WO1993014125A1 - Novel lymphocytic antigen, corresponding antibody and applications thereof - Google Patents

Abstract

Antigenic protein consisting of a 150 ± 10 kDa glycoprotein determined by electrophoresis of the SDS-PAGE type in reducing conditions, expressed on the surface of T-cell lymphocytes in human blood. The present invention also concerns the protein in its dimeric form having a disulphide bond and a molecular weight of 300 ± 20 kDa. The invention further relates to antibodies directed against a protein of the invention, hybridoma lineages and the diagnostic and therapeutic application of proteins and antibodies according to the invention.

Description

 NEW LYMPHOCYTATIVE ANTIGEN, ANTIBODIES

 CORRESPONDENT AND THEIR APPL ICATIONS

The present invention relates to a new lymphocyte antigen as well as antibodies corresponding to this antigen and their applications.

Lymphocyte activation induces or increases the expression of several surface structures, some of which, such as the IL2 and transferrin receptors, are directly involved in cell growth. In particular, the activation of human lymphocytes leads to phenotypic modifications appearing either immediately or at variable times after the disturbance of the antigen recognition structures. Phenotypic changes can be attributed to the expression on the cell surface of inducible molecules such as CD25 (1, 2) and CD71 (3), or to the increased expression of surface antigens such as CD26 (4, 5), CD 29 (6.7), CD45RO (8.9), or to an epitopic modulation as in the case of CD2 (10.11), or to changes in affinity as reported for the molecules CD 1 1a / CD18 (12) and LAMK13). It is not surprising that these inducible molecules include receptor subunits of high affinity for various cytokines (2). Most often, they are detected using a monoclonal antibody (mAb) recognizing a putative receptor for an unknown ligand. For example, the hgands of CD5 (14,15), CD43 (16) and CD69 (17,18) were very recently identified, whereas the use of mAb as agonists against these structures had made it possible to identify their crucial role in the activation of the T cell (15, 17, 18, 19). These mAbs have been shown to increase the cytoplasmic concentration of free calcium, if they are "cross linked" on the surface of T cells (19). Since an increase in cytosolic calcium and a simultaneous activation of Protein Kinase C (PKC) are early and essential events in the proliferation of T lymphocytes, investigations have been carried out on the effects of these various mAbs on the activation of T lymphocytes humans in the presence of PMA (Phorbol 12-myristate 13-acetate), which is an activator of PKC (20). In addition, the use of mAb has confirmed that a large number of leukocyte surface antigens and in particular those induced during activation are common to B and T cell lines (2,3,14).

 In order to identify new structures characteristic of activated lymphocytes, monoclonal antibodies have been defined according to the invention against functionally defined clones of human T cells.

 According to the present invention, a monoclonal antibody called BB 18 was first isolated, recognizing, on the cell surface, a new lymphocyte antigen composed of subunits of 150 ± 10 kDa, the expression of which on T lymphocytes increases rapidly. after their activation by various stimuli including iectins. On the other hand, in the presence of phorbol 12-myristate 13-acetate (PMA), the expression on the cell surface of this structure of 150 ± 10 kDa undergoes an even earlier negative modulation than the molecules of CD3. Biochemical studies as well as phenotypic analysis have revealed that this structure is different from all the molecules previously identified on the cell surface of lymphocytes.

 Other monoclonal antibodies (BD 16, BM4, BM71 and BM63), obtained according to the invention, recognize the same surface structure as i'Acm BB18. These antibodies are IgG1 which were obtained after immunization of Balb / c mice with a human thymic clone and fusion with the NSI cell line. They immunoprecipitate from the surface of human peripheral blood lymphocytes or radio-labeled iodine T clones, the same molecular structure. These antibodies react with different epitopes of this structure. Thus, there is no inhibition of the binding of the antibody BB18 in the presence of an excess of the antibody BD 1 6 and vice versa.

The present invention therefore firstly relates to an antigenic protein consisting of a phosphorylated glycoprotein with a molecular weight of 150 ± 10 kDa determined by electrophoresis of SDS PAGE type under reducing conditions, protein expressed on the surface of human T lymphocytes preferably activated. The present invention also relates to an antigenic protein consisting of the same phosphorylated glycoprotein but in the form of a disulfide-linked dimer with a molecular weight of 300 ± 20kDa determined by SDS PAGE electrophoresis under non-reducing conditions, protein expressed at surface of preferably activated human T lymphocytes, composed of subunits of 150 ± 10 kDa as defined above.

 A subject of the invention is also derivatives of the protein in monomer or dimer form, characterized in that the protein is in non-glycosylated and / or non-phosphorylated form or in that it comprises non-natural glycosilations or phosphorylations or consisting of a fragment of this protein comprising the essential antigenic sites of the protein or non-glycosylated and / or non-phosphorylated derivatives and / or comprising non-natural glycosylations and / or phosphorylation.

 The present invention also relates to the monoclonal antibodies recognizing an epitope of a protein according to the invention, in particular the monoclonal antibodies BD 16, BB18, BM4, BM71 and BM63, as well as hybridoma cell lines producing these antibodies, in particular the lines deposited at ECACC under the numbers 92010801 for the antibody BD16, 92010802 for the antibody BB18, 93 01 1201 for the antibody BM4, 93 01 1202 for BM71 and 93 011203 for BM63.

The protein, its fragments or derivatives according to the invention can be obtained from the natural protein itself obtained by purification from producer cells by immunoprecipitation techniques, in particular with the antibodies BB18, BD 16, BM4, BM71 and BM63 or other techniques. The protein according to the invention, its fragments and derivatives according to the invention can however also be obtained by recombinant DNA techniques of genetic engineering from the DNA coding for the said protein incorporated in expression vectors of that this in eukaryotic or prokaryotic cells, in which case the glycosilation of the recombinant protein obtained can differ from the natural protein as is known to those skilled in the art. A protein or an antibody according to the invention can be labeled with a detectable label with a view to their assay in vitro or their localization in vivo or ex-vivo.

 The invention indeed also relates to the use of proteins and antibodies according to the invention for the diagnosis of pathological states or the monitoring of the treatment of ailment or as a warning lamp for the appearance of these ailments. In particular, the present invention relates to the use of these proteins and antibodies for the rapid diagnosis of a state of early activation of lymphocyte T cells.

 Finally, the present invention relates to the use of a protein, antibody or antibody fragment according to the invention as a medicament in particular for the treatment of diseases with signs of immune deficiency or of inflammatory or autoimmune pathology and in in vivo or ex vivo treatment of malignant lymphoid proliferation.

 Other advantages and characteristics of the present invention will become apparent in the light of the description which follows.

 In the attached figures,

 - Figure 1 shows the kinetics of expression on the cell surface of the structure recognized by BB18 after activation of PBMC (mononuclear cells of peripheral blood) using PHA. The peripheral blood mononuclear cells were labeled before (DO), 3 days (D3), or 9 days (D9) after coculture with PHA 1 μg / ml, for indirect immunofluorescence with BB18, CD25 "BC96" or an antibody monoclonal negative IgGl. The fluorescence profiles are shown as histograms.

 - Figure 2 shows the PMA inducing an early decrease in reactivity with BB18. Peripheral blood T cells were incubated with PMA 1 μg / ml for 6, 10, 24 or 144 h. The cells were washed and treated for indirect immunofluorescence with various monoclonal antibodies. At each measurement time, a negative control made of cells incubated under the same conditions but without PMA was carried out. As no modification of the control cells was observed, the control tested after 6 h of culture is presented. The fluorescence profiles are shown as histograms.

- Figure 3 shows the SDS-PAGE profile of an immunopreopity BB 18 obtained from a lysate of cloned T cells. The human thymus T cell immunizing clone B12 was surface labeled and lysed in NP40 1%. The immunoprecipitates obtained with BB 18, CD 18 "M232" or an irrelevant IgGI monoclonal antibody were analyzed on a 7.5% SDS-PAGE gel under non-reducing conditions. The positions of the molecular weight markers have been indicated on the left in kDa. - Figure 4 shows the two-dimensional gel analysis (non-reducing / reducing) of a BB18 immunoprecipitate obtained from lysates of cloned T cells. The immunoprecipitates obtained with the monoclonal antibody BB18 were first separated under non-reducing conditions (N. Red.) On a 7.5% gel using a PHAST system. Then the gel strip containing the immunoprecipitate was cut and subjected to electrophoresis in the second dimension, as recommended by the manufacturer, after reduction (Reduction) on a 7.5% SDS-PAGE gel. The positions of the molecular weight markers have been indicated on the right in kDa.

- Figure 5 shows the kinetics of PBMC proliferation induced by the BB18 monoclonal antibody. Mononuclear peripheral blood cells were incubated with the purified CD3 "X3" 5 μg / ml (white triangles), the purified monoclonal antibody BB18 5 μg / ml (empty squares) or 10 μg / ml (white circles) in the presence 0.2 ng / ml PMA for 2 to 8 days. The incorporation of (H) TdR was measured by "drawing" the culture for the last 16 hours. The results are expressed as the average of triplicate samples and the SEM has always been less than 10% of the average. Under these conditions, PMA alone did not induce significant proliferation at any of the measurement times.

- Figure 6 represents the autoradiography of SDS-PAGE at 8.5% immunoprecipitates of BB18 and BD 16 under reducing conditions, and treated or having undergone a simulated treatment with endo-F. The positions of the molecular weight markers are indicated on the right in kilodaltons.

- Figure 7 shows the autoradiography of SDS-PAGE at 12% of BB18 and BD 16 immunoprecipitates under reducing conditions, and treated or having undergone a simulated treatment with V8 protease directly in the gel. The positions of the molecular weight markers are indicated on the right in kilodaltons.

- Figure 8 shows the co-modulation of the PBL cell surface structure at 37 ° C for 8 hours, with an excess of Acm BB 1 8 or 24 hours with mAb BD 16. Next, the cells were carefully washed and labeled with a biotiny mAb or an FITC-labeled goat anti-mouse Ig, as a control.

 - Figure 9 represents a competitive binding experiment between mAbs BB18 and BD 16. The second antibody (2nd mAb) was biotinyié, as indicated in the section "Materials and methods", and its binding was revealed by streptavidune- phycoerythrin.

- Figure 10 shows the SDS-PAGE analysis of BB18 and BD16 immunoprecipitates obtained from ³² P metabolic labeling of cloned T cells. The immunoprecipitates were analyzed under non-reducing conditions (left-hand part) and under reducing conditions

(right part). The positions of the molecular weight markers are indicated on the left in kilodaltons.

- Figure 11 represents the SDS-PAGE analysis of BB18 and BD 16 immunoprecipitates obtained from metabolic labeling with ³² P (left part) or surface labeling with ¹²⁵ 1 (right part) cloned T cells. Immunoprecipitates were analyzed under non-reducing conditions on both sides. The positions of the molecular weight markers are indicated on the right in kilodaltons. In addition, a CD 18 mAb was used as a control.

- Figure 12 represents the effect of mAbs BB18 (curve a) and BD16 (curve b) on the intracellular Ca ²⁺ concentration of Jurkat cells. The arrows indicate the nature and time in addition to the MCA. Each trace is representative of at least three experiences. The Y axis represents the intracellular Ca ²⁺ concentration in nM, while the X axis corresponds to time in seconds.

- Figure 13 represents the kinetics of the proliferative responses of PBL to a CD2 pair of mAb (CD2X11 + D66) in the presence or absence of mAb BB18 or BD 16. Acm BB18 or BD16, soluble and purified, with a final concentration of 5 μg / ml. In contrast, incubation of PBMC with PMA induces rapid negative modulation followed by complete re-expression within 24 hours. This molecule is not specific for the T cell because it is also expressed in EBV-transformed B cell lines. In addition, cells proliferate when this dimer has been stimulated in the presence of submitogenic concentrations of PMA.

I. MATERIALS AND METHODS

1. Monoclonal antibodies.

 The monoclonal antibodies used such as CD 1 "L404",

CD2 "X1 1", CD2 "D66", CD2 "GT2", CD3 "OKT3", CD4 "0516", CD8 "L533",

CD8 "OKT8", CD 18 "M232" and CD25 "BC96" were either locally produced (23) or purchased commercially. BB18 monoclonal antibodies and

BD16 were prepared by immunization of Balb / c mice with the thymic clone B12, CD4 ⁺ CD8 ^{+ (22)} (three intravenous injections with 20x10 ⁶ cells). Spleen cells from immunized mice were fused to the NS I cell line five days after the last injection. The initial screening by indirect immunofluorescence and flow cytometry using a Facstar (Becton Dickinson, Mountain View, CA) retained all the supernatants of hybridomas reacting with the immunizing cells but not or weakly with the mononuclear cells of the peripheral blood at rest. Cultures containing the above-mentioned monoclonal antibodies were cloned twice by limiting dilution. Serial passages of the cloned hybridoma were performed by ip injection in Balb / c mice sensitized with pristane. The ascites fluids were collected, ultracentrifuged and affinity purified, if necessary, on a protein A-Sepharose column. 2. Isolation of cell populations.

Human PBMCs were prepared by Ficoll-Isopaque density gradient centrifugation. The unfractionated population was separated into E rosette-less (E-) and E rosette-plus (E-) populations by interaction with 5% sheep red blood cell (SRBC). The mixture was deposited on Ficoll-Isopaque and the E- cells were recovered from the interface while the E ⁺ cells were obtained from the pellet after hypotonic lysis of the SRBC.

 The monocytes were obtained from E- cells by adhering to the glass overnight. The peripheral blood mononuclear cells CD2 + CD3 were obtained by complement-dependent lysis with a CD3 mAb in the E + cell fraction. Normal and leukemic samples were obtained from the Saint-Louis Hospital Blood Bank and cryopreserved as previously described (23).

 3. Cell cultures.

 Human T cell clones were obtained as described elsewhere (22) and cultured in RPMI-1640 medium (GIBCO, Paisley, Scotland) containing 2mmol / liter of L-glutamine, penicillin (100 U / ml) , streptomycin (100 μg / ml), 10% heat-inactivated human serum and recombinant interleukin 2, kindly provided by Roussel-Uclaf (Romainvilie, France) (30 U / ml.) The cloned cells have been restimulated every 7 days with feeder cells in the presence of purified PHA (Wellcome, Beckenham, UK) at 1 μg / ml and of recombinant interleukin 2. The feeder cells were a mixture of irradiated aliogenic LSPs from three donors (the same donors being used for several months). Cultures of EBV-transformed leukemia or B cell lines and hybridoma cell lines were mycoplasma free and maintained logarithmic in RPMI containing 10% selected heat-inactivated fetal calf serum and antibiotics.

 4. Immunofluorescence studies.

Indirect immunofluorescence studies have been performed using anti-mouse Ig conjugated to isothiocyanate fluorescein (FITC) or phycoerymthrin (PE) from Meloy Laboratories (Springfield, VA) as previously described (22). The fluorescence was read using a Facstar microfluorometer (Becton Dickinson, Mountain View, CA).

For cross-biocage experiments, the cells were first incubated with an excess of a first mAb for 30 min., Then they were centrifuged and incubated with the appropriate dilution of a biotinylated mAb. Controls included an incubation during the first stage with the same unconjugated mAb or with an inadequate mAb. After the final washes, the cells were resuspended in 0.3 ml of a 1% formalin solution in PBS. The purified antibodies were labeled with biotin using a standard protocol. Briefly, after dialysis in carbonate buffer (pH 8.8), the antibody was incubated (1 mg / ml) for 15 min. at room temperature with act-biotin (IBF, Villeneuve-La-Garenne, France). The reaction was stopped by addition of NH ₄ Cl IM, and the free biotin was removed by gel filtration. The conjugated mAbs were kept frozen at -70 ° C until use. The fluorescence emissions of FITC and PE were discriminated using band pass filters at 530 ± 15 and 575 ± 12.5.

5. PMA-induced modulation of expression at the cell surface.

 PBMCs were incubated with PMA (Sigma Chemical

Co., St. Louis, MO) at 1 μg / ml in a culture medium containing 10% of

FCS during the times indicated. After complete washing, the cells were labeled by indirect immunofluorescence using a battery of monoclonal antibodies against various surface molecules.

 6. Labeling and immunoprecipitation of cell surface proteins.

Cloned human T cells were labeled on the surface with lactoperoxidase and four additions of H ₂ O ₂ as described above. The cells were then lysed in a standard 10 mM Tris-HCl lysis buffer (pH 7.4), containing 1% (PV) of Nonidet P40, 0.15M of NaCl, 1 mg / ml of BSA and 2 mM of PMSF (Phenyl Methyl Sulfonyl fluoride), 20 mM iodoacetamide, and aprotinin at 1 lU / ml as protease inhibitors. Immunoprecipitation and analysis by SDS-PAGE were performed as described (22). Analyzes by electrophoresis on two-dimensional non-reducing / reducing gel diagonally were performed using PHAST (Pharmacia) gels of 7.5% acrylamide for the first and second dimensions according to the manufacturer's recommendations (Pharmacia, Uppsala, Sweden ). Autoradiography of dried gel plates was performed at -70 ° C. Molecular weights were calculated with reference to the mobility of standard proteins.

 In certain experiments, following SDS-PAGE under reducing conditions, the bands separated by autoradiography of the dried gel were visualized, they were excised, digested with protease V8 in a buffer sample (at 1 mg / ml) , and the fragments were analyzed in a second SDS-PAGE experiment. The nitrogen-bridged sugars were eliminated by treatment of the specific immunoprecipitates with endo-beta-N-acetylglucosaminidase-F (endo-F) (Boehringer Mannheim, Meylan France). The digestion was carried out by bringing the BB18 and BD 16 immunoprecipitates to the boil for 4 minutes in 50 μl of 100 mM potassium phosphate buffer, pH 6.5 containing 50 mM EDTA, 0.5% SDS and 1% beta-mercaptoethanol. 100 μl of 100 mM potassium phosphate buffer (pH 6.5) containing 50 mM EDTA, 1% NP40 and 1% beta-mercaptoethanol was added before the addition of 12 μl (0.6 IU) of endo-F. After 24 h of incubation at 37 ° C, the reaction was stopped by adding a buffer sample.

7. Proliferation tests.

50,000 PBMC were cultured in triplicate in 96-well rounded bottom plates (Costar, Cambridge, MA) in a total volume of 0.2 ml RPMI 1640, supplemented with 10% fetal calf serum in one volume 0.15 ml total of RPMI 1640, supplemented with 10% heat-inactivated human serum. Various concentrations of purified antibodies were added with 0.2 ng / ml PMA. After various periods of time, the wells were individually radiolabelled with 1 μCl (1 Cl = 37 Gbq) of ( ³ H) Tdr and harvested 16 hours later. The incorporation of ( ³ H) Tdr was measured using a liquid scintillation counter (Rac Beta 121 1/1212 LKB, Finland). 8. Phosphate marking and immunoprecipitation

The phosphorus labeling was carried out as follows. Cloned T cells were washed twice in phosphate-free medium containing 5% dialyzed human AB serum and then incubated in the same medium at 50 x 10 ⁶ cells in 1 ml for 30 minutes at 37 ° C.

Then 1 mCi of [ ³² P] -orthophosphoric acid was added.

After 3 hours of labeling, the cells were washed twice in PBS before their lysis in 2-3 ml of buffer containing 10 mM Tris, pH 8.2, containing 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mg / ml of BSA,

10 mM NaF, 1 mM PMSF and 10 mM iodoacetamide. Immunoprecipitation and SDS-PAGE analysis were performed as described (25).

9. Ca ²⁺ measurements.

The intracellular Ca ²⁺ concentration was measured as previously described (26). Briefly, Jurkat cells were washed twice in medium containing 25 mM HEPES (pH 7.2), 125 mM NaCl, 5 mM KCl, 1 mM Na ₂ HPO ₄ , 1 mM Ca Cl ₂ , 0.5 mM MgCl ₂ , and 1% glucose (all subsequent incubations and washes were performed in the same medium) and they were resuspended at 30x106 cells / ml in the presence of 3 μM of Fura 2-AM (Calbiochem, meudon,

France) for 30 minutes at 37 ° C in the dark. After this incubation period, the cell suspension was diluted twenty times and incubated for an additional 30 minutes under the same conditions. The cells were then washed twice and resuspended at 2 × 10 ⁶ cells / ml in a cuvette at 37 ° C. for the Ca ²⁺ measurements.

II. RESULTS A) STUDY OF AMC BB18

In an attempt to define activation-specific cell surface glycoproteins, we have, according to the invention, used mAbs obtained by repeated immunizations with highly functional human T cell clones (21). In particular, an mAb was initially identified on the clone called B12 (22) of human thymus T cells. called BB18, recognizing a new disulfide-linked dimer composed of subunits of 150 ± 10kDa. This structure, which is weakly expressed in normal peripheral blood lymphocytes, is quickly found to be strongly expressed during activation.

 A.1. BB18 mAbs react strongly with cloned T cells and weakly with resting human T cells by flow cytofluorometry.

In order to obtain monoclonal antibodies defining the stages of T cell activation, six-week-old Balb / c mice were immunized with cloned human thymic cells (22). An mAb of the IgG1 isotype, named BB18, was thus isolated. Table 1 summarizes the reactivity of the aforementioned mAb with many normal human and hematopoietic malignant cells. The molecule recognized by this mAb is weakly expressed on resting T cells but not at all on B cells from peripheral blood or lympoid organs. Among the leukemic lymphoid cell lines tested, all T cells were strongly labeled, while the Burkitt lymphoma cell lines were not reactive. Other malignant cell lines such as K562 (erythroleukemia), U937 (monocytic), HL60 (promeylocytic) or SKNSH (neurobiastoma) were not reactive. In contrast, long-activated T cells (IL2-dependent T cell clones) or B (EBV-transformed cell lines B) were found to be positive with this mAb. In addition, BB 18 strongly marked the YT cell line of activated NK, CD3 negative tumor cells and its CD 25 mutant, YT2C2. The structure recognized by BB18 appears to be strongly expressed immediately after cellular activation as shown by the representative kinetic experiment carried out with mononuclear cells from the peripheral blood of a normal individual stimulated with PHA (FIG. 1). The early increase in its expression occurred in parallel with that of C25 during the first days of activation of the T cell, but it remained persistent. A similar increase in reactivity to BB18 was also observed when peripheral blood lymphocytes (PBL) were stimulated with either allogenic cells, a CD3 mAb, or IL2 (data not shown). Therefore, the increased reactivity with the BB18 Antibody clearly defines the activation state in T cells. TABLE 1

 Reactivity of BB 18 Antibody with populations of hematopoietic cells

 a Reactivity determined by flow microfluorometry

b All cells were weakly labeled with mAb BB 18 A.2. The intensity of the reactivity with the BB18 Antibody is reduced shortly after the incubation with PMA.

 As the expression of several activation-inducible structures is upregulated by PMA, we wanted to determine whether PMA was capable of rapidly increasing reactivity with the BB18 Antibody. Surprisingly, after a 6 hour incubation with PMA at 1 μg / ml, the labeling with the BB18 Antibody was no longer detectable. As shown in the representative experiment presented in FIG. 2, the reactivity of the T cells to CD3, to CD4 and to Antibody BB18 decreases when PMA is added to the cultures. More precisely, after 6 hours with PMA, the downregulation of the molecule reacting with the Antibody BB 18 is maximum while a large fraction of the cells had lost the expression of CD4. The modulation of the CD3 molecule reaches its maximum after 24 hours while the modulation of CD4 is completed after 10 hours. However, as previously reported after 24 hours of culture in the presence of PMA, CD25 is induced and the expression of CD8 is not modified by this treatment. A.3. Biochemical analysis of the immunoprecipitated molecule by mAb BB18 after labeling the cell surface.

In order to define the molecule identified by mAb BB18, clones of human T cells were labeled with Iodine 125 on their external face, according to the lactoperoxidase method, and iysed. The BB 18 immunoprecipitates obtained from these lysates of labeled cells were then analyzed by SDS-PAGE. The autoradiography shown in Figure 3 indicates that mAb BB18 immunoprecipitates from a representative T cell clone. A predominant band migrating under non-reducing conditions with a molecular mass of approximately 300 kDa. CD18 "M232" mAbs used as immunoprecipitate control of three protein bands from the same lysate characteristic of CD 18 molecules and two different CD 11 molecules, while no protein band is obtained with an inappropriate IgG1 type mAb . In addition, as shown in Figure 4, the immunoprecipitate obtained with the BB18 Antibody consists mainly of homodimers linked by disulfide bridges of approximately 300 kDa in the first non-reducing dimension, which resolves in a second reducing dimension into a predominant protein band of approximately 150 kDa below the diagonal of the two-dimensional gel.

4. Stimulation with soluble BB18 mAbs induces proliferation of LSPs in the presence of a submitogenic concentration of PMA.

 It has been shown previously that the stimulation of certain surface molecules, with their agonist antibody, in conjunction with PMA, induces a proliferation of T lymphocytes (24, 25, 26). Therefore, the possibility that cell proliferation could be induced by submitogenic doses of PMA and purified BB18 mAbs was tested. The results of a representative kinetics experiment, shown in FIG. 5, indicate that BB18 mAbs, used either at 5 or at 10 μg / ml with 0.2 ng / ml of PMA, induce a strong proliferative response on PBMCs freshly isolated peaking on day 4. Under similar experimental conditions, a CD 1 "L404" mAb of identical isotype has been shown to be incapable of triggering lymphocyte proliferation, as previously reported , a CD3 mAb vigorously stimulates cell proliferation.

We therefore identified a new surface structure of a human lymphocyte cell with the mAb called BB 18. The immunoprecipitated molecule using BB18, from lysates of cloned T lymphocytes marked on the surface, appeared to be a homodimer linked by bridges. disulfides comprising subunits of approximately 150kDa. This structure is poorly detectable on resting peripheral blood T cells, while its expression increases rapidly after activation of the T cell and remains at this high level for some time after initial triggering. In addition, the BB18 Antibody reacts strongly with all the cloned T cells tested regardless of their CD4 / CD8 phenotype and their TcR (data not shown). Cell distribution indicates that mAb BB18 does not react with resting B cells, while long-standing EBV-transformed B cells are weakly labeled. In contrast, Burkit's lymphoma cell lines (Daudi and Namalva) clearly lack responsiveness. Furthermore, leukemic but not myelomonocytic lymphoid cell lines are reactive to BB18. Non-cellular distribution, as well as biochemical analysis of the structure recognized by BB18, have established that this molecule is different from all the surface structures of leukocytes previously known.

 The functions of other surface molecules, such as LFA-1, CD4 and CD8 have been demonstrated by the ability of certain specific mAbs to inhibit the natural or cytotoxic killer activities of T lymphocytes. Attempts to inhibit, with a BBI8 mAb purified, the cytotoxic function of allosensitized lymphocytes in vitro 6d., of cytotoxic cloned T lymphocytes or of NK cells. have not been successful. The results suggest that the epitope recognized by mAb BB18 is not involved in cytotoxic functions, any more than in the adhesion of the effector to the target cell.

 Of particular interest is the fact that cocultures of PBMC with submitogenic concentrations of PMA and purified mAb BBI8 resulted in a vigorous proliferative response, reaching a maximum on day 4. However, the comitogenic response measured under these experimental conditions does not reach the magnitude of the response observed with a CD3 trigger. It should be noted that the reactivities to CD3 and to BB18 both decrease after incubation of the LSPs with PMA. However, the kinetics of the decrease in reactivity is different for the two structures.

Several molecules have been described which, when reacted with an agonist mAb under appropriate conditions, are capable of transducing comitogenic signals with PMA. The CD28 molecule, a 44 kDa disulfide-bridged homodimer, preferentially expressed on a T cell subpopulation, was the first structure described to induce costimulatory signals (25, 26). The effect has been shown to regulate the stability of the mRNA encoding lymphokines (27). Other examples of costimulatory signal mediator molecules include the molecule CD26 (4, 5), identifying dipeptidylpeptidase IV, which is a 120 kDa structure; CD69, a very early activation heterodimer with disulfide bridges, of 28.32 kDa (17, 18); the specific T cell molecule identified by mAb 10D 1, which is a 90 kDa disulfide bridge homodimer (28); CD60 "UM4D4" recognizing a hydrocarbon structure expressed on various gangliosides (29). The CD43 mAbs are particularly interesting, identifying a highly sialylated glycoprotein of 95 kDa, which similarly to the CD3 and CD2 mAbs can directly trigger the activation of T cells in the presence of monocytes. It has been recently reported that a monoclonal antibody reactive with CD5, unlike the previously described CD5 mAb, is capable of inducing T cell proliferation in the presence of monocytes (15). This raised the possibility that only certain epitopes of the CD5 molecule, as in the case of CD2, are involved in the activation of T cells. Finally, it should be mentioned that, unlike the epitope recognized by the Antibody BB18, the surface expression of the majority of signal transduction structures is not increased during activation of the cell.

 Although human T cells can be activated through several distinct surface molecules, these activation pathways appear to share common signal transduction mechanisms. It is well known that activation of the T cell via CD2 or CD5, but not via CD43 (16) is dependent on the expression of CD3-TcR. Although no physical link of the BB18 molecule with CD3-TcR has been demonstrated according to the invention, (the AMC has labeled YT2C2 CD3-TcR negative lymphocytes), links are possible with components such as CD3 chain.

B) STUDY OF THE BD16 ANTIBODY

 B.1. BD 16 and BB 18 mAbs recognize an identical structure on human T cells

In order to obtain monoclonal antibodies defining the stages of T cell activation, we repeatedly immunized 6 week old Balb / c mice with cloned human thymic cells (22). It was reported above that an IgG1 isotype mAb, named BB18, recognized a molecule expressed at a low level on resting T cells, but not at all on B cells or peripheral blood monocytes or lymphoid organs. In addition, the structure identified by BB18 appeared to be strongly expressed immediately after cellular activation. Then, in a different fusion, we isolated a second mAb, named BD16, presenting a similar reactivity motif in immunofluorescence studies and immunoprecipitating a dimeric structure with disulfide bridges of 300 kDa originating from the surface of the T cell. to determine if BB18 and BD 16 identified the same structure, an additional biochemical analysis of the molecule immunoprecipitated by the two mAbs was performed after labeling the cell surface. Human T cell clones were labeled on their outer surface, according to the lactoperoxidase method, and lysed. The BB18 and BD16 immunoprecipitates obtained from these labeled cell lysates received treatment or simulated treatment with endo-F before being analyzed under reducing conditions by SDS-PAGE. FIG. 6 represents an autoradiography revealing an identical band of approximately 120 kDa in the two immunoprecipitates treated with endo-F, while the characteristic band of 150 kDa was obtained with the samples having received a simulated treatment. These results strongly indicated that the two mAbs react with an identical structure of the cell surface. Additional proof was obtained after digestion with protease V8 of the 150 kDa band immunoprecipitated with BB18 and BD16, isolated from a one-D gel. The results, shown in Figure 7, revealed that BB18 and BD 16 were split into three main polypeptides, which resulted in an identical band pattern after V8 protease digestion.

B.2. The surface structure identified by mAbs BD 16 and BB18 undergoes a negative regulation on PBLs during a preincubation with BB18

In order to determine whether the expression of the reactive structure with the BB18 BD 16 mAbs was modified by an interaction with these mAbs, PBLs were incubated for variable periods with concentrations. saturating either with mAb or with irrelevant IgGl as a control. Figure 8 shows that after 8 hours of incubation with a BB18 mAb, the reactive structure with BB18 or BD16 was no longer detected by a BB18 or BD 16 biotiny mAb. The use of a goat anti-mouse Ig labeled with FITC revealed that mAb BB18 was not present on the cell surface of cells preincubated with mAb BB18 and could not be responsible for the lack of reactivity of biotiny BB18. It will be noted that the modulation of the reactive molecule for ieBB18 did not modify the reactivity of a CD 18 "M232" mAb vis-à-vis the treated cells. The same figure indicates that preincubation of PBLs with saturated concentrations of BD16 produced only a partial modulation of the BD 16 / BD 18 reactive molecule, even after 24 hours of preincubation. In addition, the results presented in Figure 8 suggested that 3B18 and BD16 could identify distinct epitopes with an identical surface structure. Biotinyie mAb BB18 marked a cell number comparable to that of cells labeled with FITC-labeled goat anti-mouse immunoglobulin (GAM) after 24 hours of pre-incubation of cells with mAb BD16, while fixation of mAb BD16 was completely inhibited. To confirm the fact that the two mAbs identified distinct epitopes, we carried out an additional experiment, under conditions which prevented negative regulation of the cell surface structure reactive to BB18 (Figure 9). While a saturating amount of BB18 inhibited the binding of biotiny BB 18 and not that of biotiny BD16, on PBLs, a saturated amount of BD16 did not prevent the binding of biotiny BB18. Taken together, these results indicate that BB18 and BD16 identify two distinct epitopes of the same structure of the lymphocytic surface.

B.3. The 150 kDa cell surface molecule reactive to mAbs BB18 and BD16 is a phosphoprotein and the mAb BD16 co-precipitates a phosphorylated structure of 100 kDa.

Additional biochemical characterization of the BB 18 and BD16 reactive structure was performed after metabolic labeling with 32 P of cloned T cells. Under non-reducing conditions, a single 300 kDa band was obtained for the two immunoprecipitates (Figure 10), and under reducing conditions, a main phosphoprotein band of 150 kDa was detected (Figure 10 and Figure 11, left part). ). This protein has migrated to exactly the same molecular weight apparent that the 150 kDa protein bands observed after labeling the cell surface (FIG. 11, right part). It will be noted that an additional phosphorylated band of 100 kDa has only been regularly observed in the BD16 immunoprecipitates under reducing conditions (FIG. 10, right part and FIG. 11, left part). This additional band has never been observed in BD 16 immunoprecipitates from single-sided labeled cell lysates. Furthermore, the discovery that this 100 kDa phosphorylated band was not visible in the BD16 immunoprecipitates analyzed under non-reducing conditions (Figure 10, left part), suggests that this additional band formed multimers of molecular mass. higher not entering the gel.

 B.4. Acm BB 18, unlike Acm BD 16, induces weak but significant intracytoplasmic calcium mobilization in Jurkat cells

It has previously been reported that mAb BB18 triggers PBL proliferation when used in conjunction with submitogenic doses of PMA (30). Under similar experimental conditions, the AMC BD 16 failed to activate the PBLs (data not shown). In order to explore the possible differences in biochemical signaling responsible for these contrasting results, the intracellular Ca ²⁺ concentrations were measured after incubation of the Jurkat T cell line with a purified BB18 or BD 16 mAb. The results of a representative experiment are shown in Figure 13 and indicate that a small but reproducible increase in intracellular Ca ²⁺ concentration was obtained only with mAb BB18. In addition, a subsequent addition of purified Acm CD3 after Acm BB18 resulted in the expected rapid mobilization of Ca ²⁺ (Figure 12, curve a). It should be noted that the mobilization of Ca induced by mAb CD3 after the BD 16 incubation (Figure 12, curve b) regularly had a magnitude lower than that obtained after the BB18 trigger. Cross-linking of the mAb with rabbit anti-mouse antiserum did not change the result. B.5. The proliferation of PBL induced by CD3 is inhibited by mAb BD 16 but not by mAb BB18.

 In order to further study the difference between the roles played by the two epitopes recognized by mAbs BB18 and BD 16, we studied their effect on the proliferative response of PBL to a soluble or immobilized CD3 "OKT3" mAb. As indicated in Table 2, a very strong proliferative response of the PBLs was obtained with the CD3 mAb immobilized on plastic, with a response peak on day 4, while the LSP response to the soluble CD3 mAb "OKT3" was of a lower magnitude. In this representative kinetic experiment, Acm BB18 did not modify the proliferative response of PBLs to AcmCD3, whether in soluble or immobilized form. On the other hand, the presence of purified Acm BD 16 in the culture induced a strong inhibition of the proliferative responses induced by CD3. This inhibition was observed at all measurement points for both proliferation induced by soluble and immobilized CD3, and it represented approximately 50% inhibition.

B.6. CD2-induced PBL proliferation is greatly increased by mAb BD 16 but not by mAb BB18.

In addition to Acm CD3, some pairs of Acm CD2 have been shown to be highly comitogenic for LSP. This effect manifests itself in the presence of variable numbers of accessory cells (31). The functional effects of BB18 and BD 16 mAbs were tested on CD2-induced LSP proliferation. Figure 13 shows that after the proliferation peak expected in response to DC2X1 1 and D66 on day 4, the response decreased very quickly on days 5 and 6 (31). In this representative kinetic experiment, mAb BB 18 did not modify the kinetics of the proliferative response of PBLs. Surprisingly, in the presence of purified mAb BD 16 in the culture, the maximum proliferative response was prolonged until day 5, and then decreased. This resulted in a doubling of the cpm on day 5. Finally, this prolonged proliferative response to CD2, induced by mAb BD 16, was observed independently of the pair of mAb CD2 used. As shown in Table 3, on day 5 of culture, when proliferative responses to various pairs of CD2 declined, AMC BD 16 induced increased proliferation with each combination of CD2. AT again, note that this amplification effect was limited to mAb BD16 and was not observed with mAb BB18 or with solitary CD2 mAbs (data not shown).

TABLE 2

Acm BD 16 inhibits CD3-induced PBL proliferation

Freshly isolated PBLs were incubated for various durations with or without mAb, as described in the "Materials and Methods" section. The soluble and purified BB18, BD 16 and CD3 mAbs were used at final concentrations of 5 μg / ml.

* cpm incorporated by PBL. The results are expressed as the average of tripled samples for which the ESM has always been less than 10% of the average. TABLE 3

 MAb BD 16 enhances the proliferative response of PBL to various pairs of

 CD2

 The PBLs were cultivated for 5 days in the medium, CD2 mAbs alone with or without BB18 or BD16 as a control (results not exposed) or a combination of CD2 mAbs with or without BB18 or BD16 mAbs. Significant proliferation was observed only in wells containing pairs of CD2 mAbs. CD2 mAbs were used in the form of diluted ascites fluid, while BB18 and BD16 were purified mAbs used at final concentrations of 5 μg / ml.

* cpm incorporated by PBL. The results are expressed as the average of tripled samples for which the ESM has always been less than 10% of the average.

 DEPOSIT OF ANTIBODIES

 Antibody-producing hybridoma cell lines

BD16 and BB18 have been deposited in the European Collection of Animal Cell

Cultures (Porton Down, Salisbury, Wiltstrive SP4 03G, England), January 7

1992 and received the numbers 92010801 for the antibody BD16 and 92010802 for the antibody BB18.

 Antibody-producing hybridoma cell lines

BM4, BM 71 and BM63 have been deposited at 1 "European Collection of Animal

Cell Cultures (Porton Down, Salisbury, Wiltstrive SP4 03G, England) on January 12, 1993 and received the numbers 93 01 1201 for BM4, 93 01 1202 for BM71 and 93 01 1203 for BM 63.

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Claims

1. Antigenic protein consisting of a phosphorylated glycoprotein with a molecular weight of 150 ± 10 kDa determined by electrophoresis of SDS PAGE type under reducing conditions, expressed on the surface of T lymphocytes of human blood preferably activated.

 2. Antigenic protein in the form of a disulfide-linked homo-dimer with a molecular weight of 300 ± 20 kDa determined by SDS PAGE type electrophoresis under non-reducing conditions, expressed on the surface of preferably activated human blood T lymphocytes, composed of subunits according to claim 1.

 3. A protein derivative according to claim 1 or 2, characterized in that it is the protein or a fragment comprising the essential antigenic sites of the protein, in glycosylated or non-glycosylated form or comprising non-natural glycosylations .

 4. Derivative of a protein according to one of claims 1 to 3 characterized in that it is the protein or a fragment thereof comprising essential antigenic sites, in phosphorylated or non-phosphorylated form or comprising unnatural phosphorylations.

 5. Protein or derivative according to one of claims 1 to 4 characterized in that it comprises a marking allowing its assay in vitro or its location in vivo or ex-vivo.

 6. Antibody directed against the protein according to one of claims I to 5.

 7. Monoclonal antibody recognizing an epitope of a protein according to one of claims 1 to 5.

8. Antibody according to claim 6 or 7 characterized in that it is an immunoglobulin of subclass IgG ₁ .

 9. Antibody according to one of claims 6 to 8 characterized in that it is produced by a hybridoma cell line deposited at the ECACC under the number 92010801 for the antibody BD 16.

10. Antibody according to one of claims 6 to 8 characterized in that it is produced by a hybridoma cell line deposited at the ECACC under the number 92010802 for the antibody BB18.

1 1. Antibody according to one of claims 6 to 8 characterized in that it is produced by a hybridoma cell line deposited at the ECACC under the number 93 01 1201 for the antibody BM4.

 12. Antibody according to one of claims 6 to 8 characterized in that it is produced by a hybridoma cell line deposited at the ECACC under the number 93 01 1202 for the antibody BM71.

 13. Antibody according to one of claims 6 to 8 characterized in that it is produced by a hybridoma cell line deposited at the ECACC under the number 93 01 1203 for the antibody BM63.

 14. Antibody according to one of claims 5 to 13 characterized in that it comprises a marking allowing its assay in vitro or its location in vivo or ex-vivo.

 15. Hybridoma cell line, characterized in that it is capable of producing the monoclonal antibody according to one of claims 6 to 14.

 16. Use of a monoclonal antibody according to claims 9 to 13 for immunoprecipitating proteins according to claims 1 or 2.

 17. Diagnostic kit comprising a protein or one of the antibodies according to one of claims 1 to 14.

 18. Use of a protein or an antibody according to one of claims 1 to 14 for the rapid diagnosis of a state of early activation of lymphocyte T cells.

 19. Medicament comprising, as active principle, a protein or an antibody or their fragments according to one of claims 1 to 14.