA F₂) , VCA= Viral Capsid Antigen (BFRF₃+ BdRFj)

Elevated levels of granzymes were also detectable in plasma samples collected from a group of HIV-1 infected patients during the asymptomatic phase: GrA (114; 1-1683) 22

and GrB (20; 1-74) . Analysis of the results obtained with HIV-1 or EBV infected persons revealed a significant correlation between soluble GrA and GrB (Figure 7) .

Longitudinal studies in EBV or HIV-1 infected persons

To establish whether levels of soluble granzymes fluctuated with the stage of viral infection, levels were measured in plasma samples taken during the acute and convalescent phases . Longitudinal plasma samples from four EBV and one HIV-1 infected patients were available for analysis (Figure 8) . Although both GrA and GrB levels were increased during the acute phase of the infection, levels of GrA were consistently higher than those of GrB (not shown) . Moreover, increases of GrA were associated with markers of early viral infection, namely IgM anti-VCA in EBV infection and p24 antigen in HIV-1 infection (Figure 8) . In the period following the acute infection the granzyme levels decreased to the (high) normal range. Thus, during viral infection the levels of both GrA and GrB increased during the acute phase and then decline during resolution.

Levels of GrA-ATIII complexes

To discriminate between the measurement of pro-GrA (which may originate from constitutive release of GrA by CTL or NK cells) and active GrA (which likely reflects degranulation) , we also developed an ELISA that specifically detects active GrA. As active GrA in vivo will form complexes with its major inhibitor ATIII, this ELISA was based on the detection of these complexes. Circulating levels of GrA-ATIII complexes in healthy volunteers are within the same range as those of GrA (or GrB) as is shown in Figure 9A. Patients receiving immunotherapy with interleukin-2, which therapy aims at stimulation of CTL or NK cells to improve their anti-cancer effects, had higher levels of circulating GrA- ATIII complexes than normal volunteers, as did patients 23

undergoing a graft versus host disease as a consequence of the attack of the host cells by the cytolytic T cells and NK cells in the transplantate (Figure 9B) .

Together these results indicate that soluble granzymes circulate at detectable levels in healthy volunteers and that these levels may be substantially higher upon stimulation of CTL or NK cells in patients with viral infections, or in patients with transplant rejection or cytolytic attack of cancer.

Claims

24Claims

1. A method for determining a granzyme in a liquid sample, comprising subjecting said liquid sample to an immunoassay for detecting the presence or measuring the level of said granzyme, which immunoassay uses a monoclonal antibody capable of specifically binding said granzyme.

2. A method according to claim 1, wherein said immunoassay is a sandwich immunoassay for determining granzyme, in which catcher antibody and labeling antibody are different granzyme-specific monoclonal antibodies.

3. A method according to claim 1 or claim 2 , wherein said granzyme is human granzyme A (GrA) or human granzyme B (GrB) .

4. A method according- to claim 1, wherein said immunoassay is a sandwich immunoassay for determining a complex of granzyme and granzyme inhibitor, in which a granzyme- specific monoclonal antibody is used as catcher antibody and a granzyme inhibitor-specific monoclonal antibody is used as labeling antibody, or vice versa.

5. A method according to claim 4 , wherein said complex of granzyme and granzyme inhibitor is a complex of human granzyme A and antithrombine III (GrA-ATIII) .

6. A method according to any one of claims 1 to 5 , wherein the immunoassay is a sandwich Enzyme-Linked Immuno Sorbent Assay (ELISA) .

7. A method according to any one of claims 1 to 6 , wherein said liquid sample is derived from a body fluid.

8. A method according to claim 7 , wherein the level of said granzyme, or the level of said complex of granzyme and granzyme inhibitor, in the sample is used as a marker for degranulation of CTL and/or NK cells.

9. A method according to claim 7, wherein the level of said granzyme, or the level of said complex of granzyme and 25

granzyme inhibitor, in the sample is used as a marker for activation of the immune system.

10. A granzyme-specific monoclonal antibody capable of binding the granzyme in non-denatured state.

11. A monoclonal antibody according to claim 10, which is capable of binding soluble human granzyme A.

12. A monoclonal antibody according to claim 10, which is capable of binding soluble human granzyme B.

13. A monoclonal antibody according to any one of claims 10 to 12, which is capable of binding t╬▓e granzyme in a soluble complex with an inhibitor of the granzyme.

14. A monoclonal antibody according to claim 13, which is capable of binding a dissolved complex of human granzyme A and antithrombin III.

15. A monoclonal antibody capable of specifically binding a soluble complex of a granzyme and an inhibitor of said granzyme.

16. An immunoassay kit for determining a granzyme in a liquid sample, which kit comprises a monoclonal antibody capable of specifically binding said granzyme.

17. An immunoassay kit according to claim 16, which is a sandwich immunoassay kit for determining granzyme, in which catcher antibody and labeling antibody are different granzyme-specific monoclonal antibodies.

18. An immunoassay kit according to claim 16, which is a sandwich immunoassay kit for determining a complex of granzyme and granzyme inhibitor, in which a granzyme- specific monoclonal antibody is used as catcher antibody and a granzyme inhibitor-specific monoclonal antibody is used as labeling antibody, or vice versa.

19. An immunoassay kit according to any one of claims 16 to 18, which is a kit for a sandwich ELISA.

20. A method for diagnosing activation of the immune system in an animal, comprising preparing a liquid sample of a body fluid and subjecting said liquid sample to an immuno- 26

assay to therein detect the presence or measure the level of a granzyme, wherein said immunoassay uses a monoclonal antibody specifically binding said granzyme.

21. A method according to claim 20, wherein said immunoassay detects the presence or measures the level of a complex of said granzyme and an inhibitor of said granzyme .