Note: Descriptions are shown in the official language in which they were submitted.
~~~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.
1
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
2
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
3
CA 02108533 2001-03-09
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
4
~~~~~~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
5
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.
6
21~533
A summary of the purification of the cytosolic NDP-
kinase from HL60 cells is set out in Table 1.
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
7
210853
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.
8
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-
9
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
11
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
12
2103J33
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.
TABLE 2
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
13
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)
14
_. 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);
2108533
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:
16
2108J33
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-
17
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.
18
