CA2108533C - Monoclonal antibodies directed against ndp-kinase - Google Patents

Abstract

A monoclonal antibody against cytosolic nucleoside diphosphate kinase (NDP-kinase: EC 2.7.4.6) from HL60 cells has been produced. The monoclonal antibody solely recognizes the active oligomeric form of NDP-kinase and does not inhibit NDP-kinase activity. Reduction and alkylation of NDP-kinase oligomer performed under denaturing conditions fully destroyed the antigenic site recognized by the monoclonal antibody.
Western blot analysis after non-denaturing polyacrylamide gel electrophoresis (PAGE) revealed that a single protein oligomer is recognized by the MAb. The protein band showed the same electrophoretic mobility of pure cytosolic NDP-kinase detected by a specific NDP-kinase enzymic staining assay performed after nondenaturing PAGE. Collectively, the results suggest that the MAb recognizes the native form of NDP-kinase.
Searching for the reactivity of the MAb against membrane-bound NDP-kinase it was found that the MAb selectively reacts with a cell-membrane component of monocytes (93%), granulocytes (97%) and 14% of lymphocytes. Two color immunofluorescence showed that the MAb reacted only with CD19+ B cells (93% of CD19+ B
cells), which suggest that NDP-kinase may play an important role in normal B lymphocyte function.

Description

~~~8~33 This invention relates to a monoclonal antibody (MAb) directed against NDP-kinase, and in particular against the cytosolic and membrane bound NDP-kinases of human HL60 cells.
NDP-Kinase activity has been related to various physiological processes including DNA and RNA synthesis, production of cyclic AMP, superoxide metabolism and activation of the enzyme complex involved in DNA repair. In general the activity of NDP-Kinase has been paralleled to cellular proliferation, i.e. enhanced cytosolic NDP-Kinase activity is detected during cell proliferation. Moreover, the nm23 gene for which RNA levels are reduced in tumorous cells of high metastic potential, possesses a high degree of homology with the gene encoding the NDP-Rinase.
At present, there is a limited number of cell markers Which can be used to identify both tumor progression and metastasis. In this respect, it is worth noting that monoclonal antibodies have provided an important tool for following up the expression of such markers in various pathological tissues. The present inventors have found that by taking advantage of the similarities between membrane-bound and cytosolic NDP-kinase, there can be produced monoclonal antibodies against both purified proteins. Monoclonal antibodies raised against cytosolic NDP-kinase activity recognize the membrane bound form of the enzyme and vice versa.

21~~ X33 In accordance with one aspect of the present invention, there is provided a monoclonal antibody (MAb) directed against NDP-kinase, and specifically a clone which secretes immunoglobulins that recognize the active oligomer of membrane-bound NDP-kinase.
In accordance with another aspect of the invention there is provided a monoclonal antibody which can be used to detect tumor development and abnormal proliferation of B-cells. Immunoperoxidase staining of pathological tissues in combination with flow cytometric studies using the MAb are a potential clinical level for the detection of aberrant growth of various cellular tissues.
In accordance With another aspect, the invention provides a kit which can be used for detecting membrane bound activity of NDP-kinase in different cell extracts in laboratory clinics which do not possess flow cyometers.
The nucleoside diphosphate-kinase facilitates the intracellular conversion of both deoxy and ribonucleotide diphosphates to their phosphorylated forms. Although the majority of the nucleoside diphosphate kinase (NDP-kinase) activity is found in the cytosol of various cell types, the enzyme is also present in other cell constituents such as the multimeric microtubule protein rings, isolated plasma membranes of human and rabbit platelets, and beef brain particulate material. The wide distribution of NDP-kinase reflects its active role in various fundamental cellular processes related to nucleotide and superoxide metabolism, synthesis of cyclic AMP and in general to cell proliferation and differentiation.
In early studies [Cheng et al, Biochemistry 12:5 (1973)] it was suggested that the NDP-kinase might exist as various isoenzymes with characteristics isoelectric points and molecular weights. By contrast, studies (enzyme kinetics, peptide mapping and immunoblotting) performed on both the membrane-associated and cytosolic NDP-kinase [Kimura et al, J
Biol Chem 263:4647 (1988)], have shown that both enzymes are identical monoisozymic forms of the NDP-kinase. The inventors have found that the active cytosolic NDP-kinase purified from HL60 cells is an oligomer of about 67 kDa composed of two distinct subunits of.approximately 17 kDa and 33 kDa respectively, and that the cytosolic NDP-kinase is monoisozymic and the various apparent isozymes are the result of different states of phosphorylation of the protein, and not the product of NDP-kinase proteolysis.
In accordance with the present invention, we have prepared monoclonal antibodies against purified cytosolic NDP-kinase from HL60 cells. One of the monoclonal antibodies (MAb4A12) can selectively react with the active oligomer of the NDP-kinase. After denaturation, the monomeric components of this enzyme are no longer recognized therefore, native NDP-kinase conformation is required for binding of the MAb4A12 antibody. NDP-kinase has been found in both the cytoplasm and as a membrane associated complex. Furthermore, identical kinetic and physiochemical properties have been reported for NDP-kinase in both sites. These observations prompted the inventors to analyze the reactivity and distribution of MAb4A12 in human peripheral blood cells. It has been found that both monocytes and granulocytes react with MAb4A12 and that only a fraction of lymphocytes react with the antibody.
Further analysis shows that only B lymphocytes react with the anti-NDP-kinase antibody which suggests that NDP-kinase may play an important role in normal B lymphocyte function.
The preparation and characterization of a monoclonal antibody directed against NDP-kinase is described in the following:
MATERIALS
All reagents for electrophoresis, Sephacryl S-200, Protein-A Sepharose 4B and Protein G Sepharose 4 FF (MAb Trap G) were from Pharmacia LKB Biotechnology (Piscataway, NJ., USA). Low molecular weight protein markers for SDS-PAGE were from Bio-Rad Laboratories (Mississauga, Ont. Canada).
Freund's complete adjuvant, RPMI 1640 medium, penicillin, streptomycin, trypan blue, glutamine, sodium pyruvate and fetal calf serum (FCS) were purchases from Gibco (Grand Island, NY, USA). All nucleotides, 6-phosphogluconic dehydrogenase (yeast), glucose-6-phosphate-dehydrogenase and phenylmethylsulfonyl fluoride (PMSF) were purchases from Boehringer Mannheim (Montreal, Quebec, Canada). Ninhydrin, ~' Denot es trademark ~~~~~~cgD
hydrindantin, -D(+)-glucose, -nicotinamide adenine dinucleotide phosphate ( -NADP),3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT), phenazine methosulfate, dimethylamidobenzide (DMAB), pristane, dimethylsulfoxide (DMS), 8-azoguanine, aminopterin, HAT and HT media were obtained from SIGMA (St. Louis, M0, USA). Nitrocellulose membranes, Partisil 10 SAX analytical column (4.6mm I.D. x 25 cm), DEAE cellulose (DE52), DEAF ion exchange paper (DE81) and phosphate cellulose (P11) were from Whatman International (Maidstone, Kent, U.K.); phycoerythrin (PE) or fluorescein isothyocyanate (FITC) conjugated monoclonal antibodies were obtained from Becton Dickinson Immunocytometry Systems (San Jose, Ca., USA); Immunoprep (erythrocyte lytic agent, leukocyte stabilizer and cell membrane fixative) was obtained from Coulter Diagnostics (Hialeah, FL., USA). Horseradish peroxidase-conjugated rabbit antimouse IgG was purchased from DaRopatts, (Hamburg,. Germany); 3HdATP (40.0 Ci mmol.) was from NEN Research Products (Boston, Mass. USA).
PURIFICATION OF CYTOSOLIC NDP-KINASE
Cytosolic NDP-kinase from HL60 cells was purified as described by Pulido-Cejudo et al [FASEB J 3:60$a (1989)).
Briefly, the cytosolic fraction of cell extracts obtained from 15g (wet weight) of phosphate-buffered saline (PBS) washed HL60 call pellets was applied to a phosphate cellulose column (P11) (1.6 cm x 28.0 cm) previously equilibrated in phosphate buffer A [50mM potassium phosphate pH 7.0, 2mM MgCl2, 1mM

2i0~533 PMSF, 1mM DTT and 10% glycerol]. The column was washed with 300 ml of the same buffer at a flow rate of 0.10 ml/min.
Unbound protein was concentrated to 10 ml by ultrafiltration using YM5 membrane (5000 M.W. cutoff, Amicon Div., Danvers, Ma., USA). The concentrate was then applied to a DEAE
cellulose column (2.6 cm x 28.5 cm) equilibrated and washed with Tris buffer (50mM Tris-HC1 pH 7.5; 2mM MgCl2; 1mM PMSF;
1mM DTT and 10% (V/V) glycerol]. NDP-kinase was eluted using a linear gradient (0 to 1M NaCl in Tris buffer) at a flow rate of 0.50 ml/min. Fractions containing NDP-kinase activity were pooled and subsequently concentrated by ultrafiltration. NDP-kinase concentrate was loaded into a Sephacryl S-200 column (2.6 cm x 51 cm) pre-equilibrated in Tris buffer and the active material recovered after this purification step was immediately frozen at -20~C.until further analysis. NDP-kinase activity was determined by measuring the amount of dATP
consumed with dADP as phosphate acceptor and a fixed amount of enzyme. DEAF ion exchange paper (DE81) was used to separate ~idATP from the remaining 3HdADP after NDP-kinase mediated phosphate transfer to unlabelled dADP.
Protein concentration was estimated by the method of Lowry et al (J Biol Chem 193:265 (1951)] using bovine serum albumin as a standard. When assaying solutions with low protein content this was determined by the ninhydrin-hydrindantin method after alkaline hydrolysis.

21~533 A summary of the purification of the cytosolic NDP-kinase from HL60 cells is set out in Table 1.

Summary of Purification of the NDP-Kinase from HL60 Cells Total Specific Step Proteins Activity2 Activity2 Fold Yield (mg) (nmole/min) (nmole/min) g Homogenate 875 125 0.14 1 100 Supernatant 623 102 0.16 1.15 81.60 Cellulose Phosphate 135 100 0.74 5.21 80.00 Cellulose DEAE 1.18 14 11.86 83.52 11.20 Sephacryl S-200 0.006 6 1000 7042 4.80 1- the amount of protein was determined after alkaline hydrolysis and ninhydrin staining.

2- measured the amount of dATP hydrolysed in the as presence of a five fold excess of dADP over dATP added.

PURIFICATION OF NUCLEOTIDES

Befor e use, all deoxyribonucleotides and ribonucleotideswere purified by anion exchange chromatography.23mM solutions of each nucleotide were loaded onto a PartisillOSAX analytical column equilibrated with 0.4M

NHiHZPO~, pH . Isocratic elution of the two samples 3.9 was performed at flow rate of 1 ml/min. and at a pressure a of 1700 to 1800 i (Waters HPLC system Model 510). The ps pH of the purified material was adjusted to 7.0 With ammonia, and the final concentration determined spectrophotometrically at 260 nm.
MONOCLONAL ANTIBODY PRODUCTION AND PURIFICATION
The protocols for antigen preparation for immunization, preparation of spleen cells from immune animals, fusion of spleen cells with myeloma cells and plating of fused cells in selective HAT medium was conducted following the detailed guidelines described by Campbell [Burdon RH, Knippenberg PHV (eds): Laboratory Techniques in Biochemistry and Molecular Biology, Amsterdam, Elsevier, p219 (1984)] and by Lietzke and Unsicker [Lietzke R. Unsicker K: A Statistical Approach to Determine Monoclonality After Limiting Cell Plating of a Hybridoma Clone, J Immunol Methods 76:223 (1985)]. Contrary to most standard procedures used for the production of monoclonal antibodies, the primary immunization was performed with highly purified NDP-kinase (7000 purification fold - see Table 1) and not with crude or partially purified enzyme preparations. The production of ascitic fluid was achieved by priming BALB/C mice with 500 1 of pristane one week before intraperitoneal injection of 3x106 hybridoma cells. Ascitic fluid was collected after 20 days by draining the peritoneal cavity. Purification of IgG
immunoglobuline from ascitic fluids was performed by affinity chromatography on a 3m1 Protein G Sepharose 4 FF column (Pharmacia, Uppeala Sweden) following the manufacturer's protocol.

Screening of monoclonal antibodies against NDP-kinase was performed by dot blot immunostaining on nitrocellulose. Briefly, 0.5 to 1 g of NDP-kinase (5 1) was spotted onto a nitrocellulose membrane dried and blocked for one hour at 37~C with PBS/3% BSA (blocking solution). The mouse monoclonal antibodies (1/100 and 1/500 dilutions) were prepared in blocking solution and reacted with immobilized NDP-kinase at 37~C for 17 hours and then detected using an immunoperoxidase method.
POLYACRYLAMIDE GEL ELECTROPHORESIS i(PAGE) Slab polyacrylamide gel electrophoresis was performed under reducing conditions using Tris-glycine buffer containing 0.1% SDS. Samples were diluted in Tris-HC1 pH 6.8, 2% SDS, 25% glycerol, 1% -mercaptoethanol and 0.001%
bromophenol blue and boiled for 5 min. at 100~C.
Electrophoresis was carried out at 50mA for 3 hours at 4~C.
After electrophoresis, the gels were fixed in 5% methanol, 12%
acetic acid for 30 min. and thereafter stained with silver nitrate according to the method described by Merril et al [Science 211:1437 (1981)].
Protein electrophoresis under non-denaturing conditions was performed on 7% polyacrylamide gels in the absence of sodium dodecyl sulphate. Both 3% stacking and 7.0%
running gals were prepared in Tris-glycine buffer (25mM Tris, 192 mM glycine pH 8.3). Samples were dissolved in Tris-z~og~~3 glycine buffer, 15% glycerol, 0.001% bromophenol blue and run at a constant current of 50mA at 4~C for 3 hours.
Western blot analysis was performed by electrotransfer of 40 to 50 g of native or reduced and alkylated NDP-kinase. After PAGE, the gels were electrotransferred to nitrocellulose membranes and stained using an immunoperoxidase method. In some experiments, NPD-kinase was reduced and alkylated by the method described by Christie and Gagnon [Biochem J 201:555 (1982)].
DETECTION OF NDP-KINASE ACTIVITY AFTER
NON-DENATURING PAGE
Slab gels containing NDP-kinase were run under non-denaturing conditions and stained for NDP-kinase activity as described by Lam and Packham [Biochem Pharmacol 35:4449 (1986)]. Briefly, gels containing 5 to 10 g of NDP-kinase were put in a glass container wrapped in aluminum foil and immersed in a solution containing: 1mM ADP; 1mM GTP; O.lmM
NADP; 7.5mM MgCl2; 1.25mM ethylenediaminetetraacetic acid (EDTA); O.1M Tris-acetate, pH7.2; 2mM -D(+) glucose; 0.22 U/ml hexokinase; 0.22 U/ml glucose-6-phosphate dehydrogenase and 0.15 U/ml 6-phosphogluconate dehydrogenase. Just before staining the gels, phenazine methosulfate (0.075 mg/ml) and MTT-tetrazolium (0.2 mg/ml) Were dissolved in the staining solution. Gels ware incubated overnight in the dark at 37 C
with constant gentle stirring and then washed twice with deionized water and twice with 7% acetic acid. The activity 21~3~33 of NDP-kinase is detected through the formation of an insoluble colored product (MTT-formazan) that precipitates in the location of the enzyme.
IMMUNOPRECIPITATION
Samples containing NDP-kinase were immunoprecip-itated with the monoclonal antibody MAb4A12 previously adsorbed to protein-A Sepharose beads (4 mg of antibody per 1 ml of packed protein-A Sepharose beads). First, protein-A-Sepharose-MAb4A12 IgG beads were blocked for 2 hours with 10%
FCS-RPMI 1640, and washed with lOmM Tris-HC1 pH8. Samples (100 1) with a known NDP-kinase activity were incubated either with protein-A-Sepharose beads alone or with protein-A-Sepharose-MAb4A12 beads, and incubated overnight at 4~C with constant stirring. After incubation, the NDP-kinase activity in the supernatant was determined by the radio-isotopic assay described above.
INDIRECT IMMUNOFLUORESCENCE STAINING OF
HUMAN PERIPHERAL BLOOD
Whole blood cells were stained using a two color immunofluorescence method. Briefly, 20 1 of MAb4A12 antibodies (200 g/ml) or control IgGl was added to 100 1 of blood. After 30 min, incubation, the cells were washed with cold PBS and resuspended in 100 1 of PBS. The cells were then incubated with goat-antimouse IgG-FITC-conjugated for 20 min.
Cells were washed and resuspended in 100 1 of PBS. Blood cells were thereafter incubated with 20 1 of one of the 21a~~~3 following phycoerythrin-conjugated mouse antibodies: IgG,-PE
(control); Leu 3-PE (antiCD4); Leu 2-PE (antiCDB); Leu 4-PE
(antiCD3); Leu 12-PE (antiCDl9); Leu llc-PE (antiCDl6); Leu 19PE (antiCD56). After 15 min. incubation, the erythrocytes were processed in an automated Q-prep workstation (Coulter Electronics Inc. Hialeah, FL., USA) which sequentially delivers an erythrocyte lytic agent, a leukocyte stabilizer and a fixative. Then erytrocyte-free unlyzed cells were washed twice with PBS at 4~C and fixed with lml of 2%
paraformaldehyde. The samples were analyzed in a Profile II
flow cytometer (Coulter Electronics Inc., Hialeah, FL., USA) equipped with an air-cooled argon ion laser operating at 10 mwatt. Simultaneous excitation of FITC and PE conjugates is achieved by fixing the excitation wavelength at 488 nm. Data analysis Was performed with the Elite Workstation software version 2.1 (Coulter Electronics Inc.) and a minimum of 10,000 cells were collected in List Mode. Lymphocytes were clearly distinguished from monocytes and granulocytes on the basis of their forward light scatter (FAS) and side scatter light (SS) on a bivariant display. An electronic gate set around the cell population bearing the light scatter characteristics of granulocytes, monocytes and lymphocytes were activated for analysis.
CTERIZATION OF MONOCLONAL ANTIBODIFS
As shown above in Table d, the cytosolic NDP-kinase used to immunize BALB/C mice had a purification of 7000-fold and no adenosine triphosphatase activity in the absence of a phosphate donor was observed. Searching for non-inhibiting anti-NDP-kinase antibodies in positive hybridoma cultures, a monoclonal antibody identified as MAb4A12 was found to possess a strong reactivity against pure native NDP-kinase. This monoclonal antibody does not inhibit NDP-kinase activity and, as shown in Table 2, no significant change in the Km values obtained for different nucleotides was detected.

Effect of MAb(MAb4A12) on the Ligand Constants of NDP-Rinase Alone MAb4A12(200 g/ml Ligand Km' Apparent Kml Apparent ( M) Vmax ( M) Vmax dGTP 105.4 3.1x10-5 102.0 2.9x10-5 GTP 163.6 4.5x10-5 164.1 5:0x10-5 dCTP 433.0 3.3x10-' 430.2 3.1x10 CTP 446.0 6.7x10 4 443.2 6.2x10-~
1- Corrected Rm values were estimated as described by Mourad and Parks [J Biol Chem. 241:271 (1966)]. Briefly, 1/Vo values expressed as nmoles of dATP hydrolysed/min were plotted against reciprocal of the substrate concentration and the extrapolation to the X axis, estimated by linear regression analysis, yielded -(1/Rm). Ligand constants were determined using isotopic assay.
However, the monoclonal antibody MAb4A12 bound to protein-A Sepharose can effectively precipitate NDP-kinase 3s activity. Meatsrn blot analysis of NDP-kinase following non-denaturing PAGE showed a single homogeneous band when .the anti-NDP-kinase MAb4A12 antibody was used. A protein doublet 21a8~33 with a mobility identical to pure cytosolic NDP-kinase oligomer was also detected by Western blot when a crude cytosolic extract from HL60 cells was analyzed in parallel.
The protein doublet of NDP-kinase was found only in fresh enzyme preparations and may reflect different levels of protein phosphorylation. Further experiments were performed to study the identify of the protein bands recognised by MAb4A12 on crude cell extracts. Equal amounts of enzyme were subjected to non-denaturing PAGE and transferred to nitrocellulose. One sample was stained for NDP-kinase activity and the second reacted with the anti-NDP-kinase monoclonal antibody. Protein bands containing NDP-kinase activity, identified by the typical MTT formazan insoluble precipitate, ran at exactly the same position as those identified by Western blot using the monoclonal antibody MAb4A12. Collectively, these results confirm the specificity of the monoclonal antibody MAb4A12 and suggest that the latter antibody recognizes the active NDP-kinase cytosolic oligomer.
In this respect, there is increasing evidence suggesting that . in different tissues, the NDP-kinase occurs as a monoisozymic oligomer [J Biol Chem 256:10769 (1981) and Anal. Biochem 137:8 (1984)] rather than as isozymic multimers with characteristic isoelectric points as proposed by Cheng et al [Biochemistry 12:5 (1973)]. It is still uncertain, however, the identity and stoichiometric arrangement of the subunit(s) within the active NDP-kinase oligomer [Biochem Pharmacol 35:4449 (1986) _. 210533 and J Biochem 95:925 (1984)]. Pulido-Cejudo et al [FASEB J
3:608a (1989)) have reported that after full reduction and alkylation of cystine residues, the cytosolic oligomer purified from HL60 cells is composed of monomers with distinct molecular weights of about l7KDa and 33KDa respectively.
Subsequently, the ability of the monoclonal MAb4A12 was tested to recognize individual subunits of the NDP-kinase oligomer. Treatment of the NDP-kinase with denaturing agents (such as 6M guanidinium chloride followed by disulfide reduction and carboxymethylation) produced two major components with approximate molecular weights of l7KDa and 33RDa respectively. None of these two monomers showed a positive immunostaining after Western blot analysis of the electroblotted material, suggesting that the native oligomeric conformation of the enzyme is required for the recognition of the antigenic sites) by the monoclonal antibody MAb4A12.
IMMIJNOFLUORESCENCE STAINING OF NORMAL PERIPHERAL
BLOOD CELLS WITH ANTI-NDP-RINASE MAb4A12 ANTIBODY
Specific non-inhibiting monoclonal antibodies against NDP-kinase are of particular interest for studies concerning structure-function relationships of the NDP-kinase. It has been proposed that the membrane-bound NDP-kinase might modulate the activity of adenylate cyclase by replenishing GTP
levels required for the activation of G proteins, Gs and G1, v~hich in turn mediate hormonal stimulation and inhibition of adenylate cyclase respectively [J Hiol Chem 258:2278 (1983);

Biochem Biophys Res Commun 134:928 (1986) and Ann Rev Biochem 56:615 (1987)]. Moreover, it appears that cytosolic and membrane-associated NDP-kinase oligomers show identical physicochemical and structural properties (J Biol Chem 263:4647 (1988)] where the membrane-associated NDP-kinase activity is enhanced by piconilic acid, an isomer of nicotinic acid which is known to enhance intracellular cyclic A~IP levels [J Biol Chem 258:12609 (1983) and J Biol Chem 254:5588 (1979)]. These observations prompted the inventors to determine the reactivity of pheripheral blood cells towards the anti-NDP-kinase MAb4A12 antibody.
Indirect immunofluorescence staining of fresh peripheral blood cells from normal volunteers revealed that on average, 97% of gated granulocytes, 93% of monocytes and 14%
of lymphocytes stained positive using MAb4A12 as first antibody. The low percentage of gated lymphocytes that were labelled with anti-NDP-kinase monoclonal antibodies suggested that NDP-kinase may be restricted to a subset of lymphocytes.
To test this hypothesis two color immunofluorescence studies were done using MAb4A12 antibodies and various phycoerythrin conjugated monoclonal antibodies, recognizing characteristic epitopes expressed by different subsets of lymphocytes. Fresh peripheral blood cells were double stained using anti-NDP-kiaase antibodies and one of the following markers:

CD4(T-helper/inducer cells); CD8(T-cytoxic/suppressor cells;
NK-subset); CD3(T-cells, T-cell receptor complex);
CD19+H-cells; CD16(NK cells/neutrophils) and CD56(NK
cells/cytotoxic).
Two fluorometric methods were used to analyze double labelled cell populations. As the first approach, a conventional bivariant dot plot based on dual light-scattered parameters was used to identify and gate lymphocyte cells.
Alternatively, bivariant dot plot analysis was performed by plotting fluorescence intensity versus side scatter. The latter analysis served to identify specific lymphocyte subsets labelled with both phycoerythrin-conjugated monoclonal antibodies against various lymphocyte markers and with anti-NDP-kinase monoclonal antibodies. It was found that only lymphocytes, carrying the CD19 integral membrane glycoprotein characteristic of B-cells, were simultaneously labelled with anti-CD19 and anti-NDP-kinase monoclonal antibodies. In additional, practically all B-lymphocytes (93%) expressed the antigen recognized by MAb4A12. None of the other possible lymphocyte populations were selectively labelled with the monoclonal antibody raised against cytosolic NDP-kinase.
Hence, based on the identical structure and physiochemical characteristics of the membrane-associated and cytosolic NDP-kinase (J Biol Chem 263:4657 (1988)] and on the reactivity of the monoclonal antibody MAb4A12 against this enzyme, it appears that the former monoclonal antibody solely labels B-CA 02108533 2001-03-09 ,' lymphocytes, most likely by, reacting with the membrane-associated NDP-kinase oligomer. Upon activation of B-lymphocytes with IL4( 10'6M) the percentage of CD19+/MAb4A12' double labelled cells decreased from 98.5$ (control) to 7.8$
(activated). By contrast, following IL4 stimulation, the percentage of CD23 positive cells increased five fold.
Collectively these observations suggest that MAb4A12 mostly labels resting CD19+ circulating B-cells. Finally, the expression of the epitope recognized by MAb4A12 in various B-cell-IL6-transfected hybridomas with various~inetastatic potential is decreased in those cell lines that possess the greatest metastatic ability. Similar studies have been performed with brain tumours of glial origin whose decrease immunoreactivity against MAb4A12 has been detected in parallel with an increase tumour agressiveness. In these two cases, the decrease and/or lack of reactivity of MAb4A12 has been useful as prognosis of cellular invasiveness.
The MAb4A12 has been deposited within the American Type Culture Collection, under ATCC number CRL 11634.

Claims (5)

1. ~A monoclonal antibody have ATCC number CRL 11634 which specifically and strongly reacts with cytosolic and membrane bound NDP-kinase of human HL60 cells, the monoclonal antibody labelling resting CD19 + B-cells, upon IL4activation of such cells the labelling decreases, the expression of the epitope that is recognized by the monoclonal antibody decreases in those cell lines with greater metastatic potential, and the antibody exhibiting no significant changes in Km ligand constant values towards different nucleotides of NDP-kinase; said antibody being produced from a hybridoma cell line formed by purifying cytosolic NDP-kinase at least 7000 fold; immunizing mice~
with the thus purified NDP-kinase; fusing immunized spleen cells from the mice with myeloma cells to produce hybridomas; culturing the hybridomas; selecting a hybridoma that produces an antibody specific for NDP-kinase; and culturing the selected hybridoma.

2. ~A monoclonal antibody according to claim 1, wherein said cytosolic NDP-kinase is purified 7042-fold.

3.~The hybridoma cell line which produces a monoclonal antibody having ATCC number CRL 11634 which, specifically and strongly reacts with cytosolic and membrane bound NDP-kinase of human HL60 cells, the monoclonal antibody labelling resting CD19+B-cells upon IL4 activation of such cells the labelling decreases, the expression of the epitope that is recognized by the monoclonal antibody decreases in those cell lines with greatest metastatic potential, and the antibody exhibiting no significant changes in Km ligand contanst values towards different nucleotides on NDP-kinase; said cell line being produced by purifying cytosolic NDP-kinase at lease 7000 fold; immunizing mice with the thus purified NDP-kinase; fusing immunized spleen cells from the mice with myeloma cells to produce hybridomas; culturing the hybridomas;
selecting a hybridoma that produces an antibody specific for NDP-kinase; and culturing the selected hybridoma.

4. ~A method of producing a monoclonal antibody against cytosolic NDP-kinase from HL60 cells, comprising the steps of purifying a sample of cytosolic NDP-kinase at least 7000 fold; immunizing mice with the thus purified NDP-kinase; isolating the thus immunized spleen cells; fusing the immunized cells with myeloma cells to form hybridomas; culturing the hybridomas; selecting a hybridoma that produces an antibody specific for NDP-kinase; and culturing the selected hybridoma to produce the monoclonal antibody.

5. ~A method according to claim 4, wherein said cytosolic NDP-kinase is purified 7042 fold.