MXPA97009446A - Method for preparing a monoclonal antibody, monoclonal antibody, a pharmaceutical composition and a diagnost reagent - Google Patents

Abstract

The present invention relates to an efficient method for producing monoclonal antibodies against cell surface antigens and viruses. The method is adapted for antigens, which are present only in relatively small amounts, or antigens of which only very small amounts are available or antigens that easily lose their conformation in vivo. Thus, the method according to this invention comprises a series of steps, comprising a step in which the B cells of a mammal injected with a material comprising surface antigen are enriched with respect to the relative number of specific B cells, and a step that involves a small scale fusion technique

Description

METHOD FOR PREPARING A MONOCLONAL ANTIBODY, MONOCLONAL ANTIBODY, A PHARMACEUTICAL COMPOSITION AND A DIAGNOSTIC REAGENT The present invention relates to a method for preparing a monoclonal antibody against a surface antigen, and in particular such a surface antigen, which constitutes only a negligible amount of the total antigen injected into a mammal and a surface antigen, which easily loses its conformation live. The problem of an insignificant amount of antigen relative to the total amount of antigen, for example, ensues when the antigen material used to inject a mammal is derived from other mammalian species. The problems associated with the above specified antigens become clear when, for example, it is desired to make monoclonal antibodies against the specificity that determines part of a T cell receptor (TCR). Such antibodies (monoclonal) are known as anti-clonotype antibodies, since they recognize the antigen-specific part (or cloneotype structure) of a T cell receptor of a particular T cell clone. While monoclonal (murine) anti-clonotype antibodies against T cell receptors (TCR) have only been reported occasionally, there are still fewer monoclonal (murine) anti-clonotype antibodies known against human cell receptors. T cells are difficult to grow, making it difficult to obtain and purify a sufficient amount of antigen, ie, T cell receptor protein. This in turn makes it difficult to obtain an immune response and also makes classification difficult. In one of the few cases where a monoclonal anti-clonotype antibody is available against a human TCR, these problems did not arise because the antigen was present in Jurkat leukemia cells (ref 1). As the leukemia cells can be grown in unlimited amounts, the problem of a shortage of antigen did not arise. Accordingly, until now there has not been a method available to prepare monoclonal antibodies against rare and / or highly unstable antigens without having to unfavorably classify large numbers of hybridoma clones. The aim of the present invention is to provide a method for preparing a monoclonal antibody against a cell surface antigen under unfavorable circumstances as described above, said method dramatically reducing the number of hybridoma clones to be classified, the method being according to to the most efficient or even successful invention where methods according to the state of the art fail. To this end, the method according to the invention comprises the steps of 1) injecting a mammal with material comprising cell surface antigen, choosing said material from the group consisting of i) whole cells and ii) a membrane fraction obtained when treating whole cells; 2) isolating a fraction of cells containing B cells from the spleen of said mammal; 3) enriching said cell fraction obtained in step 2 in B cells specific for said cell surface antigen, by contacting the fraction of the cell with material bound to a cell carrier related to said complete cells, said bound material the carrier of cells lacking said cell surface antigen, and separating the B cells bound to the carrier-linked material from the related cells of the enriched fraction of B cell-containing cells for use in the next step; 4) subject the enriched fraction of cells containing B cells obtained in the previous step to limit the dilution, followed by clonal expansion; 5) select a B cell clone and immortalize said selected B cell clone, using a small scale fusion technique; Y 6) select and clone a hybridoma capable of producing antibodies, which specifically bind said cell surface antigen, followed by the isolation of a fraction comprising monoclonal antibody from the supernatant of said hybridoma. For the purpose of maintaining the description and claims legible and intelligible, the term cell is used herein not only to indicate mammalian cells but also viruses, and in particular viruses comprising membrane. The term "material bound to the cell carrier" encompasses complete or intact cells (and viruses), membrane fractions of said whole, intact cells (and viruses), or substantially purified surface antigens or complexes thereof. Unless otherwise specified, the term "whole cell" refers to cells having the surface antigen of interest. The term "related cells" refers to cells that differ in preference only in that they lack the surface antigen of interest, or more specifically, at least, they lack the immunological determinant against which it is desired to obtain a monoclonal antibody. Surprisingly, it proves to be possible to enrich the cell fraction in B cells specific for the cell surface antigen without the use of cell surface antigen by contacting the cell fraction containing isolated B cells with cell material from the cell surface. the same species, since the material comprising cell surface antigen was derived from, but lacking the cell surface antigen. In other words, by bringing the B cells into contact with irrelevant cell material or related cells, and removing the bound B cells, enrichment was achieved. Thus, it is possible to prepare a monoclonal antibody against a lesser cell surface antigen, even if said cell surface antigen suffers from conformational instability. European patent EP 0 488 470 and ref. 3, which is based on this European patent, describes a method in which I) a mammal is injected with an antigen, II) a fraction containing B cells is isolated, III) B cells are selected specific for the antigen, IV ) the enriched fraction is subjected to clonal expansion and then V) select a clone of B cells and immortalize using a small scale fusion technique and, VI) the hybridoma is selected and cloned followed by the isolation of a fraction comprising antibody monoclonal In step III, B cells are selected by ligating them on a plastic coated surface antigen, or by branching with antigen coated with paramagnetic beads. Non-specific B cells are removed by washing. Thus, apart from other differences, this method depends on the wide availability and use of antigen -during the selection 2 mg / ml of antigen is used to cover the plates with antigen-, while the present invention solves the problem for the case in which the antigen is available in very limited quantities and even highly impure. A preferred embodiment is characterized in that the mammal injected with surface antigen is of a species different from the mammalian species from which the surface antigen originates. The method according to the present invention is very suitable for preparing monoclonal antibodies when many antigens capable of causing an immune response are present. This situation exists in particular, when the surface antigen has a constant section and a variable section, wherein at least a part of said variable section defines a specificity that determines part of said surface antigen. According to a preferred embodiment, a material comprising receptor molecules is used as the material comprising cell surface antigen. The method according to the invention is particularly suitable for preparing monoclonal antibodies against the specificity that determines part of a receptor molecule. The specificity that determines part of the receptor molecule alone is a minor part of the receptor molecule, which in itself is a minor constituent of all molecules - and therefore antigens - on the surface of a cell. A preferred object according to the invention is a T cell clone, said T cell clone is used to prepare the material comprising receptor molecules. A T cell can be used as a whole cell, or used for the preparation of a membrane fraction thereof. Thus, in the previous case, the term prepares can, here, be related to the simple isolation of a T cell or to redispersing in a different medium. According to the preferred embodiments, the membrane fraction in step 1 is obtained by mechanical treatment of the whole cells, and the material comprising cell surface antigen is injected into the mammal in the absence of an auxiliary. Both measures help to prevent the loss of the in vivo conformation of the surface antigen. In addition, the enriched cell fraction containing B cells is preferably further enriched (step 3) by contacting the cell fraction with material comprising cell surface antigen bound to the carrier chosen from the group of j) whole cells, jj) a membrane fraction obtained from said complete cells, and jjj) substantially purified cell surface antigen, and subsequently separates the B cells not bound to said material bound to the carrier of the B cells bound to said carrier-bound material comprising the additionally enriched cell fraction.

This additional enrichment can be designed as a positive selection technique, specifically selecting specific B cells. Thus, although any antigen is hardly available, additional enrichment can be achieved. According to a preferred embodiment, paramagnetic beads are advantageously used as the carrier. The use of paramagnetic beads during enrichment facilitates the separation of desired and unwanted B cells. A negligible amount of antigen also presents a problem during sorting. According to a preferred embodiment, selection in at least one of steps 5 and 6 is conducted using an agglutination test, wherein the supernatant of the B cell clone is contacted with a carrier coated with antibodies capable of binding antibodies of the species of the injected mammal used in step 1 and whole cells that support the cell surface antigen and agglutination is detected. Simply by mixing the B-cell culture supernatant, the whole cells and a carrier coated with antibodies capable of binding antibodies of the mammalian species used in step 1, allow rapid and very sensitive detection of suitable clones, while not it requires washing. Advantageously, whole cells related to whole cells but lacking surface antigen can be used as a control.

This allows rejection of false positive clones and saves time by avoiding superfluous small-scale mergers. According to the preferred embodiment of the method according to the invention, the B cell clone selected in step 5 is mixed with myeloma cells and subjected to mini-electrofusion. The mini-electrofusion allows the efficient fusion of very small numbers (for example hundreds) of cells. The invention also relates to a pharmaceutical composition comprising a monoclonal antibody prepared according to the invention, mixed with a suitable excipient. In addition, the present invention relates to a monoclonal antibody reagent with the clonotypic structure of a T cell receptor. Such a monoclonal antibody can be used for diagnostic purposes, as well as for the preparation of a pharmaceutical composition. More specifically, the T cell receptor is a T cell receptor associated with an autoimmune disease, and in particular with rheumatoid arthritis. Preferably, the monoclonal antibody is reactive with the T cell receptor of a HC gp-39 reactive T cell clone, and in particular with the T cell clone H243 (Access ECACC No. 96103122). Specific examples of suitable monoclonal antibodies are those produced by a hybridoma selected from the group consisting of TCR 69 (Access ECACC No. 961031 18), TCR 70 (Access ECACC No. 961031 19), TCR 72 (Access ECACC No. 961031 20) and TCR 83 (Access ECACC No. 96103121).

As indicated above, the invention also relates to a pharmaceutical composition comprising a monoclonal antibody according to the invention, mixed with a suitable excipient, suitable for the treatment of rheumatic arthritis. Finally, the invention relates to the diagnostic use of a monoclonal antibody selected from the group consisting of a monoclonal antibody prepared using the method according to the invention. Also a diagnostic reagent comprising the antibody is a modality of the invention. Now, the invention will be explained in more detail with reference to the following examples, said examples showing the best mode of carrying out the invention applied to the preparation of a murine monoclonal antibody specific for a cloneotype of a human T cell receptor.

LEGENDS FOR THE FIGURES Figure 1: Identification of H.243 T cell surface molecules immunoprecipitated by Mab directed against H.243. SDS / PAGE was performed under non-reducing conditions (channel 2, 3 and 4) or under reducing conditions (channel 5, 6 and 7) in a 10% gel. Channel 1: 10kD scale marker; Channel 2 and 5: Control Mab; Channel 3 and 6: TCR 83; Channel 4 and 7: anti-CD3, OKT3.

Figure 2: Anti-clonotype MAb recognizes TCRab in "Western blotting". Immunoprecipitated TCR / CD3 complexes of H.2233 were resolved on a 10% SDS-PAGE gel under non-reducing conditions and subsequently transferred to PVDF membranes. The membranes were incubated with Mab and finally the ligated Mab was detected using a second antibody of phosphatase-conjugated goat anti-mouse Ig. Channel 1: scale marker of 10kD, line 2: TCR 64, channel 3: TCR 66, channel 4: TCR 69, channel 5: TCR 70, channel 6: TCR 72, channel 7: TCR 73, channel 8: TCR 76 , channel 9: TCR 78, channel 10: TCR 79, channel 1 1: TCR 83, channel 12: Control Mab, channel 1 3: control medium.

Figure 3: Stimulated proliferation of immobilized anti-TCR MAb of human T cell clone H243. The proliferation induced by MAb directed against H.243 (TCR 64 to TCR 83) was compared with the control MAb as indicated and with TCR, which is an anti-clonotype MAb directed against another TCR. Each value represents the average count for 5 min of crops in quadruplicate +/- the standard deviation.

Figure 4: Pre-incubation with anti-TCR MAb inhibits or blocks the targeted antigen proliferation of the human T cell clone H.243. a) Inhibition by MAb directed against H.243 (TCR 64 to TCR 83) was compared to the control MAb as indicated and to TCR 44, which is an anti-clonotype MAb directed against another TCR. Each value represents the mean count for 5 min of cultures in quadruplicate +/- the standard deviation, b) dose response curves of strong MAb inhibition; TCR 64 (empty circles), TCR 70 (filled triangles), TCR 78 (empty squares), TCR 83 (filled squares) and MAb control 1 (filled circles). Each value represents the average count for 5 min of cultures in triplicate.

Figure 5: Anti-clonotype MAb induces anergy in human TH1-clone H.243 human. The immobilized anti-clonotype MAb or control MAb 1 were incubated overnight with H.2243 T cells. After removing the cells from the anergic stimulus, the cells were given a complete stimulus to increase the concentrations of peptide and DRB1 * 0401 coupled to APC. Proliferation was assessed by incorporation of 3H-thymidine. Each value represents the average count for 5 min of cultures in triplicate +/- the standard deviation.

Figure 6: Atrial H.243 T cells suppress the response of non-anergic H. 243 T cells. The immobilized TCR 76 or the MAb 1 control were incubated overnight with H.243 T cells. Then, both populations of T cells were used in a proliferation assay with increasing concentrations of peptide (or PHA) and APC using 2 x 10 4 per well. In addition, variable amounts of anergic cells resulting from incubation with TCR 76 were mixed with 2 x 104 non-anergic cells resulting from incubation with Mab 1 and a proliferation assay was performed. A / N proportion of anergic cells against non-anergic cells; 1 = 2 x 104 cells. Proliferation was assessed incorporation of 3H-thymidine. Each value represents the average count for 5 min of cultures in triplicate + standard deviation.

EXAMPLES Materials and methods Reagents Culture medium: DMEM / HAM's F12 (Gibco cat. No. 32500) supplemented with 2500 mg / l sodium bicarbonate, 2.3 mg / l 2-mercaptoethanol, 55 mg / l sodium pyruvate, 1 .22 mg / l of ethanolamine, 360 mg / l of L-glutamine, 4.5 x 10"4 mg / l of sodium selenite, 62.5 mg / l of sodium penicillin and 62.5 mg / l of streptomycin sulfate. The medium was supplemented with 1 3.61 mg / l of hypoxanthine and 3.83 mg / l of thymidine, this medium is referred to as DMEM / HAM's F12 / HT.The selection of hybridomas was performed in DEMEM / HAM's F12 / HT supplemented with 1% (v / v) of IL-6 containing supernatant of a T24 human bladder carcinoma cell line (T24CM) and 0.4 mM of aminopterin Melting medium: 280 mM inositol, 0.2 mM calcium acetate, 0.5 mM magnesium acetate and 1 M histidine, specific resistance: 1.1 1 104 W cm The ingredients were dissolved in Milli-Q water and the cond was subsequently adjusted uctivity at 90 mS / cm with Milli-Q water or a solution containing 1 mM of calcium acetate and 5 mM of magnesium acetate.

Cell cultures The H.243 T cell clone was derived from a patient suffering from rheumatoid arthritis. This T-cell clone similar to Th 1 recognizes the epitope RSFTLASSETGVG (SEQ ID NO: 1) of the Human Cartilage gp-39 (HC gp-39) in the context of DRB1 +0401 (belongs to MHC class II). The TCR of this clone is characterized as Va8 and Vb9 positive. Cells were routinely cultured in DMEM / HAM's F12 supplemented with 10% FCS, 20 U / ml IL-2, 5 U / ml IL-4 and periodically re-stimulated with antigen and Histocompatible Antigen presenting cells (APC) or with phytohemagglutinin (PHA) and feeder cells. For the proliferation assays, Fetal Calf Serum (FCS) was replaced with Normal Human Serum (NHS).

Preparation of T cell lysate and beads loaded with complexes TCRyCD3 Ten to fourteen days after restimulation, the T cells were washed with PBS and solubilized upon incubation (30 min, 0 ° C) at a concentration of 10 8 cells / ml in extraction buffer according to Oettgen et al. (ref 2) (10 mM triethanolamine, 150 M NaCl, 1 mM Na2EDTA, 1% digitonin, 10 mg / ml leupeptin, 10 mg / nl aprotinin, 1 mg / ml pepstatin, 1 mM PefablocR AEBSF and 1.8 mg / ml iodoacetamine of pH 7.8). The soft detergent digitonin allows the extraction of intact TCR / CD3 complexes. Cell and nuclear debris were removed by centrifugation at 16,000 g for 15 min.

At 4 ° C and the supernatant was stored in aliquots at -20 ° C until used for the selection of B cells or immunoprecipitation. For the selection of specific B cells, the paramagnetic beads were loaded with TCR / CD3 complexes.

Briefly, 100 ml of sheep anti-mouse Ig coupled in paramagnetic beads (SAM-beads; Dynaperlas® 1 10.02) were incubated (overnight, 4 ° C) with 22 mg of anti-CD3, OKT3, in PBS with 0.1% of BSA. After washing the beads with PBS / BSA, 1.4 x 108 T cell lysate equivalent cells and an equal volume (1.4 ml) of PBS / BSA were added with 1% normal mouse serum. The latter was added to prevent B cells from binding to free SAM ligation sites in the beads. The bead suspension was incubated for 2 to 4 hours at 4 ° C and washed with PBS / BSA before use.

Immunization Six week old BALB / C female mice were immunized at 3 to 7 week intervals with either 5 x 1 06 complete T cells (invention) or paramagnetic beads loaded with TCR / CD3 complexes of 5 x 10 7 equivalent cells (control experiment) ). Different routes of administration were used as indicated in Table I. In case an auxiliary was used (control experiment), the first injection was performed with complete Freund's assistant, subsequent injections with incomplete Freund's assistant and final reinforcement without auxiliary Generation of anti-clonotype MAb Female BALB / C mice, six weeks old, were injected intraperitoneally 4 times at intervals of 3 to 7 weeks, each time with 5 x 106 remaining T cells in PBS. Five days after the final injection, the mice were sacrificed and spleen cell populations emptied of monocytes and erythrocytes were prepared as described above (refs 23, 24). This resulted in a cell fraction enriched in B cells, but did not result in enrichment of B cells specific for said cell surface antigen with respect to other non-specific B cells according to step 2 of the present invention. To predepure these populations of spleen cells for B cells reactive to human CD3 or the constant region of TCRab, approximately 3 x 107 were incubated twice with 20 ml of SAM beads loaded with TCR / CD3 complexes from an levant T-cell clone ( positive T cell clone Va3.Vb14, SCRO.08a, but no other levant T cells will work). Subsequently, B cells specific for the TCR variable region were selected by incubating the resulting cell suspension with SAM beads loaded with TCR / CD3 complexes from the T cell clone of interest (H.2243). Each incubation was performed for 90 min. at room temperature in DMEM / HAM's F12 supplemented with 10% of Calf Serum (Hyclone ") and 0.2% of normal murine serum., the suspension was thoroughly homogenized every 5 min. After the final incubation, the beads were carefully washed five times with DMEM / HAM's F12, 10% Calf Serum and resuspended in the same medium. Hybridomas producing monoclonal antibodies were generated from these B cells selected by clonal expansion and mini-electrofusion as previously described (23) Briefly, selected B cells were mixed with T cell supernatant (TSN) and 50,000 B5 EI cells -4 irradiated (2500 rad) in a final volume of 200 ml of DMEM / HAM's F12 supplemented with 10% FCS in tissue culture plates with 96-well flat bottom. On day 8, the supernatants were examined in an assay of one-step T cell agglutination as described below, using either the T cell clone of interest or an irrelevant T cell clone. The B cell cultures that produce discriminant MAb were subjected to a mini-electrofusion procedure. The contents of these B cell cultures were mixed with 106 NS-1 myeloma cells (25) and were serum free when washed with DMEM / HAM's F12 / HT. Then, the cells were treated with pronase solution for 3 min. and subsequently washed with fusion medium. The electrofusion was carried out in a 50 ml fusion chamber by means of an alternating electric field of 30 s, 2 MHz, 400 V / cm followed by a high change pulse, square of 10 ms, 3 kV / cm and again an electric field alternating 30 s, 2 MHz, 400 V / cm. Finally, the contents of the fusion chamber were transferred 20 ml of selection medium and placed in a 96-well microtitre plate. On day 8 after fusion, the cultures were examined for hybridoma growth and classified in a one-step T cell agglutination assay again.

T cell algutination assay The hybridoma culture classification for T cell antigen antibodies and the determination of cross-reactivity of MAb with other T cells was performed in a one-step agglutination assay. Using microtitre plates from half the area, 50 ml of hybridoma supernatant was mixed with 20 ml of bead / suspension of beads containing 2 x 10s of paramagnetic beads with covalently ligated sheep anti-mouse Ig and 5 x 10 4 cells. T of interest in DMEM / HAM's F12. After 2 to 3 hours of incubation at 37 ° C, the algutination was examined under a microscope looking for aggregates of cells and beads.

Immunoprecipitation and "Western Blotting" Ten to fourteen days after restimulation, the T cells were washed and the cell surface proteins were labeled with biotin. Briefly, the T cells were incubated in a concentration of 5 x 106 cells / ml with 0.5 mg / ml of ImmunoPure® sulfo-NHS-biotin in PBS for 30 min. at room temperature. Subsequently, the cells were washed again and solubilized as described above. Before being used in an inmonoprecipitation experiment, the cell lysate was predeployed once upon incubation with paramagnetic SAM beads. Immunoprecipitation was performed by incubating 10 7 cells equivalent to pre-purified lysate for 3-4 h with 15 measuring paramagnetic SAM beads preloaded with MAb (1 ml of hybridoma supernatant). Then, the immunoprecipitates were washed three times with PBS, boiled in a sample buffer and subjected to SDS-PAGE on a 10% gel either under reducing conditions or non-reducing conditions, using the Laemmli method (ref. 5) and the Mini-protean II system (Biorad). Immunoblotting was performed according to Towbin et al. (ref 6) using PVDF membranes. Non-specific binding sites were blocked by incubating the membranes with 5% skimmed milk in PBS supplemented with 0.5% Tween 20. After washing, the blots were probed with streptavidin-alkaline phosphatase in PBS, 0.5% Tween 20, 1% BSA, 1% normal goat serum for 1 h at room temperature. Finally, the blots were developed with BCI P and NBT as a chromogenic substrate. For "Western blot" studies, the paramagnetic SAM beads were loaded with TCR / CD3 complexes as described above using OKT as the immunoprecipitating antibody and normal T cell lysate. After SDS-PAGE in 10% PAA-gel under reducing or nonreducing conditions, the blots were incubated with 2.5 ml of hybridoma supernatant. The ligated antibodies were detected using goat anti-mouse Ig conjugated with alkaline phosphatase and BCIP / N BT as described above.

Proliferation of antibody mediated T cells The flat bottom microtiter wells were covered (overnight, room temperature) with 100 ml of sheep anti-mouse Ig at 40 mg / ml in PBS. The wells were washed once and then incubated with 100 ml of MAb at a concentration of 2 mg / ml in PBS for 2 h. The excess free MAb was removed by washing and 2.5 x 104 of remaining T cells were added in 200 ml DMEM / HAM's F12 supplemented with 10% NHS. After two days of incubation at 37 ° C, the cells were pulsed with 0.5 mCi [3 H] -thymidine and incubated for another 16 to 18 h. Finally, the cells were harvested on glass fiber filters and [3H] -thymidine incorporation was measured by beta counting in a Matrix 96 ™ (Packard). Each variable was examined in quadruplicate.

Antigen-Induced Proliferation Antibody-Mediated Inhibition Flat-bottom microtitrelation wells were inoculated with 20 ml of hybridoma supernatant, 2 x 104 of remaining T cells and 1 x 10s of histocompatible MNC in 150 ml of DMEM / HAM's F12 supplemented with 10% NHS. After a 3 h incubation at 37 ° C, 50 ml of RSFTLASSETGVG peptide (16 mg / ml) was added to the cultures. The cultures were incubated for another two days at 37 ° C and the incorporation of [3 H] -thymidine was determined as described above. Each variable was examined in quadruplicate.

Induction of anergy using specific clonotype MAb Twenty-four cavity culture plates were covered (overnight, room temperature) with 1 ml of sheep anti-mouse Ig at 40 mg / ml in PBS. The wells were washed once and then incubated with 1 ml of MAb at a concentration of 2 mg / ml in PBS for 2 h. The excess of free MAb was removed by washing and 2 x 106 of washed remaining T cells were added in 2 ml of DMEM / HAM's F 12, and 10% of N HS Where indicated cyclosporin A (Sandoz, Basel, Switzerland) was added. rl L-2, cycloheximide (Sigma, San Luis, MO, EU) or APC coupled to H LA-DRB1 +0401 (3000 irrad irradiated), at the time of culture initiation After incubation overnight, the plates were cooled in ice and the T cells were resuspended by pipetting. The cells were washed once with complete culture medium and used for a T cell proliferation assay, cytokine analysis and FACS analysis as described below. The antigen-specific proliferation response of the T cells was assessed in flat-bottomed microcavity cultures containing 2 x 104 T cells, 1 x 105 HLA-DRB1 * 0401 coupled, irradiated (3000 rad) PBMC and variable antigen concentrations in 200 ml of DMEM / HAM's F12, 10% NHS. After two days of incubation at 37 ° C, the cells were pulsed with 0.5 mCi [3 H] -thymidine and incubated for another 16 - 18 hours. Finally, the cells were harvested on glass fiber filters and [3H] -thymidine incorporation was measured by gas scintillation in a Matrix 96 (Packard, Meriden, CT, USA). Each variable was examined in triplicate.

Results // 7/77 < and / 7 / Zac / ones The immunizations using auxiliary (control experiment) did not yield any serum containing antibodies capable of discriminating between H.243 and irrelevant T cells (Table 1; Immune group II, l l and IV). The title of the sera of group V was low (1: 100 in the agglutination test), while those of group I and I I were much higher (1: 1200).

Generation of anti-clonotype MAb Using group I spleen cells, incubation with TCR / CD3 complexes from an irrelevant T cell clone, here H .258, charged paramagnetic beads resulted in many branched cells, indicating a successful removal of many cells B reactive CD3- and TCRab. When the unbranched cells underwent a second pre-purification with irrelevant TCR / CD3-complexes from the same T-cell clone, hardly any branched cells were visible. Microscopic examination after incubation with paramagnetic beads with the T-cell clone of interest, did not result in visible branching formation. This was not surprising given the expected frequency of specific B cells. After limiting dilution and clonal expansion, it was found that 36 supernatants of B cells agglutinate H.2233 and not the irrelevant T cell clone H.258 (Table I I). Some of the B cell supernatants were able to bind both clones, indicating that they escaped the pre-purification procedures. Additionally, it can be deduced from Table II that most of the specific B cell cultures are not clonal, as also the B cells that had grown do not react with any of the T cells. However, this was not a problem because Hybridomas of these non-specific B cells can be selected after mini-electrofusion. Eighteen of the specific B cell cultures were subjected to mini-electrofusion. Some fusions did not result in specific hybridomas of T cells and some others could not be cloned into stable cell lines. Finally, stable hybridomas were obtained in 1 1 of 18 mini-electrofusions. These 1 1 MAb were further characterized. The isotypes of these MAbs are shown in Table III.

MAb specificity To further investigate the specificity of the MAb, T-cell algutination and immunoprecipitation tests were performed as well as "Western blots". SDS / PAGE under non-reducing and denaturing conditions showed that all MAbs were able to immunoprecipitate a band greater than about 85 kD and a minor band of 21 kD of a digitonin lysate of T cell clone H.243. This is consistent with the molecular weight of the TCRab complex and CD3 e / d chains, respectively. Under reducing conditions the 85kD band dissociates into two bands of 40 and 50 kD, which is consistent with the molecular weight of TCR a- and b chains, respectively. The immunoblot of one of the specific anti-H.243 MAbs is shown in Figure 1, together with OKT3 as a positive control and an irrelevant MAb as a negative control. Table 11 shows cross-reactivity of the MAb with six Vb9 positive T cell clones, two Va8 positive T cell clones and eight other T cell clones with different Va and Vb. The TCR 74 antibody must be considered as specific Vb9 since it binds all the Vb9 positive T cells and not the others. The other TCR antibodies only react with H. 243 so that it is very possible that these antibodies are directed against the antigen binding part of H.243, since the reaction with Va8, Vb9, TCRab constant region, CD3 and other Common T cell surface molecules are excluded. Figure 2 shows that the antibodies of the hybridoma clones TCR 64, TCR 66, TCR 69, TCR 70, TCR 73, and TCR 76 MAb are able to stain the TCR in an unreduced Western blot (85 kD band). ). The nonspecific heavy bands in the upper part of the figure descended from immunoprecipitating antibodies SAM and OKT3. No dye was obtained with the anti-clonotype MAb in a "Western blot" run under reducing conditions (the results are not shown), suggesting that the MAbs recognize a conformation epitope formed by the intact TCRab complex.

T-cell proliferation induced by MAb To investigate whether MAbs are able to induce proliferation of H.243 T cells, MAbs were immobilized in flat-bottomed microtiter plates and incubated with T cells. MAb, Figure 2 shows that all can induce the proliferation of H.2243 cells. Similar proliferation for anti-CD3 (OKT3) was obtained with TCR 64, TCR 66, TCR 70, TCR 76 and TCR 79. Proliferation was not obtained with TCR 44, which is an anti-clonotype MAb for another TCR.

MAb-mediated inhibition of T-cell antigen-induced proliferation In addition, it was investigated whether the MAb panel could inhibit antigen-directed proliferation of T-cell H.2243. In this experiment, the MAbs were previously incubated with T cells and APCs and three hours later, they were pulsed with different concentrations of peptide. Figure 4 shows that TCR 64, TCR 70, TCR 76, TCR 78 and TCR 83 strongly inhibit antigen-directed proliferation. Up to 98% inhibition was observed with TCR 83. Dose response curves show that maximum inhibition can be obtained with as little as 100 ng / ml of TCR 64, TCR 70 and TCR 83. The TCR 78 was approximately ten times less powerful Inhibition patterns were not dependent on whether saturation or suboptimal peptide concentrations were added (results not shown). An anti-clonotype MAb directed against another TCR (TCR 44) showed no inhibition.

Anti-clonotype MAb induces indifference of clone H-243 from human T cells to subsequent stimulation with antigen and APC. The occupation of the T cell receptor in the absence of co-stimulation is known to induce anergy. Previously, we found that the anticlonotype MAb immobilized to H.243 can functionally trigger the TCR of this clone. Consequently, it was interesting to investigate whether the same antibodies could induce anergy. For this purpose, 10 different anti-clonotype MAbs were immobilized in 14-well plates and incubated overnight with H.243 T cells to give the anergic stimulus. Then, the T cells were removed from the plates and tested if they could respond to increasing concentrations of HC gp-39262"277 (SEQ ID NO: 3) presented by APC coupled to irradiated DRB1 * 0401. Figure 5 shows that this The response was completely abolished by 8 of the 10 MAb, while the H.2433 cells incubated with control MAb1 still responded well to the peptide presented by APC The response of T cells incubated with TCR 69 and TCR 83 was significantly reduced but not canceled completely at higher peptide concentrations.

Anergic cells suppress the response of non-anergic cells We also investigated whether the amergic .243 H cells were able to suppress the response of non-anergic H.2233 cells. The anergy was induced with TCR 76 and the non-anergic cells were obtained from the incubation with control MAb 1. Subsequently, proliferation assays were performed with HC peptide g? -39261 '275 (SEQ ID NO: 2) using different viz T cell populations. 2 x 10 4 anergic T cells per well, 2 x 10 4 non-anergic T cells per well or mixtures of decreasing concentrations of anergic T cells (4 x 104, 2 x 104, 1 x 104 and 0.5 x 104) and 2 x 104 non-anergic T cells per cavity. The response of non-anergic cells was significantly reduced in a custom dose related by the addition of anergic cells (Figure 6). Approximately 90% of the proliferation reduction was obtained at an antigen concentration of 1 mg / ml and at the ratio of anergic cells against non-anergic cells of 2: 1. At higher antigen concentrations, less proliferation reduction was observed (72% at 5 mg / ml and 39% at 25 mg / ml). Thus, this example shows that it is possible to generate anti-clonotype monoclonal antibodies against human T cell receptors. This experiment resulted in ten hybridomas, four of which were deposited with the ECACC (Salisbury, United Kingdom), as indicated in Table ll. The strategy did not require large numbers of T cells or large amounts of purified TCR. The method according to the invention is an attractive alternative for tedious recombinant methods in which TCR soluble TCR expressed in syngeneic mouse cells are generated for use in immunization procedures. Thus, possible problems with conformational stability are also avoided. As the monoclonal antibodies showed different responses in a test for T-cell proliferation induced by antibody and a test for antibody-mediated inhibition of antigen-directed T cell proliferation (up to 98% inhibition was observed), this suggests that different epitopes they are recognized in the same clonotypic structure. If they were intertwined, all monoclonal anti-clonotype antibodies were able to induce the proliferation of H.2243 T cells. It was shown that small amounts of immobilized anti-clonotype MAb can induce anergy in the autoreactive clone. Following the anergic stimulus, the T cells failed to proliferate upon restimulation as a result of a lack of transcription of gene I L-2. In addition, a decreased I FNg production was found. FACS analysis of the anergic cells indicated that the anergy was not the result of low modulation of TCR or the absence of free TCR. In cocultivation experiments, it was found that the anergic T cells suppress the response of reactive cells of the same clone. This surrounding deletion led to 90% inhibition of proliferation. The data demonstrate the anergization potential of clone-specific antibodies in vitro. Such MAb can be used for the induction and maintenance of tolerance of antigen-specific T cells in rheumatic arthritis. In sera from mice immunized by injecting preferably pure TCR / CD3 immune complexes coupled to paramagnetic beads (Table I; immunization group IV). The sera of the mice proved to be more positive for the specific T cell, which was used for immunization than an irrelevant T cell. However, it was not possible to obtain hybridomas that produced anti-clonotype monoclonal antibodies. Although in the sera of mice immunized with small amounts of complete T cells could not demonstrate specificity for the T cell of interest against an irrelevant T cell, the method according to the invention yielded hybridomas capable of producing anti-clonotype monoclonal antibodies. It goes without saying that the antigen binding fragments can be made of the monoclonal antibodies according to the invention, for example, using papain. Also the monoclonal antibodies or antigen binding fragments thereof can be labeled to facilitate their detection and the scope of the present invention encompasses all of these forms. Nor is it necessary to say that the irrelevant cells used to remove non-specific B cells are preferably as similar as the cell surface antigen-bearing cells.

Table I: Immunization inventory and serum characterization Groups of two BALByC mice were immunized with either complete T cells or TCR and CD3 complexes linked to paramagnetic beads. Immunizations were performed in the absence or presence of helper as indicated. Different immunization routes were followed specifically: ip = intraperitoneally; my = intramuscular; se: subcutaneously; is = intrasplenically. The sera of the mice were characterized by binding in a T cell agglutination assay, FACS staining and "Western blotting". The functional characterization was carried out in an antibody-mediated T-cell proliferation assay and in an antibody-mediated inhibition test of T-cell-directed proliferation. antigen. * T 1 cell = T cell clone used for immunization; T 2 cell = irrelevant T cell clone. * + means inhibition of T cell proliferation.

Table II: Excretion of anti-T cell specific B cells in B-cell cultures EL-4 28 x 10 lymphocytes were subjected to an immunobeel selection procedure for the selection of anti-clonotype specific B cells. The selected B cells were expanded in different plate densities in a B-cell culture system EL-4 and on day 8, the agglutination of antibodies with the T cells of interest (H.243) and the irrelevant T cells was evaluated. (H.258) x: undetermined number of selected cells Table III: Agglutination of different types of T cells with anti-TCR MAb 1) H.243: Va8 and Vb9 positive LITERATURE 1. Moretta, A., et al. (1985) Selection and characterization of monoclonal antibodies to the idiotype-like structure of an interleukin-2-producing human leukaemia T-cell line. Int. J. Cancer 36: p. 253-259. 2. Oetgen, H: C: et al. (1986) A T3-like protein complex associated with the antigen receptor on murine T cells. Nature 320: p. 272-275. 3. Steenbakkers, P.G. A., et al. (1994) Efficient generation of monoclonal antibodies from preselected antigen-specific B cells. Molecular Biology Reports 19, p. 125-134. 4. Steenbakkers, P.G. A et al (1992) A new approach to the generation of human and murine antibody producing hybridomas. J. Immunol. Methods 152, p, 69-77. 5. Laemmli, UK (1970) Cleavage of structural proteins during the assembly of the head of a bacteriophage T4. Nature 227: p. 680-685. 6. Towbin, H. et al. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications: Proc. Nati Acad. Sci. USA 76: p. 4350-4354.

Abbreviations PBS Phosphate buffer saline BCIP salt 5-bromo-4-chloro-3-indoylphosphate-p-toluidine NBT p-nitro blue chloride tetrazolium BSA Bovine serum albumin FCS Fetal calf serum SAM anti-mouse sheep TSN T cell supernatant MAb monoclonal antibody (antibodies) Producers Biorad, Richmond, CA, E.U. Gibco, Paisley, Scotland Hyclone, Logan, E.U. Packard, Meriden, CT, E. U.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Akzo Nobel N.V. (B) STREET: Velperweg 76 (C) CITY: Arnhem (E) COUNTRY: The Netherlands (F) POSTAL CODE: 6824 BM (G) TELEPHONE: 041266379 (H) TELEFAX: 0412650592 (i) TITLE OF THE INVENTION: Method for prepare a monoclonal antibody, monoclonal antibody, a pharmaceutical composition and a diagnostic reagent (ii) NUMBER OF SEQUENCES: 3 (iii) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: IBM PC compatible ( C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PACKAGE: Patentln Reeléase # 1.0, Version # 1.30 (EPO) (1) INFORMATION FOR SEQ ID NO: 1: (¡i) CHARACTERISTICS OF THE SEQUENCE: ( A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) FILAMENTS: simple (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: internal (ii) DESCRIPTION OF SEQUENCE: SEQ ID NO : 1: Arg Ser Phe Thr Leu Wing Being Ser Glu Thr Gly Val Gly 1 5 10 (11) INFORMATION FOR SEQ ID NO: 2: (Ü) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) FILAMENTS: simple (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (v) FRAGMENT TYPE: internal (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Phe Gly Arg Ser Phe Thr Leu Wing Ser Ser Glu Thr Gly Val Gly 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 3: (Ü) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) FI LAMENTS: simple (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: internal (ii) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: Gly Arg Ser Phe Thr Leu Wing Ser Ser Glu Thr Gly Val Gly Wing Pro 1 5 10 15

Claims (21)

1. CLAIMS 1 .
2. The method for preparing a monoclonal antibody against a cell surface antigen, comprising the steps of 1) injecting a mammal with material comprising cell surface antigen, said material being selected from the group consisting of i) whole cells and ii) a membrane fraction obtained by treating whole cells; 2) isolating a cell fraction containing B cells from the spleen of said mammal; 3) enriching said cell fraction obtained in step 2 in B cells specific for said cell surface antigen by contacting the fraction of the cell with material bound to the carrier of cells related to said whole cells, said material related to the carrier ligated cells lacking said cell surface antigen, and separating B cells bound to the carrier-linked material of the related cells from the enriched cell fraction containing unbound B cells for use in the next step; 4) subject the enriched fraction of cells containing B cells obtained in the previous step to limit the dilution followed by clonal expansion; 5) selecting a B cell clone and immortalizing said selected B cell clone using a small scale fusion technique; and selecting and cloning a hybridoma capable of producing antibody, which specifically binds said cell surface antigen, followed by the isolation of a fraction comprising monoclonal antibody from the supernatant of said hibpdoma.
3. The method according to claim 1, characterized in that the The mammal injected with the surface antigen is of a species different from the mammalian species from which the surface antigen 3 is originated.
4. The method according to claim 1 or 2, characterized in that the surface antigen has a constant section and a cross section. variable, wherein at least a part of said variable section defines a specificity determining part of said surface antigen.
5. The method according to claim 3, characterized in that the material comprising the receptor molecule is used as the material that It comprises surface antigen 5 The method according to the claim 4, characterized in that a T cell clone is used to prepare the material comprising the receptor molecule 6.
6. The method according to any of the preceding claims, characterized in that the membrane fraction of step 1 is obtained by mechanical treatment of the cells.
7. The method according to any of the preceding claims, characterized in that the material comprising cell surface antigen is injected into the mammal in the absence of an auxiliary
8. 8. The method according to any of the preceding claims characterized in that the enriched cell fraction containing B cells of step 3, which is used in step 4, is further enriched by contacting the cell fraction with cell surface antigen ligated to the carrier chosen from the group of j) whole cells, jj) a membrane fraction obtained from said complete cells, and jjj) substantially purified cell surface antigen, and subsequently separating B cells not bound to said material bound to the carrier of the cells bound to said carrier-bound material, said B cells bound to said carrier-bound material comprising the cell fraction further enriched for use in step 4.
9. The method according to any of the preceding claims, characterized in that the Paramagnetic beads are used as the carrier.
10. The method according to any of the preceding claims, characterized in that the selection in at least one of steps 5 and 6 is conducted using an agglutination test, wherein the supernatant of the B cell clone is contacted with a carrier covered with antibodies capable of binding antibodies of the species of the injected mammal used in step 1 and whole cells that support the cell surface antigen, and agglutination is detected.
11. The method according to claim 10, characterized in that the whole cells related to said complete cells but lacking said cell surface antigen, are used as a control.
12. The method according to any of the preceding claims, characterized in that the B cell clone selected in step 5 is mixed with myeloma cells and subjected to mini-electrofusion.
13. 13. The monoclonal antibody reactive with the clonotypic structure of a T cell receptor.
14. The monoclonal antibody according to claim 1, characterized in that the T cell receptor is a T cell receptor associated with an autoimmune disease. .
15. 15. The monoclonal antibody according to claim 14, characterized in that the autoimmune disease is rheumatic arthritis.
16. 16. The monoclonal antibody according to claim 15, characterized in that said monoclonal antibody is reactive with the T cell receptor of a reactive T cell clone HC gp-39.
17. 17. The monoclonal antibody according to claim 16, characterized in that the T cell clone is H.243 (Access ECACC No. 96103122).
18. 18. The monoclonal antibody according to claim 17, characterized in that it is produced by a hybridoma selected from the group consisting of TCR 69 (Access ECACC No. 961031 18), TCR 70 (Access ECACC No. 961031 19), TCR 72 ( Access ECACC No. 96103120) and TCR 83 (Access ECACC No. 96103121).
19. 19. The pharmaceutical composition comprising a monoclonal antibody prepared according to any of the conclusions 1 to 12, mixed with a suitable excipient.
20. 20. The pharmaceutical composition comprising a monoclonal antibody according to any of claims 13 to 18 mixed with a suitable excipient, suitable for the treatment of rheumatic arthritis.
21. 21. The diagnostic reagent comprising a monoclonal antibody selected from the group consisting of a monoclonal antibody prepared according to any of claims 1 to 12 and a monoclonal antibody according to any of claims 1 to 18.