CA2346068A1

CA2346068A1 - A novel lymphocyte population expressing intracellular mo2

Info

Publication number: CA2346068A1
Application number: CA002346068A
Authority: CA
Inventors: Boris Tartakovsky; Israel Yust
Original assignee: Individual
Current assignee: Tel-Aviv Medical Center Research & Development Fund
Priority date: 1998-10-04
Filing date: 1999-10-04
Publication date: 2000-04-13
Also published as: JP2002526105A; AU5997199A; WO2000020562A1; IL126452A; EP1121418A1; IL126452A0

Abstract

The present invention concerns a novel population of lymphocytes that expres s intracellularly, an antigen recognized by MO2 MAbs and do not express this antigen on their surface. Such cells are termed "MO2+ cells". The level of MO2+ cells was shown to be higher in blood samples of individuals suffering from various infections such as an HIV infection as compared to the level of the same cells in healthy individuals and thus, these cells are useful in detecting individuals having a high probability of having such an infection. In addition, the level of MO2+ cells in the blood of an individual is useful in determining the efficacy of various anti-infectious treatments.

Description

WO 00/205b2 - 1 - PCT/IL99100524 FIELD OF THE INVENTION
The invention concerns a novel population of lymphocytes and further concerns use of said lymphocytes for various diagnostic assays.
PRIOR .ART
The following is a list of prior art publications referred to in the present specification.
1. Simmons, David, L., et al., Blood, 73:2,84-289, 1988.
io 2. Goyert, S.M.; et al., Science, 239:497-~E00, 1988.

3. Haziot A., et al., J. Immunol.,141:547-552, 1988.
is 4. Lauener, R.P, WO Patent Application No. 93632418.
5. Hans, et al., WO Patent Application No. 9525117.
6. Dietzh, et al., German Patent Application No. DE 4237502.

7. Landsorf, P. and Thomas, T, Canadian Patent Application No.
2191655.
8. Calder, V L. et al., 5'h International Conference on Human Leukocyte 25 Differentiation Antigens, Boston, Mass., U.S.A., 1993 9. Bernard, A., et al., Leukocyte Typing: i3erlin, 108:425-426, 428, 1984.

WO 00!20562 - 2 - PCT/IL99/00524 10. McMichael A.J., Leukocyte ~'yping Ltl, White Cell Di, f,~'erentiation Antigens, 586-589, 1987.
11. Todd, R.F., et al., Hybridoma, 1:329-33'l, 1982.
12. Todd, et al., J. Immunol.,126:1435-144:2, 1981.
13. Todd, R.F., and Schlossman, S.F., Blooci; 59:775-786; 1982.
14. Todd, et al., Leukemia Res., 5:491-4.95, 1981.
The acknowledgement herein of the above art should not be construed as an indication that this art is in any way relE;vant to the patentability of the invention as defined in the appended claims.
The above publications will be acknowledged in the following by indicating their number from the above list.
BACKGROUND OF THE INVENTION
Identification of specific cell types and populations is necessary and 2fl desired fox correct diagnosis of various disorders and diseases and enables more correct and e~cient prognosis and treatment of such conditions.
Many specific populations and sub-populations of lymphocytes are known today which are identified typically by use of monoclonal antibodies that are capable of specifically recognizing antigens expressed by such cells.
25 CD 14 is a glycosyl-phosphatidylinositol-linked single-chain surface membrane glycoprotein with a molecular weight of about 55 kDa~~~. It is expressed to a high extent on monocytes, macrophages and to a low extent on neutrophils. It has been shown to be very weakly expressed on the outer membrane of certain B-cell lymphocytes, but is absent from T-cells, null ;a cells, red blood cells and platelets. CD14 was also described to be expressed on the membrane of Langerhans cells, follicular dendritic cells and histiocytes, and to be present in a soluble farm in serum. CD 14 has been WO 00/20562 - 3 _ PCT/IL99/00524 shown to be a receptor for lipopolysachharide (LPS) and is considered to be a myelomonocytic differentiation antigen~2'3}.
Anti CD 14 MAbs have been shown to induce II,-10 secretion from CDi4 expressing cells or by inducing 't-cell tolerance or anergy~4~.
Anti-CD 14 antibodies were also described for use in the prevention or treatment of various bacterial infections such as toxemia associated with gram-positive bacteria infection, especially se~psis~s~. Medicaments containing anti-idiotype CD14 antibodies having high endotoxin binding capacity were described for the treatment of infection and other disorders~d~.
io Compositions comprising a combination of a number of antibodies including anti-CD 14 antibodies have also been described for use in the production of enriched human blood cell preparations as well as for the enrichment of non-hematopoietic metastatic tumor cells~~~.
A number of CD14 epitopes havf; been reported, each being recognized by a specific MAb. Such Mabs are, for example, My4~g' to>, CMRF-3 I~g~ and M02 ~9-14~ (hereinafter "the 11~I02 MAbs "). Intracytoplasmic CD 14 protein recognized by the My4 and CMRF-31 antibodies was reported in some non myeloid cells~g~.
The M02 MAb has been shown to react with 58%-84% of peripheral blood monocytes. The M02 antigen was sho~Hn to be expressed on 6%-16%
of bone marrow cells, on mature monocytes and on certain macrophages, but is not known to be expressed by T or B lymphocytes or by granulocytes.
SUMMARY OF THE INVENTION
In accordance with the present invention, a novel population of 2s lymphocytes was surprisingly discovered comprising cells that express intracellularly, an antigen recognized by the M02 Mab. Such cells will be referred to hereinafter as "M02~ cells ", and the antigen expressed intracellularly by such cells will be referred to as "M02 antigen ".

WO 00120562 . - 4 _ PCT/IL99l00524 The novel population of cells was dftected using the known M02 antibody and the expression of this antigen in these cells was detected following their fixation and permeabilization. No expression of the M02 antigen was detected on the cell surface of these cells. Therefore, unless specifically stated otherwise, reference to the expression of the M02 antigen by cells will refer to intracellular expression of the antigen.
The novel cell population of the invention was found to comprise two subpopulations. One subpopulation of cells, in addition to expressing the M02 antigen internally, was also found to express the general T-cell antigen to marker CD3 and therefore this population of cells is considered to be a T-cell population of lymphocytes and will be referred to hereinafter as CD3+ M02+
cells. In accordance with the invention, it was also found that a large percent of T-cells in peripheral blood which express the CD8 antigen (characterizing mainly suppressor and cytotoxic T-cells) also express the M02 antigen i5 intracellularly. Only a small percent of peripheral blood T-cells expressing the CD4 antigen (characterizing T-helper cells) were found to express the M02 antigen intraceliularly.
T-cells expressing the gamma/delta T-cell receptor (TCR) are believed to be involved in the etiology of infectious diiseases. In accordance with the 2o invention, it has been shown that most of these T-cells expressing the gamma/delta TCR also express the M02 antigen intracellularly.
The second novel subpopulation of lyrrrphocytes which, in accordance with the invention were found to express the MO2 antigen internally, are characterized as non-T, non-B cells. At least some of these cells express the 2s CD 16 antigen marker (and thus may be NK or NK-like cells).
The present invention thus provides a novel population of lymphocytes which express intracellularly a protein or polypeptide which binds to an antibody being a member of the group consisting of:
(i) anti MO2 MAbs;

WO 00/20562 _ $ - PC'TIIL99/00524 (ii) fragments of the antibodies of (i;) above which essentially retain the antigen binding characteristics of the non f ~agmented anti M02 MAb;
(iii) antibodies which bind to the aJltigenic epitope bound by any one of the antibodies of (i) or (ii) above;
said lymphocytes having no expression of the M02 antigen on their surface.
By one embodiment, the novel population of M02+ cells in accordance with the invention are T-cells expressing the CD3 antigen on their cell surface and expressing the M02 antigen intracellularl.y, hereinafter CD3+ M02+. The to expression of the CD3 antigen on these cells can be detected by any of the methods kriown in the art using any of the anti-CD3 MoAbs available such as, for example, the MoAb manufactured by Coulter Immunology. The M02+
T-cells comprise additional subpopulations wJhich express, in addition to the M02 antigen, also CD4 (helper cells), CD8 (suppressor cells) or gammaldelta i 5 antzgen.
The present invention further provides a novel population of non-T
non-B M02+ lymphocytes. Such cells may be detected by their positive reaction after fixation and permeabilization with the anti MO2 MAb, and by their negative reaction with anti-CD3 Mabs (a general marker of T-cells) as 2o well as their negative reaction with anti-CD22 MAbs (a general marker of B
cells) Sorne of these CD3M02+ cells were :Pound to react with anti CD16 MoAb (a marker of natural killer (NK) and NI<:-like cells.) Moreover, in accordance with the invention, a significant increase was shown in the percent of M02+ T-cells and or in the level of M02 expression 2s in peripheral blood cells of individuals suffering from infections of viral or bacterial origin as compared to the percent of such cells and/or level of intracellular M02 in the same cells in periphe~.°al blood of healthy individuals.
Further analysis showed that in many cases th.e most significant increase was WO 00/20562 _ 6 _ PCT/IL99100524 in the M02~ CD3+ cells (including both CD~l+ M02+ cells and CD8t M02+
cells} of the infected individuals. .
The present invention further provides a method fox detection of an individual with a high probability of having an infection comprising the steps of (i) obtaining a peripheral blood sample from said individual;
(ii) separation of mononuclear cells from said peripheral blood;
{iii) fxation and permeabilization of'said mononuclear cells;
(iv) incubation of said fixed and permeabilized mononuclear cells ~ o with MAbs which bind to the M02 antigen under conditions enabling binding of the Mabs to said antigen;
(v) detecting binding of said antibodies in said cells, and determining the number of cells in said sample expressing the MO2 antigen internally and/or the intracellular level of M02 is expression in said cells;
(vi) calculating a cutoff value based on an average number of M02+
cells or average level of M02 expression in samples obtained from healthy individuals; and (vii) comparing the number of M02~ cells and/or level of 2o expression of M02 in said cells measured in (v) above to said cutoff value, a measured number of M02+ cells andlor level of expression of M02 in said cells higher than the cutoff value, indicating a high probability of the existence of an infection in said tested individual:
2s The term "healthy individual " relates to an individual which does not have a bacterial or viral infection or any other detectable disorder or disease.
The level of expression of M02+ ce;Ils in healthy individuals will typically be determined by obtaining a blood sample from a large number of healthy individuals, determining the number of M02+ cells and/or level of WO 00/20562 _ 7 _ PCT/IL99/00524 M02 expression in each sample as described above, calculating an arythmetic average of the values determined in each sample and adding a value of one standard deviation to said calculated average to obtain a "cutoff value " (one based on the number of M02* cells and ,another on the level of MO2 expression). The value of the measured number of M02+ cells and/or level of MOZ expression in cells of the tested individual are then compared to the corresponding Cutoff value. Where the measured value is higher than the cutoff value, there is a high probability of the existence of an infection in the tested individual.
Io The cutoff value may be predetermined for a certain set of conditions such as the kind of assay used, the reagents used etc., and should correlate with the conditions used in the method. Thus, it will be clear to a person versed in the art that, under certain conditions it may be necessary to add, for example, more than one standard deviation value to the average value to is obtain the correct cutoff value.
In accordance with a preferred embodiment of the above aspects of the invention, following, or simultaneously with the incubation of the fixed and permeabilized mononuclear cells with anti M02 MAbs, the cells are also incubated with MAbs capable of binding to additional T-cell antigens (such 2o as CD3, CD4 and CD8) under conditions enalbling binding of these MAbs to said antigens. Following double staining of the cells, the number of M02+
cells which also bind the additional MAbs (hereinafter "double positive M02+ cells) in said sample as well as the level. of the M02 expression in such cells are determined; a higher number of double positive cells andlor a higher 2s level of M02 expression in such cells compared to the number of double positive (which bind the same additional Mab) M02+ cells andlor the level of M02 in such cells in control non-infected individuals, indicating a high probability of the existence of an infection in said tested individual.

WO 00/20562 _ g _ PCTIiL99100524 As explained above, the level of M02 expression in healthy individuals will typically be expressed as a cutoff value calculated as explained above and the measured value of the double positive cells will be compared to the cutoff value.
s Where necessary, the significance of the difference between the measured value and the cutoff value may be analysed by any of the known Statistical tests such as, for example, Students' t-Test.
In accordance with the invention, the term "infection " relates to any infection of a bacterial origin such as, for example, pneumonia or urinary 1o track infection (L1T2), which often results in sepsis. In addition, an infection may be of a viral origin such as, for example, an HIV infection.
The separation of mononuclear cells from peripheral blood obtained from a tested individual may be carried out by any of the methods known in the art such as for example separation on a Ficoll-Hypaque gradient.
t s The detection of the M02 antigen in accordance with the invention may be carried out by using any antibody capable of binding to the M02 antigen such as, for example, the M02-RD 1 antibody used in the examples below. The antibody used may be directly labeled with a detectable marker such as radioactive or fluorescent marker. Alternatively, the anti M02 zo antibody may be detected by using a secc>ndary antibody comprising a detectable label.
In accordance with the invention, the; anti M02 antibody used for detecting the M02 antigen may be a whole antibody or alternatively, fragments of the anti M02 Mab having similar binding properties. Such Mab 2s fragments may be for example, an Fab fra,~nent or an F(ab'~ fragment obtained by cleavage with appropriate enzymes as known in the art.
Fragments which may be useful in accordance with the invention may be screened by competitive binding assays with the respective whole anti M02 MAb and those which are capable of competing with such binding, i.e. bind WO 00!20562 - 9 - PCT/IL99100524 to the M02 antigen may be used. The MAbs in accordance with the invention may belong to any of the various immunoglobulin classes.
The binding of the anti M02 MAb may be detected in accordance with the invention by any of the methods known in the art such as FACS, RIA, ELISA, immunoblotting, etc. For detection of intracellular expression of the M02 antigen in the cells in accordance with tl~e invention, the tested cells are fixed and permeabilized prior to their stainiing with the relevant MAb by methods known in the art.
The detection of the additional T-cell antigen in the tested cells may be carried out by using any of the anti-T-cell specific antibodies known in the art (such as anti-CD3 for detecting T-cells, anti-CD4 for detection of helper T-cells or anti-CD$ for detection of suppressor T-cells) as well as any fragments thereof or labeled antibodies that essentially maintain the binding characteristics of the whole non labeled antibody. The detection of the ~s binding of such antibodies to the cells may be carried out by any of the methods known in the art such as those mentioned above.
In addition, in some cases cells were found in accordance with the invention to be in a "reactive " state. The term "reactive " relates to any kind of reaction of the cells which results in intracellular changes as compared to zo the cells in theix resting form. The reactive state may be of various kinds including, for example, an apoptotic reaction of the cells. The internal expression of the M02 antigen in the cells may be a result of an internal signal in the cells or may result from the cells' response to an external signal of some kind (e.g. soluble M02 antigen) and this signal may, in some cases, 2s be taken up by the cells from their environment. In accordance with the invention, it has been shown that the intracellular expression of the MO2 antigen in M02+ cells may be detected even in relatively slightly reactive cells, (such as, for example, was shown in gamma/delta positive M02+ cells following their growth in culture (see example. below).

w0 00/20562 ~ _ 1 ~ _ PCT/IL99/00524 In accordance with an additional aspect of the invention, it has been shown that the level of M02+ cells may be a useful marker for monitoring the efficacy of a certain treatment administered to an in individual suffering from an infection: As mentioned above, infected individuals show a higher level of s M02t cells before receiving treatment as compared to the level of M02~ cells in healthy individuals. In accordance with thf: invention, it was shown that, following treatment, the effect of the treatrr~ent may be monitored in the infected individuals on the basis of the level of their M02~ cells following treatment. Routinely, the effect of treatment of a viral infection is monitored io by measuring the viral load in the treated individual. In accordance with the invention, it has been shown that even when tile viral load of treated infected individuals decreases in all the treated individuals, in some, the level of the M02+ cells remains significantly higher than the level of the cells in healthy individuals, while in other individuals followi~c~g treatment, the level of is cells is reduced to the level of these cells in healthy individuals. Thus, the level of M02+ cells in treated infected individuals may be used as a basis for determining, on a cellular level, the effect of the treatment on the treated individual.
Thus the present invention further provides a method for monitoring 2o the efficacy of a treatment in an infected individual comprising:
(i) obtaining a peripheral blood sample from said individual prior to administration of said treatment;
(ii) separation of mononuclear cells from said peripheral blood;
(iii) fixation and permeabilization of said mononuclear cells;
2s (iv) incubation of said fixed and petmeabiiized mononuclear cells with MAbs which bind to the :M02 antigen under conditions enabling binding of the MAbs to~ said antigen;
(v) detecting binding of said antibodies in said cells, and determining the number of cells in said sample expressing the WO 00/20562 _ 11 _ PCTliL99100524 M02 antibody internally and/or the intracellular level of M02 expression in said cells;
(vi) administering said treatment to the individual;
(vii) at various periods of time following said treatment obtaining a peripheral blood sample from said treated individual;
(viii) determining the number of cells in said samples expressing the M02 antigen internally and/or the intercellular level of M02 expression in said cells as in step (ii) - (v) above;
(ix) comparing the number of said cells and/or said level of to expression of M02 in the cells :in the sample obtained in (i) to the number of said cells and/or the level of M02 expression in samples obtained from said individual following treatment, a significantly different number of M02+ cells or a significantly different level of expression of M02 in cells present in samples obtained from said treated individual as compared to the number of M02+ cells or the level of M02 expression in cells in a sample obtained prior to said treatment indicating efficacy of the treatment.
The term "significantly d~erenP' relates to either the number of M02+
2o cells or to the level of expression of M02 in the cells which may be higher or lower than the number of M02~ cells or the level of expression of M02 in healthy individuals. The significance of the difference will be determined by known Statistic tests. The measured value obtained from the tested individual may be compared to a cutoff value calculated from samples of healthy 2s individuals as explained above, By another of its aspects; the present invention provides a kit comprising antibodies which bind to the M02, antigen together with reagents necessary for fixation, and permeabilization of the tested cells and, optionally, additional antibodies capable of binding to 'T-cell antigens and means for WO OOI20562 _ 12 _ PCT/IL99100524 detecting said binding of said MAbs to the tested cells. Optionally, the kit will also comprise cutoff values to which the measured level of M02+ cells and/or level of M02 expression are compared.
EXAMPLES
The invention will now be illustrated further by the following examples with occasional reference to the figures. The examples are intended for illustration purposes only and are not to be construed as limiting.
BRIEF DESCRIPTION OF THE FIGURES
to Fig. 1 shows the results of fluorescence analysis by FACS of mononuclear cells obtained as described below which were first fixed and permeabilized as explained below, and then stained using one or more of the following MAbs:
Fig.1A MY4-FITC
Fig.1B M02-RD1 Fig. 1C IgG2-FITC {control for MY4-IaITC); and Fig.1D IgM-RD 1 (control for the MO;?-RD 1 MAb).
The figure shows the level of fluorescence on the stained cells as well as the size of the cell. The cells are considered to be positively stained 2o wherein the level of fluorescence of the cells is above the fluorescence level of the same cells after their incubation with thc~ control MAbs.
Fig. 2 shows FACS analysis of cells stauned by the MAbs mentioned in Fig. 1 without fixation or permeabilization o:f the cells prior to staining, i.e.
the membrane expression of the antigens.
2s Fig. 3 shows results of FACS analysis of lymphocytes stained with the control IgM-RD 1 Mab or M02-RD 1 MAb either after fixation only {Figs. 3A
and 3C) or after fixation and permeabilization (Figs. 3B and 3D).

Fig. 4A shows results of FACS analysis of lymphocytes following double staining of the cells with the MO2-RDI MAb and anti CD3-FITC
MAb;
Fig. 4B shows FACS analysis of the; lymphocytes stained with the control MAb IgM-RD I and the anti CD3-FITC MAb.
Fig. 5 shows the results of FACS analysis of double staining of lymphocytes with the M02-RD 1 MAb and anti CD8-FITC MAb (Fig. 5A}.
IgM-RD 1 was used as control MAb (Fig. 5B}.
Fig. 6 shows results of FACS analysis of double staining of the m lymphocytes with the M02-RD I MAb and an anti CD4-FITC MAb {Fig. 6A) the IgM-RD I antibody was used as control MAb (Fig. 6B).
Fig. 7 shows FACS analysis of double staining of lymphocytes with the M02-RD 1 MAb and anti gammaldelta T'CR-FITC MAb (Fig. 7A). The IgM-RD 1 antibody was used as control (Fig. 7B).
is Fig. 8 shows FACS analysis of double staining of lymphocytes with the M02-RD l MAb and an anti CD22-FITC MAb (Fig. 8A}. The IgM-RD 1 MAb was used as a control Mab (Fig. 8B).
Fig. 9 shows the results of FACS analysis of lymphocytes stained with the anti CD16-FITC MAb and M02-RD1 TJlAb (Fig. 9A). The IgM-RD1 2o MAb was used as a control MAb (Fig. 9B).
Fig. 10 is a schematic representation showing the percent of the lymphocytes expressing the M02 antigen intracellularly in mononuclear blood cells obtained from individuals suffering from various kinds of infections (black bars in the fig.) as compared to the presence of M02+ cells 2s obtained from healthy control individuals (grey bars in the f g.). The expression of the M02 antigen was detected. using the M02-RD 1 MAb on fixed and permeabilized cells as described below. The results show the total M02+ lymphocyte population, the M02+CD3~~ lymphocyte population and the M02~ CD3' lymphocyte population in both above groups of individuals.

w0 00/20562 PCT/IL99/00524 Fig. 1.~ is a schematic representation showing the percent (arid mean pezcent + S.D.) of Ivi02~' .CDBt cells in four individuals suf~erin.g from various kinds of infections as compared to the percent (and rxxean percent +
S.D.) o~MU2+ CD4y cells in the same individuals.
Fig. 12 is a schematic representation showing the percent of i1~I02~"
CD3~ cells in ten individuals suffezing from various kinds of infections as compared to the percent of these cells in healthy individuals.
MATERIALS AND METHODS
Fixation and permeabilz~.ation of Ivmphoc~rtes:
Peripheral blood is obtained fzvm :individuals to be tested and mononuclear cells are separated on a ficoll-hypaque gradient. ?he cells are then washed in phosphate buffered saline (PBS) and then fixed in 4%
paraformaldehyde in PBS for 30 n~ins. at 4°~C. The cells are then washed ~s again in P13S and permeabilized in PBS supplemented with O.l.% saponin and 1% bovine serum albumin for 30 rains. at r~oorr~ temperature.
Staining:
The cells prepared as described above, are then divided into staining 2o tubes and processed in the saponin buffer for 1 or 2-color. fluorescent staining:
The cells are incubated with the tested maizoclonal antibody or with the control monoclonal antibody for 30 rains: at zoom temperature. Staining can be done either by dizect staining, i.e. ~y sncuba~:ing the cells with a labeled Ab, ox indirectly, i.e. by incubating the cells with first non-labeled Ab and then zs with a secondary labeled Ab. Following incubation., the cells are .washed once in saponin buffer anal twice in PBS. The level of staining is then determined using the FRCS as described below.

WO 00120562 - 15 _ PCTlIL99100524 Monoclonal antibodies:
The MAbs used to determine the expression of various antigens in the tested cells are the following:
The anti-CD 14 MAbs anti-MY4 and anti-M02 as well as the s Anti-CD3, Anti-CD4, Anti-CD8, and Anti-CD16 were all obtained from Couiter Immunology. The anti-TCR, alpha beta, and anti-TCR gamma delta MAbs were obtained from Immunotech.
Fluorescence-activated cell Sorter-FACS
io The analysis of the intracellular or membrane expression of the M02 antigen is performed with the use of the FACS (model FACSCAN
manufactured by Becton Dickinson) or FACS CALIBUR.
Cells of the lymphocyte or monocyte lineage can be detected and characterized according to their light-scattering properties. Their scatter is positions, determined by their different forward and side laser- light scatters, enable to analyze both populations independently, by applying a "gate " or window on the cells of interest. In addition, the instrument is capable of measuring the fluorescence of the cells, either the green fluorescence, emitted by FITC, or the red fluorescence, emitted by RD 1 - two different dyes p conjugated to the MAbs. On the basis of thc~ fluorescence of the cells, data can be obtained on the number of fluorescent: cells, above background, in the population of cells under analysis, and also on the level of intensity of the fluorescence, a measurement directly correlated with the amount per cell of the antigen detected by the conjugated MAb.
as RESULTS:
Example 1: Intracellular expression of M~D2 in lymphocytes As seen in Fig. 1, the novel population of lymphocytes which express the MO2 antigen internally was detected using the M02-RD1 antibody on WO 00/20562 - 1( - PGT/IL99/00524 f xed and permeabilized mononuclear cells which were obtained as described above. No expression of an antigen recognized by the MY4-FITC antibody was detected on the same cells. As seen in Fig: 2, no expression of the M02 antigen or the MY4 antigen was detected in the same cells which were not fixed and permeabilized, i.e. the M02 antigen. which was expressed internally on the cells was not detected on their surface.
As seen in Figs. 1 and 2, the M02 antigen was expressed to some extent on the cell surface of monocytes.
to Example 2: Detection of intracellular expression of M02 on lym~hacytes by fixation and~ermeabilization.
Mononuclear cells obtained as explained above, were divided into the following two groups:
1 s 1. Cells that underwent fixation only as described above;
2. Cells that were fixed and also permeabilized as described above.
Both groups of cells were incubated with the M02-RD1 MAb and the expression ofM02 was analzyed by FACS.
20 As seen in Fig. 3, the M02 antigen was detected only in the above group 2 lymphocytes and no antigen was detected in cells of group 1. The above results show that the cells express the 1'vI02 antigen internally only and that detection of the M02 antigen in these cells by the Anti-M02 MAbs requires permeabilization of the cells.
. Example 3: The M02~ cells comprise two subnopulations:
3.1 Mononuclear cells obtained a..~ explained above were double stained with an anti-M02-RD 1 antibody and an anti-CI~3-FITC
MAb and the level of fluorescence was measured by FACS as 3o explained above. As seen in Fig. 4, the M02+ cells comprise WO 00/20562 _ 17 - PCT/IL99/00524 two subpopulations: those which do riot express the CD3 antigen {non T cells, hereinafiter CD3' M02+ (Fig. 4A) and those cells expressing the C;D3 antigen (T lymphocytes, hereinafter CD3+ M02+ (Fig. 4A).
3.2 Characterization of the CD3+ M02+ T lymphoc es:
3.2. i Mononuclear cells were double stained with the anti M02 MAb and an anti-CD8 MAb {a marker of cytotoxic and suppressor cells). A:> seen in Fig: 5, a large percent of the CD8+ cells also express the M02 antigen to internally.
3.2.2 The mononuclear cells vcrere double stained with the anti M02 MAb and an anti-C:D4 Mab (a marker for T helper cells). As seen in Fig. 6, only a very small pecent of the CD4+ cells expressed the M02 antigen internally.
3.2.3 The mononuclear cells v~rere double stained with the anti M02 Mab and with a MAb recognizing the gammaldelta T-cell receptor (about 2~% of peripheral blood T cells).
As seen in Fig. 7, moss; of the T cells expressing the gamma/delta type T cell receptor also expressed the 20 M02 antigen internally.
3.3 Characterization of the non T M02+ CD3' cells:
3.3.1 Mononuclear cells obtained as explained above were double stained with tlhe anti M02 MAb and an anti-CD22 MAb (whiclh is a marker for mature B
zs lymphocytes). As seen F'ig. 8, none of the CD22+ cells (mature B lymphocytes) were positive for M02. The above results show that none of the B cells express the MOZ antigen internally. 'These results also show that the WO 00120562 _ 1$ - PCTIIL99100524 CD3~ cells which express the M02 antigen internally (see 3.1 above) are non T- non B cells.
3.3.2 The cells were double stained with the M02 Mab and with the anti-CD 16 antibody (NK-like cells). As seen in Fig. 9, a large percent of the NK-like cells (CD I 6+) also express the M02 antigen :internally.
Example 4: Expression of M02+ cells in individuals sufferinE from infection:

4.1 The percent of the cells expressing the M02 antigen internally (M02~ in individuals suffering from various infectious diseases of bacterial and viral origin were determined and compared to the percent of the M02+ cells in healthy ~s individuals. As seen in Fig. 10, the percent of M02+
lymphocytes in individuals suffering from infections was substantially higher than the percent of these cells found number of the cells in healthy individuals. As may be seen, the most substantial rise is in the percent of CD3~ M02+ cells.
4.2 As seen in Fig. 11, the level of 1VI02f CD3+ cells was found to be higher in individuals suffering from various kinds of infections including bacterial (sepsis) and viral (asymptomatic and symptomatic HIV+ individuals). The mean percent of M02+ CD3~ cells in healthy individuals was in the range of 2s between about 0% to I S% while the mean percent of these cells in individuals suffering from infection was in the range of between about 20% to about 60%.
4.3 In order to characterize the MO:Z~ T cells appearing in a higher level in individuals suffering from infections as compared to healthy individuals lymphocytes obtained from four individuals WO 00/20562 _ j~ _ PCTIIL99/00524 suffering from various infections were also stained with the M02-RD 1 MAb and with anti-CD8 or with anti-CD4 antibodies. As seen in Fig. 1:?, in each of the four tested individuals, the level of the MG2~ T cells expressing the CD8 s antigen was substantially higher than the percent of M02* T
cells expressing the CD4 antigen in the same individual.
Therefore, the majority of the M:02+ T cells which are found at a higher level in individuals sui~ering from infections seem to be CD8~ T cells while only the minority of these cells seem to t o be CD4+ T cells.
Example 5: Expression of intracellular 1V~02 in ~amma/delta positive cells activated by growth is cullture Is Mononuclear cells, obtained after Ficoll-Hypaque purification, vc~ere divided into two groups. Celfls in group one were fixed, permeabilized and double stained for intracellular M02 antigen and for the gamma.-delta T-cell receptor antigen, as explained above. Cells in the second group were put in culture {RPMI medium, supplemented 2o with 5% fetal-calf serum, glutamine and antibiotic mix}, in a humidified incubator, for 18 hours, at X37 degrees and 5% COz. At the end of the incubation period, cells were washed, fixed, permeabilized and double stained for intracellular M02 antigen and for the gamma-delta T-cell receptor antigen, as. explained above.
2s Results, shown in Table 1, represent the net mean intracellular M02 fluorescence of gamma-delta positive cells, before and after the incubation. The net mean fluorescence channel (attained from the FACS analysis}, correlates with the intracellular amount of the M02 antigen. As shown, in the majority of the samples analyzed, we ~o observed a significant increase in the amount of the intracellular M02 WO 00/205b2 - 20 - PCTIIL99/00524 antigen in the gamma-delta T-cells, following their stimulation by the 18 hours culture.
Table 1.
s Individual # Mean Net Fluorescence Channel (Correlates with :intracellular amount of M02 anti en Before Culture After Culture 1 2.3 4 2 2.5 11.7 3 1.5 12 4 2.7 9.7 6 I7.8 20.6 7 0.9 6.6 8 1.5 12.8 9 3 13.3 I0 2.7 13.9 Example 6: Monitoring the effect of treatment in HIV-positive individuals 6.1 The level of several populations of M02+ cells was determined in a number of healthy individuals as well as in asymptomatic untreated individuals which were determined as HIV-positive individuals on the basis of a positive serological test (based _on positive binding of anti-HIV antibodies determined by ELISA
is and Western Blot assays). The; level of three populations of M02~ cells CD3+ M02+ cells, CD4+ M02* cells and CD8+
M02+cells was determined by double staining of cells present in the samples obtained from the tested individuals with M02 MAbs, anti CD4 MAbs or anti CD8 MAbs, respectively, as 20 described above.

w0 OOI20562 - 21 - PCT/IL99/00524 As seen in Table 2, the level of each of the above three M02+
populations was significantly higher in HIV-positive individuals as compared to the llevel of the same population of cells in healthy individuals.
s Table 2 Healthy Individuals% CD3TM02'~ % CD4 M02t % CD8~M02~' Age 21-60 8.4% (SD 6.2) 0.3% (SD 0.7)14.8% (SD
5.7) n=23 n=11 n= il HIV-Pos Individuals31.3% (SD 11.3)6.4~% (SD S I.6% (SD
6.2) 11:5) A a 25-54 n - 17 n = 17 n = 17 6.2 Monitoring the effect of anti-retroviral treatment of HIV-positive individuals on the basis of the level of M02+
cells:
Four HIV positive individuals receiving anti-retroviral treatment comprised of a combination of three drugs (two nucleoside analogs and one protease inhibitor) were tested at is various periods of times after the beginning of the treatment for their total viral loads as well <~s for the level of their M02+
cells. The level of viral load was determined by the NASBA-Nuclisense Nucleic Acid Sequence based amplification {Nuclisense Organon Teknika). The level of the 2o four sub-populations of M02+ cells in the blood samples was detected by double staining of the cells as described above.
Results of the tests determining viral load in the treated individuals showed that the total viral load decreased in all four treated individuals following treatment (results not shown).
2s V~hen testing for the level of IVI02~ cells, as seen in Table 3 below, in two HIV-positive treated individuals (designated as WO 00/20562 _ 22 _ PCT/IL99/00524 AP and RA in the table) a significant decrease was seen in the level of M02~ cells following treatment of the individuals. The decrease was seen following a period of one month of the treatment. The level of the M02+ cells continued to decrease after three months and four months of the treatment. The level of the M02+ cells in these two treated individuals reached a level which was very similar (and in some cases even lower) than the level of the cells in heallhy individuals.
However, as seen in the table, in the two remaining to HIV-positive treated individuals {designated as GR and DO in the Table) the level of the 1'vI02~ cells did not decrease following treatment and in some cases the level of these cells even increased (see the level of CD4+ M02~ cells in individual GR).

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~ a r~c~o WO 00/20562 _ 24 _ PCT/IL99/00524 The above results show that the; level of M02+ cells may be a preliminary marker for HIV infection. In addition, the level of these cells may be useful to monitor the effect of an antiviral treatment on a tested individual.

Claims

CLAIMS:

1. A novel population of lymphocytes which express intracellularly a protein or polypeptide which binds to an antibody being a member of the group consisting of:
(i) anti MO2 MAbs;
(ii) fragments of the antibodies of (i) above which essentially retain the antigen binding characteristics of the non fragmented anti MO2 MAb;
(iii) antibodies which bind to the antigenic epitope bound by any one of the antibodies of (i) or (ii) above;
said lymphocytes having no expression of the MO2 antigen on their surface.

2. A population of lymphocytes in accordance with Claim 1, wherein said lymphocytes further express the CD3 antigen an their cell surface.

3. A population of lymphocytes in accordance with Claim 2, wherein said lymphocytes further express the CD8 antigen on their cell surface.

4. A population of lymphocytes in accordance with Claim 2, wherein said lymphocytes further express the CD4 antigen on their cell surface.

5. A population of lymphocytes in accordance with Claim 2, wherein said lymphocytes further express the gamma/delta receptor.

6. A population of lymphocytes in accordance with Claim 1, wherein said lymphocytes have no expression of the CD3 and CD22 antigens on their cell surface.

7. A population of lymphocytes in accordance with Claim 6, wherein said lymphocytes express the CD16 antigen on them cell surface.

8. A method for detection of an individual with a high probability of having an infection comprising the steps of:
(i) obtaining a peripheral blood sample from said individual;
(ii) separation of mononuclear cells from said peripheral blood;

(iii) fixation and permeabilization of laid mononuclear cells;
(iv) incubation of said fixed and permeabilized mononuclear cells with MAbs which bind to the MO2 antigen under conditions enabling binding of the Mabs to said antigen;
(viii) detecting binding of said antibodies in said cells, and determining the number of cells in said sample expressing the MO2 antigen internally and/or the intracellular level of MO2 expression in said cells;
(ix) calculating a cutoff value based on an average number of MO2+
cells or average level of MO2 expression in samples obtained from healthy individuals; and (x) comparing the number of MO2+ cells and/or level of expression of MO2 in said cells measured in (v) above to sand cutoff value, a measured number of MO2+ cells and/or level of expression of MO2 in said cells higher than the cutoff value, indicating a high probability of the existence of an infection in said tested individual.

9. A method in accordance with Claim 8, wherein following or simultaneously with the incubation of said fixed and permeabilized cells with MAbs which bind MO2, said cells are also incubated with MAbs which bind T-cells specific antigens and the number of MO2+ cells which also bind said anti-T-cell MAbs in said sample as well as the level of MO2 expression in said cells is determined and compared to the number of cells which bind MO2 and said anti-T-cell specific MAb or the level of MO2 in such cells in control non-infected individuals, a higher number of MO2+ cells or a higher level of MO2+ expression in said tested individual indicating a high probability of the existence of an infection in said tested individual.

10. A method in accordance with Claim 9, wherein said anti-T-cell specific antigen is CD8.

11. A method in accordance with Claim 9, wherein said T-cell specific antigen is CD4.

12. A method in accordance with Claim 9, wherein said T-cell specific antigen is the gamma/delta receptor.

13. A method for monitoring the efficacy of a treatment in an infected individual comprising:
(i) obtaining a peripheral blood sample from said individual prior to administration of said treatment;
(ii) separation of mononuclear cells from said peripheral blood;
(iii) fixation and permeabilization of said mononuclear cells;
(iv) incubation of said fixed and permeabilized mononuclear cells with MAbs which bind to the MO2 antigen under conditions enabling binding of the MAbs to said antigen;
(v) detecting binding of said antibodies in said cells, and determining the number of cells in said sample expressing the MO2 antibody internally and/or the intracellular level of MO2 expression in said cells;
(vi) administering said treatment to the individual;
(vii) at various periods of time following said treatment obtaining a peripheral blood sample from sad treated individual;
(viii) determining the number of cells in said samples expressing the MO2 antigen internally and/or the intercellular level of MO2 expression in said cells as in step (ii) - (v) above;
(ix) comparing the number of said cells and/or said level of expression of MO2 in the cells in the sample obtained in (i) to the number of said cells and/or the level of MO2 expression in samples obtained from said individual following treatment, a significantly different number of MO2+ cells or a significantly different level of expression of MO2 in cells present in samples obtained from said treated individual as compared to the number of MO2+ cells or the level of MO2 expression in cells in a sample obtained prior to said treatment indicating efficacy of the treatment.

14. A method in accordance with Claim 13, wherein following or simultaneously with incubation of said cells in said (iv) with Mabs which bind the MO2 antigen, the cells are also incubated with antibodies which bind T-cells specific antigens, the number of cells which bind said Mabs is determined before and after said treatment and the number of said cells or the level of expression of MO2 in said cells is compared to the number of said cells or the level of MO2 expression in said cells in samples obtained from said individual prior to receiving treatment; a significantly different number of said cells and/or of the level of expression of MO2 in said cells indicating .
efficacy of said treatment.

15. A method in accordance with Claim 14, wherein said additional T-cell specific antigen is CD8.

16. A method in accordance with Claim 14, wherein said additional T-cell;
specific antigen is CD4.

17. A method in accordance with Claim 14, wherein said additional T-cell specific antigen is gamma/delta receptor.

18. A kit comprising antibodies which bind to the MO2 antigen together with reagents necessary for fixation, and permeabilization of the tested cells and, optionally, additional antibodies capable of binding to T-cell antigens and means for detecting said binding of said MAbs to the tested cells.

19. A kit in accordance with Claim 18, further comprising cutoff values to which the measured number of MO2+ cells and/or level of MO2 expression are compared.

20. A population of lymphocytes in accordance with any one of Claims 1-7, wherein said anti-MO2 MAbs are MO2-RD1 MAbs, fragments -29-~
thereof retaining the antigen-binding characteristics of said MAbs or antibodies which bind to the antigenic epitope bound by the MO2-RD1 antibodies or fragments thereof.

21. A method in accordance with any of Claims 8-17, wherein the MAbs which bind to the MO2 antigen are MO2-RDI MAbs.

22. A kit in accordance with any of Claims 18 or 19, wherein the MAbs which bind to the antigen are MO2-RDI MAbs.