CH676600A5 - - Google Patents

Description

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description

The present invention is directed to ligands, namely antibody molecules or fragments of antibody molecules or other ligands, such as lymphokines, which are capable of binding a B cell surface marker with a molecular weight of 50 kDa, which is referred to below as Bp50 , which has a function in B cell proliferation, but not in early B cell activation. The present invention is also directed to the Bp50-B cell antigen itself. In a particular embodiment of the invention, the monoclonal antibody G28-5 is described which defines the Bp50 and appears to play a role in the proliferation of activated B cells, but has no detectable effect on the proliferation of the remaining B cells.

The ligands according to the invention, such as the antibodies, the lymphokines and the fragments thereof, can be used to control and regulate human B cell proliferation and / or to differentiate. In addition, the ligands according to the invention can be modified by linking them to other compounds which can be used in the treatment and / or in the detection of malignant cells which express the Bp50 antigen.

The activation of the remaining B cells from the Go phase to the Gi phase of the cell cycle and the subsequent introduction of the activated B cells for proliferation are different stages which require different regulatory mechanisms. Some drugs, including mouse B cells, that stimulate factor p1 (BSF-p1) (Rabin, et al., 1985, Proc. Nat. Acad. Sei, USA 82, 2935-2939) or lower doses of Anti-immunoglobulin (Anti-Ig) (DeFranco, et al., 1985, J. Immuno !. 135, 87-94; Wetzel, et al., 1984, J. immunol. 133, 2327-2332; DeFranco, et al ., 1982, J. Exp. Med. 155. 1523-1536; Muraguchi, et al., 1984, J. immunol. 132.176-180) are "activation" or "competence" factors. This means that they induce B cells to enlarge, for the synthesis of more RNA and occur in the Gì, but they alone cannot initiate DNA synthesis in B cells. Other "growth" factors, such as human B cell growth factor (BCGF) and interleukin-2 (IL-2), cause activated B cells to cross the cell cycle and enter the S phase, but with the remaining B cells cannot be resolved (Kehrl, et al., 1984, Immunol. Rev. 18; 75-96; Muraguchi, etat., 1984, J. Immunol. 132: 176-180; Zubler, et al., 1984, J. Exp Med. 160: 1170-1183; Jung, et al., 1984, J. Exp. Med. 160: 1597-1604).

A number of factors that promote the growth of B-lines have now been described by researchers in both the mouse and human systems. These include B cell growth factors (BCGF) derived from various sources including T cell lines or hybrodoma, B cell lines or dendritic cells. Although both interleukin (IL-1) and interleukin-2 (IL-2) have shown improvement in B cell growth, they obviously differ from certain BCGF. For example, monoclonal antibodies (mAb) block against mouse BGGF (O'Hara, et al., 1985, Nature (London) 315: 333) or against human BCGF (Ambrus, et al., 1985, J. Exp. Med. 162 : 1319) BCGF activity but not IL-1 or IL-2 activity. Although different from IL-1 or IL-2, the BCGF itself appear to be heterogeneous based on the biochemical data and different activities on different B-cell subsets or on cosfimulation tests. For example, a 60 kiiodalton (kDa) high molecular weight human BCGF, BCGF (high), was identified which differs from a 12 kDa low molecular weight form, BCGF (low) (Ambrus, et al., 1985, J. Clin. Invest. 75: 732). The cDNA encoding a 20 kDa mouse BCGF has recently been cloned and sequenced (Noma, et al., 1986, Nature, 319: 640). The recombinant lymphokine not only has BCGF activity, but can also activate the remaining B cells and induce the differentiation of IgGi-producing cells; consequently, it differs from human BCGF (high) and BCGF (low), both in its molecular weight and in its range of activity.

These activation and growth signals presumably regulate the cells by interacting with specific B cell surface structures. In addition to the antigen-specific signal from the surface Ig, other B cell surface polypeptide candidates have been identified that have some function in the activation or growth of the B cells. For example, the cell surface receptor was used for IL-1 (Dower, et al “1985, J. Exp. Med. 160: 501) and for IL-2 (Robb, et al., 1984, J. Exp. Med. 160: 1126) Functional IL-2 receptors on B cells have been characterized and recently identified (Zubler, et al., 1984, J. Exp. Med. 160: 1170; Jung, et al., 1984, J. Exp. Med. 160 : 1597; Muraguchi, et al., 1985, J. Exp. Med. 161: 181). However, receptors for B cell growth and activation factors have not been fully characterized. Various possible B-cell surface polypeptides have been identified, which could have some function in the activation or growth of B-cells. For example, Sub-barao and Mos'rer (Subbarao, et al., 1983, Immunol. Rev. 69: 81-97) have found monoclonal antibodies (mAb) against the mouse B cell antigen to activate Lyb2 B cells, and more recently evidence has been provided that Lyb2 can be the receptor for BSF-pI (Yakura, 1985, Fed. Proc. 44: 1532). Similarly, it was found that the appropriate mAb (1F5) against a 35 kDa polypeptide, Bp35 activated human B cells from Go in Gt (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766- 1770; Gollay, et al., 1985, J. Immunol. 135: 3795-3801). Aggregated C3d or antibody against the 140 kDa

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C3d receptor, Bp140, causes proliferation of B-lines which are T-cell dependent (Melchers, et al., 1985, Nature 317: 264-267; Nemerow, et al., 1985, J. Immunol. 135 : 3068-3073; Frade, et al., 1985, Eur. J. Immunol. 15: 73-76). Although BCGF has been identified in both mice and humans, the receptors for these factors have not yet been isolated. Wang and co-workers (Wang, et al., 1979, J. Exp. Med. 149: 1424-1433) produced a polyclonal antiserum which identified a 54 kDA polypeptide (gp54) on human B-lines and showed that the rabbit antiserum against gp54 induced tonsillary B cells for division. Recently, Jung and Fu (Jung, et al., 1984, J. Exp. Med. 160: 1919-1924) isolated a mAb (AB-1) against a 55 kDA antigen that was restricted to activated B cells, which blocked BCGF-dependent proliferation. However, it is not yet known whether an anti-gp54 or AB-1 recognizes a BCGF receptor.

The present invention is directed to the subjects defined in the claims. The ligands according to the invention bind Bp50, a B cell-specific surface polypeptide with a molecular weight of 50 ku (kDA) have the ability to increase the proliferation of activated B cells.

The invention is also directed to the Bp50 antigen itself, which is defined by the monoclonal antibody G28-5 and plays a role in the proliferation of activated B-lines. In addition, the invention is directed to ligands that bind Bp50, but have no biological effect or function, such as increasing the proliferation of activated B cells.

The ligands according to the invention comprise antibody molecules, monoclonal antibodies and fragments of these antibody molecules which contain the antigen-combining site or chemically modified antibodies and fragments; such fragments include FV, Fab, F (ab ') 2, Fab' and the like. In addition, the Ugandans according to the invention include lymphokines, which can include human B cell growth factors, as well as chemically modified lymphokines. The ligands according to the invention can be chemically modified, for example by connecting or coupling a compound to a ligand. Such compounds are, for example, cytotoxic agents, therapeutic agents, chemotherapeutic agents, labeling substances such as radio labels, dyes, enzymes, radio-opaque compounds and the like. The Ugands according to the invention can be used in their modified or unmodified form for controlling, regulating and modifying human B cell proliferation and / or differentiation.

The present invention is based on the discovery that two human B-cell differentiation antigens, Bp35 and the B-cell gene described here, Bp50, obviously play different roles as signaling receptors in B-cell activation. Monoclonal antibodies (mAb) against Bp35 and Bp50 show positive signs for B cells, which stimulate their transition through the cell cycle. mAb against Bp35, like anti-Ig antibodies, works principally by activating the remaining B-lines, which become able to enter the Gi phase of the cell cycle. In contrast, a monoclonal antibody described here or its F (ab ') 2 fragment against Bp50 works a 50 kDa poly peptide which is expressed by all B cells by activating the activated B lines for the traversing stimulation of the cell cycle and improved proliferation of activated B cells. Monoclonal antibodies against Bp35, such as anti-Ig antibodies, activate tonsillary B cells and initiate lower levels of B cell proliferation. In contrast, anti-Bp50 monoclonal antibodies alone neither activate B cells nor induce B cells for proliferation, but together with anti-Bp35 or anti-Ig antibodies they improve B cell proliferation. In this regard, the action of the anti-Bp50 antibody is equal to the activity of the B cell growth factor (BCGF). Only 0.05 (xg / ml of anti-Bp50 is needed to improve proliferation, and like BCGF, anti-Bp50 is effective even when it is 12-24 hours after activating B cells with anti -lg or anti-Bp35 without additional exogenous signals, anti-Bp35 and anti-Bp50 antibodies together induce strong proliferation of purified remaining B cells, and these results suggest that Bp35 and Bp50 Surface molecules are involved in the regulatory control of B cell activation and progression through the cell cycle Because of the importance of anti-Bp35 and similar molecules with regard to the effect and the action of the Ugands according to the invention, reference is expressly made to the publication by Clark, et al ., 1985, in Proc. Nati. Acad. Sei (USA) 82: 1766-1770.

Although the activity of the anti-Bp50 is the same as that of BCGF (low), since both anti-Bp50 and BCGF (low) are costimulatory with the same active ingredients, but not with one another, and both anti-Bp50 and BCGF (low) only on activated B Cells have an influence and are effective in a soluble form, the activity of anti-Bp50 can be distinguished from the activity of BCGF (low), since the proliferation of B cells, which with optimal proportions of anti-Bp50 and anti ti-Bp35 (or anti-Ig) are stimulated, can be improved by BCGF (low) and both blood B cells and certain B cell lymphomas react differently against anti-Bp50 against BCGF. For optimal activity, anti-Bp50 should be added within 12 hours after B cell activation, while BCGF (low) maintains optimal activity, even 24 hours after activation. In addition, Bp50 is expressed on all B cells, while the receptors or BCGF (low) are restricted to activated B cells. As a result, Anti-Bp50 and BCGF (low) can coordinate B cell growth, but apparently through different signals.

According to the invention, the ligands which bind Bp50 and improve the proliferation of activated B cells can be used to increase an immune response. For example, those 3

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gene ligands that form Bp50 are used as an "adjuvant" to improve an immune response against a vaccine. Alternatively, these ligands can be used to increase an immune response of an immunosuppressive individual.

In another embodiment, the ligands according to the invention can be chemically modified so that the cells to which the ligands are bound are killed. Since all B cells express the Bp50 antigen, this attempt would suppress an immune response. For example, a cytotoxic agent that is bound to a ligand according to the invention can be used in vivo to effect immunosuppression in order to overcome histocompatibility barriers in transplant patients; alternatively, these modified ligands can be used to control autoimmunity disorders.

Furthermore, ligands according to the invention can be used to treat malignant cells, such as tumor cells, which express BpSO, with a chemotherapeutic agent which is useful for the treatment of such neoplastic disorders being bound to the ligand. These modified ligands can be used in vivo to target the chemotherapeutic agent against certain types of malignant cells that express the Bp50 antigen, including cells that are not B cells but express Bp50. When using ligands according to the invention which improve B cell proliferation, a particular advantage can be perceived if the malignant B cells are treated with ligands to which a chemotherapeutic agent is bound which is more effective in killing proliferating cells. In this case, potentiation of the effect of the agent should be observed.

Alternatively, the ligands according to the invention can be used in vitro to identify or separate cells which express the Bp50 antigen and / or to test body fluid for the presence of the Bp50 antigen which is rejected or not rejected. In addition, the ligands according to the invention can be used in vivo to image cells or tumors which express the Bp50 antigen.

The purified Bp50 antigen according to the invention can be used to produce antibodies and to produce or construct other ligands according to the invention. In addition, Bp50 antigen could be used in tests such as diagnostic immunoassays. In addition, the Bp50 could be used in vitro or in vivo as a mediator for cell immunity.

The following abbreviations are used in the present description, the meaning of which is given below;

AO = acridine orange BCGF = B cell growth factor

BCGF (high) = human BCGF with a molecular weight of 60 kDa BCGF (low) = human BCGF with a molecular weight of 12 kDa

Bp35 == B cell-specific surface polypeptide (CD20) with a molecular weight of 35 kDa, which is defined by the mAb IF5

Bp50 = B cell-specific surface polypeptide with a molecular weight of 50 kDa, which is defined by the mAb G28-5

Fv = variable area or antigen-combining site of an antibody molecule, this can be any

Fragment which contains, but is not limited to, the idiotype of one of the following molecules: Fab, F (ab ') 2, Fab', and the like.

IF = immunofluorescence lg = immunoglobulin

IL-1 = interleukin 1

IL-2 = interleukin 2

kDa = kilodalton mAb = monoclonal antibody

SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis TPA = 12-O-tetradecanoyIphorbol-l 3-acetate.

Description of the figures

Fig. 1 The expression of Bp50 is restricted to Bp35 ± B cells. The two-color flow cytometer analysis of 50,000 cells was performed as described by Clark (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770). The data are plotted as the cell number against the log of green fluorescence and against the log of red fluorescence, with 4-5 points approximately doubling the fluorescence. The data are given to show the autofluorescence of negative cells. PE (red) -Anti-Bp35 (IF5) against FITC (green) -Anti-Bp50 (G28-5) staining shows that all Bp50 + cells are also Bp35 +.

2 shows the biochemical comparison of the Bp50 polypeptide with other B cell surface antigens. The immunoprecipitation of the Bp50 from the surface of tonsillary cells which were labeled with 125 l was carried out as described. The isolated antigens were subjected to electrophoresis using 10% SDS gel blocks without reduction. The gees were made visible by autadiadiography and intensification screens. Plate A: Lane 1, anti-Bp5Ö (G28-5); track

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2, anti-Bp95 (G28-8); Lane 3, Sepharose-goat-anti-mouse Ig alone, exposure time: 4 days. Plate B: Lane 1, anti-Pb50 (G28-5); Lane 2, anti-Bp45 (BLAST-2); Lane 3, anti-Bp39 (G28-1); Lane 4, anti-Bp39 (41-H16); Lane 5, Sepharose-goat-anti-mouse Ig alone. An exposure time of 2 days was selected so that the bands in lanes 2-4 were not exposed for too long and could be clearly distinguished from Bp50. One of 3 experiments.

Figure 3 shows a two-color immunofluorescence analysis of Bp50 expression. Peripheral blood or tonsillary mononuclear cells were isolated by centrifugation on Ficoll and stained with PE (red) conjugated G28-5 (anti-Bp50) in combination with fluorescein (green) conjugated reference antibodies, including 2C3 (anti-IgM); 1F5 (anti-Bp35); HB10a (anti-DR); and 9.6 (anti-CD2, E-receptor). The cells were analyzed with a FACS IV, which was provided with 4 decades of log amplifiers in the red and green dimensions. Forward and right angle light scattering was used to block the monocytes. Uncolored lines were on the back of the grid; red fluorescence on the right and green fluorescence on the left.

Figure 4 shows the dose response curves for an improvement in the proliferation of dense tonsillar Er-B cells by anti-Bp50 antibodies as indicated: medium alone; Anti-Bp50 alone; Anti-Bp35 (5 (ig / ml) alone; BCGF alone; Anti-Bp35 plus BCGF; Anti-Bp35 plus graded doses of Anti-Bp50. Major proliferation ± standard deviation of four-fold samples was measured on day 3.

Figure 5 shows that anti-Bp50 mAb was most effective for improving proliferation when added after a B cell activation sign. Dense tonsillary Er-B cells were incubated with medium only for 4 days, then anti-Bp5Q (0.5 ng / ml) was added after different times after the incubation, anti-Bp35 (5 jig / ml) was added at different times added after incubation. The anti-Bp50 was kept constant, at which later anti-Bp35 was added at different times; Anti-Bp35 was kept constant and anti-Bp50 was added to the cultures after various times. 3H-Thymidine was added over the past 10 hours and its incorporation measured.

Figure 6 shows the comparison of the ability of anti-Bp35 and anti-Bp50 to induce permanent tonsillar B cells to exit the Go stage of the cell cycle. On day 3 after treatment, medium became alone (-), anti-Bp35 alone (-); and Ig alone (....), A, no additional additives; B, anti-Bp50 (0.5ng / ml) to each group; C, 5% BCGF added to each group. The data were recorded as the relative cell count against the log of red fluorescence from the AO (RNA).

Figure 7 shows the kinetics of B cell proliferation after stimulation with anti-Bp50 against BCGF. Dense tonsillary E-B cells were stimulated with medium alone; 10% BCGF alone; Anti-Bp35 alone; Anti-Bp50 alone; Anti-Bp35 + 10% BCGF; Anti-Bp35 + Antî-Bp50 and Anti-Bp35 + Anti-Bp50 + 10% BCGF. The proliferation on the specified days was measured by an 18-hour pulse with 3H-thymidine. The proliferation was measured four times and the standard deviation was given. One of 3 experiments.

Figure 8 shows the times after anti-Bp35 stimulation where anti-Bp50 (A) or BCGF (B) optimally improves proliferation. Dense tonsillary E-B cells were stimulated as indicated and proliferation was measured by an 18 hour pulse of 3H-thymidine on day 3. Medium; Anti-Bp35 was only added at the times indicated; Anti-Bp50 or BCGF alone; Anti-Bp35 was added at the beginning of the culture, after which anti-Bp50 or BCGF was added at the indicated times; Anti-Bp50 or BCGF was added at the beginning of the culture, followed by anti-Bp35. One of 2 experiments. The proliferation was measured four times and the standard deviations are given. Doses used: Anti-Bp35, 5 ng / ml; Anti-Bp50, 0.2ng / ml; BCGF (low), 5%. Concentrations were used as follows: Anti-Bp35.5 (xg / ml; Anti-Bp50.0.2 ng / ml; BCGF, 5%.

Figure 9 shows that anti-Bp50 and BCGF have additive effects on B cell proliferation. Dense tonsillary E-B cells were stimulated with graded doses of BCGF (low), along with anti-Bp50 alone; Anti-Bp35 alone; Anti-Ig beads alone; Anti-Bp35 and anti-Bp50; or anti-Bp50 and anti-Ig. Proliferation was measured 3 days after stimulation with an 18-hour pulse of 3H-thymidine. The proliferation was measured four times and the standard deviations are given. One of 4 experiments. Doses used for 106 cells: anti-Bp35, 5 μg / ml; Anti-Bp50, 0.2 fig / ml; Anti-Ig beads, 50 ml / ml.

Figure 10 shows comparative effects of anti-Bp50 and BCGF on normal and malignant B cells. Peripheral blood E-B cells (A) or dense tonsillary E-B cells (C) were stimulated with or without TPA (75 ng / ml) in the presence of 10% BCGF or 1 ng / ml anti-Bp50. Two separate B cell lymphomas (plates B and D) were stimulated in the same way. Proliferation was measured on day 3 by the incorporation of 3H-thymidine during a 12-hour period. The proliferation was measured four times and the standard deviations are given.

The present invention is directed to ligands which a) bind Bp50, a B cell-specific surface polypeptide with a molecular weight of 50 kDa and b) improve the proliferation of activated B cells.

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The invention is also directed to the Bp50 antigen itself, which is defined by mAb-G28-5 and functions in B cell proliferation. The invention is also directed to Ugandans that bind Bp50 but do not have a biological effect or a function such as, for example, to improve the proliferation of activated B cells.

The Ugands according to the invention comprise antibody molecules, monoclonal antibody molecules and fragments of these antibody molecules which have the antigen-combining site which binds the Bp50 receptor, including modified antibodies and fragments thereof; such fragments include, for example, Fv, Fab, F (ab ') 2 or Fab'. In addition, the Ugands according to the invention also comprise lymphokines which are bound by the Bp50 receptor. These include, for example, BCGF as well as chemically modified lymphokines and the like. The ligands according to the invention can be used in their modified or unmodified form for modulating and regulating immune responses and for the therapy of malignant cells which express the Bp50 anti-gene. The uses are discussed in detail below.

If the ligand is a monoclonal antibody or a fragment thereof, the monoclonal antibodies against Bp50 can be produced using any technique which involves the production of antibody molecules by continuous cell lines in cultures. For example, Koehler and Milstein's original hybridoma technique (1975, Nature 256: 495-497) can be used as well as other more recent techniques, such as human B cell hybridoma technique (Kozborm et al, 1983, Immunology Today 4:72). and the EBV hybridoma technique for the production of human monoclonal antibodies (Cole, et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pages 77-96). Reference is expressly made to the publications mentioned above for the present invention.

Antibody fragments containing the idiotype of the molecule can be formed by known techniques. Such fragments are, for example, the F (ab ') 2 fragment, which can be formed by treating the antibody molecule with pepsin; the Fab 'fragments, which are formed by reducing the disulfide bridges of the F (ab') 2 fragment; the F (ab ') 2 fragment, which can be obtained by treating the antibody molecule with papain; and the 2Fab or Fab fragments, which can be formed by treating the antibody molecule with papain and a reducing agent to reduce the disulfide bridges.

If the ligand that forms Bp50 is a lymphokine, the lymphokine can be obtained from natural sources, or if its amino acid sequence is known or derived, the lymphokine can be prepared by chemical-synthetic methods. Alternatively, if the gene sequence is known, recombinant DNA techniques can be used to clone the gene into an expression vector, which can then be used for the transcription and translation of the gene sequence in an appropriate host cell.

Depending on the intended use, the ligand or appropriate fragments of the ugand can be chemically modified by coupling any compound to the ligand by known methods. Such techniques include, for example, the use of carbodiimide, cyanogen bromide, bifunctional reagents such as glutaraldehyde, N-succinimidyl-3- (2-pyridyidithio) proprionate (SPDP), reactions with Schiff bases, bonds to sulfhydril residues, the use of isothiocyanate or enzymatic binding processes, to name just a few. If a radioisotope has to be bound to the Ugandan, this can also be done with enzymatic means, oxidative substitution, chelation, etc. For an overview of the chemical reagents that can be used for protein modification, see Lundblad and Ijloyes, Chemical Reagents for Protein Modification, Volume III, CRD Press, Inc., Boca Raton, Florida, Ch. 5, pages 123-139 , 1984.

Chemical binding or coupling of a compound to a ligand could be directed to a site on the ligand that does not participate in the binding with Bp50. This could be done by protecting the Uganda's binding site from the coupling reaction. For example, the ligand can first be bound to Bp50 to protect the Bp50 binding site, then the coupling reaction can be carried out to carry out the desired connection to available reactive sites on the ligand-Bp50 complex. After the coupling reaction has ended, the complex can be separated again, a modified Ugand being formed to which the desired compound is bound in such a way that the Bp50 binding site of the molecule is minimally influenced. If the ligand contains a monoclonal antibody, such as G28-5, in which the Fc region of the molecule is not required for the ligand to exert its effect (see below), it may be advantageous to couple the desired compound to an Fc -Direct range of the molecule.

The following sections describe the new 50 kDa B cell surface marker, Bp50, which obviously has functions in B cell proliferation, as well as ligands that bind the new 50 kDa receptor and their uses. An example of Ugand according to the invention is a monoclonal antibody which defines Bp50 and its F (ab ') 2 fragments, which, like BCGF, increases B cell proliferation. In contrast to anti-Bp35 mAb, which can induce the resting B cells to go from Go to Gt, anti-Bp50 mAb cannot activate the resting B cells. Anti-Bp35 and Anti-Bp50 mAb together without additional exogenous signals initiate strong activation and proliferation of purified B cells.

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The experiments described below also demonstrate that the anti-Bp50 activity is the same as the BCGF activity, but that the anti-Bp50 is different from a BCGF because anti-Bp50 and low molecular weight BCGF are clearly additive and different on different B cells -streamed or malignant cells responded. Bp50 can represent a receptor for a particular BCGF or for a transmembrane signal that modulates BCGF production or BCGF receptor expression.

Method for characterizing the Bp50 receptor cell preparations:

Mononuclear cells were isolated from normal or leukaemic, heparanized, peripheral blood by Ficoll-Hypaque gradients (Pharmacia, Piscataway, NJ). Mononuclear cells from tonsillar tissue were obtained by the method described by Clark (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770). The T cells were emptied using AET-treated sheep erythrocytes with rosette formation and gradient separation using Ficoll-Hypaque. In some experiments, blood B cells were enriched by isolating the cells attached to nylon wool. The monocytes were removed by incubation once or twice on Petri dishes at 37 ° C for 45 min unless otherwise stated. Floating or dense tonsillary B cell fractions were isolated using Percoll step gradients as described by Clark (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770). Dense tonsillary B cell preparations consistently had more than 95% slg + Bp35 + cells. The preparations, which were enriched with blood B cells, had 60 to 85% sIg + cells. The B cell lymphoma cells were isolated by gently plucking lymphoma cells in the medium and then subjecting them to a Ficoll-Hypaque gradient centrifugation.

Monoclonal antibodies:

The G28-5 antibody to Bp50 was raised by immunizing BALB / c mice with human E-tonsillar lymphocytes and fusing immuno-spleen cells with NS-1 myeloma (Koehler, et al., 1975, Nature 256: 495 -497; Ledbetter, et al., 1979, Immunol. REv. 47: 63-82). Hybrid cell cultures that secrete antibodies that were reactive with tonsillar B cells but not with T cells were identified using indirect immunofluorescent (IF) and analysis with a FACS IV cell sorter; Cultures with antibodies which gave a histogram pattern which were similar to the known mAb against Pan-B cell markers (e.g. Bp35) were cloned and selected for further studies. The G28-5 clone produced an IgGt mAb which only reacted with normal or malignant B cells or B cell lines. Other mAb used in this study have been described in detail by Clark (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770; Clark et al., 1986, Human Immunol 16: 100; Ledbetter, et al., 1986, Human Immunol. 15: 30-44; Ledbetter, et al., 1985, in Per spectives in Immunooenetics and Histocompatibility. ASHl, New York, 6, pages 325-340). These include 1F5 (lgG2a) AntiBp35, HB10a (lgG2a), anti-HLA-DR, 2C3 (IgGi} anti-u chain, G19-4 (IgGi) anti-CD3, FC-2 (IgGsa) anti-Fc receptor CD16 and 9.6 (IgGga) anti-CD2 (E receptor) provided by Dr. Paul Martin (Martin, et al., 1983, J. Immunol. 131: 180). The IgGi mAbs were purified by precipitation , using 45% to 50% saturated ammonium sulfate and DEAE-Sephacryl column chromatography and purifying the IgGsa mAb using Protein A-Sepharose columns The F (ab ') 2 fragments of G28-5 were determined according to the method of Parham (Parham, et al., 1983, J. Immunol. 131: 2895), where they were purified on a 2 meter long Sephacryl S200 column and tested for unity by SDS-PAGE (Ledbetter, et al., 1985, J. Immunol. 135: 1819) The 2C3 mAb against u chains was combined with Sepharose 4B beads (Pharmacia Fine Chemicals, Uppsala, Sweden), coupling with cyanogen bromide.

Fluorescein and Phycoerythrin Conjugations:

Purified mAbs were either directly conjugated to fluorescein using fluorescein isothiocyanate (FITC; molecular samples) (green) following the method of Goding (Goding, et al., 1976, J. Immunol. Meth. 13: 215-226) or conjugated with R-Phycoerythrin (PE) (red) using SPDP (Pharmacia) according to the procedure detailed in Ledbetter, et al., 1985 in Perspectives in Immunooenetics and Histocompatibility. ASHl, New York, 6, 119-129. Lymphoid cells were incubated in round-bottom microtiter plates for 30 minutes with an appropriate dilution of green and / or red mAb, washed twice and then analyzed on a FACS IV cell sorter.

Two-color immunofluorescent:

The two-color studies were carried out using a fluorescence-activated cell sorter (FACS IV: Becton-Dickinson, Mountain View, CA), a 560 nm dichroistic mirror for

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beam and a 580 long pass filter and a 540 short pass filter (Ditric Optics, Hudson, MA) were used on the front of the red and green photomultiplier tubes. In addition, a two-color compensator (T. Nozaki, Stanford University) was used to correct minor deviations in the green and red signals. For each two-color stain, data from 40,000 cells was collected and stored on floppy disks. The data are presented as cell numbers (vertical) against the log of green fluorescence against the log of red fluorescence on a 64 x 64 point grid. Approximately 4.5 points represent a doubling of the fluorescence. Unstained cells are in the back corner of the grid; red fluorescence is on the right and green fluorescence is on the left. The cytometric system used for the two-color 1F with fluorescein and phycoerythrin has been described in detail by Ledbetter (Ledbetter, er al., 1985, in Perspectives in Immunooenetics and Histocompatibilitv. ASHl, New York, 6,119-129 and 325-340).

Cell culture:

Blood or tonsillary lymphoid cells were cultured fourfold in 5-10 x 105 ml 96-well microtiter plates containing 200 nl RPMI-1640 medium with 15% bovine serum, antibiotics, glutamine and pyruvate (R15). After 1 to 7 days the cells were pulsed with 0.5 | xCi 3H-thymidine per hole (New England Nuclear, 6.7 Ci / mmol; 1 Ci = 37) for 18 hours. The cells were then harvested on glass fiber filters harvested and radioactivity was measured on a scintillation counter. In some experiments, antibodies or factors were added at different time intervals after the cultures started; the proliferation of these experiments was measured on day 3.

Costimulatory factors:

Purified BCGF was supplied by Cytokine Technology (Buffalo, New York) and contained no detectable IL-1, IL-2 or interferon activity. This BCGF was made by the method of Mai-zel and co-workers (Maizel, et al., 1982, Proc. Nat. Acad. Sci. USA 79: 5998), who showed that the main BCGF activity of this material in a 12 kDa molecule was present, hereinafter referred to as "BCGF (lower)" (Mehta, et al., 1985, J. Immunol. 135: 3298). The purification step included DEAE preparative-scale affinity chromatography, followed by hydroxyapatite column chromatography. The IL-1, cleaned to homogeneity, was a generous dose from Dr. Steven Dower (Dower, et al., 1985, J. Exp. Med. 162: 501). The recombinant IL-2 was kindly provided by Cetus Corporation. TPA (12-0-tetra-deconoyl-phorbol-13-acetate) was supplied by Sigma.

Proof of cell activation:

Changes in cell volume that were induced by mAb and / or factors could be measured using a cell sorter and a forward angle light shaker. Changes in the cell cycle in the cellular RNA and DNA levels were measured by staining activated cells with acridine orange and the relative cellular RNA (red) and DNA (green) content using a cell sorter according to the method of Darzynkiewicz et al. were measured (Darzynkiewicz, et al., 1980, Proc. Nat. Acad, Sei. USA 77: 6697-6702). Changes in the relative content of cell surface antigen was followed using mAb conjugated directly to fluorescein and then quantified by direct IF fluorescence intensities using an Epics V cell sorter

Biochemical characterization Bp50:

Immunoprecipitation of Bp50 from surfaces with tonsillary cells marked by 125 I was performed as described by Ledbetter (Ledbetter, et al., 1985, J, Immunol. 134: 4250-4254). Isolated antigens were electrophoresed on 10% SDS polyacylamide gel blocks. The gels were visualized using autoradiography at -70 ° G and using a Cronex Lightening plus intensifying screen (Dupont).

Characterization of the BoSO receptor

The results of the experiments obtained using the above methods are given below.

Identification of a B cell-specific surface marker with a molecular weight of 50 kDa. Bp50

A mAb against Bp50 was elicited by immunizing BALB / c mice with human tonsillar lymphocytes and fusing immune spleen cells with NS-1 myeloma. One clone, G28-5, produced an IgGt mAb that did not contain the NS-1 light chain. After a

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After thorough analysis by IF analysis, it was found that G28-5 only reacts with normal or malignant B-cell or cell lines. Comprehensive screening of normal tissue using best practices (Clark, et al, 1985, Proc. Nat. Acad. Sei. USA 82: 1766-1770; Ledbetter, et al., 1986, Human Immunol. 15: 30-44; Ledbetter , 1985, in Perspectives in Immunooenetics and Histocompatibilitv. ASHL, New York, 6, pages 325-340) showed that the antibody G28-5 reacts with E-rosette negative (Er -) cells from blood or tonsils, but not with T - Cells that did not attach to nylon wool, PHA-induced T cell blasts or with blood granule cells, monocytes, red cells or platelets. He reacted strongly with all 7 B-lymphoblastoid cell lines tested and with 3 Burkitt lymphoma lines (Raji, Daudi, Namalwa), but not with 4 T cell lines (CEM, HSB-2, JURKAT and HPB-ALL). All chronic lymphocytic leukemias tested (3/3) and 90% (9/10) of the B-lymphoma tested expressed the Bp50 marker, while only 28% (2/7) of non-T-, non-B-CALLA + - acute lymphocytic leukaemias expressed Bp50.

The restricted distribution of Bp50 on normal tissues was further confirmed by quantitative two-color immunofluorescence (two-color IF) analyzes. Using an R-phy-coerythrin (PE) conjugated antibody (red) against the Pan B cell antigen Bp35 (Bl, CD20) and fluoro-eszein conjugated anti-Bp50 antibody (green), it was found that Bp50 only on Bp35 + B cells (Fig. 1) in blood or tonsils. B-cells from blood consistently expressed slightly smaller amounts of Bp50 than tonsillary B-cells; this is similar to HLA-DR expression (Ledbetter, et al., 1986, Human Immunol. 15: 30-44) of gp54 expression (Wang, et al., 1979, J. Exp. Med. 149: 1424- 1433), which are also lower in blood B cells. Bp50 was expressed in similar amounts of tonsillar B cell subsets, which were separated into floating and dense fractions by Percoll gradients. Using the PE-conjugated mAb against the T cell marker, CD3 (T3) and NK cell associated marker, CD16 (Fc receptor) (Ledbetter, et al., 1979, Immunol. Rev. 47: 63-82) , it was found that Bp50 is not expressed on T cells or NK cells. Using two-color IF, it was also found that CD3 + PHA blasts that expressed high levels of the IL-2 receptor did not express the Bp50.

The G28-5 antibody reacted with a single polypeptide on tonsillar lymphocytes, which migrated at about 50 Kd under non-reducing conditions (Fig. 2A). This molecule is larger than previously described B cell markers in the same molecular weight range as Bp39 or Bp45 (Zipf, et al., 1983, J. Immunol. 131: 3064-3072; Kitner, et al., 1981, Nature 194, 458-460; Clark, et al., 1986, in Leu-koevte Tvoina II. Editor Reinherz, et al., Springer Verlag, Berlin, Chap. 12, Vol. 2,155-167; Slovin, et al. 1982, Proc. Nat Acad USA 79: 2649-2653; Thorley-Lawson, et al., 1985, J, Immunol. 134: 3007-3012 and Fig. 2B). The exposure time for this gel was chosen so that the molecular weights of the other B cell markers could easily be compared to Bp50. In contrast to Bp50, the Bp39 marker is expressed on granulocytes and Bp45 and also in contrast to Bp50, it is restricted to B cell blasts. Antibodies against Bp39 (41-H16) and Bp45 (MNM6, BIast-1, Blast-2), which were made available through an international workshop (Clark, et al., 1986, in Leukocvte Tvoino II. Editor Reinherz, et al. , Springer Verlag, Berlin, chap. 12, volume 2, 155-167) did not block the binding of fluoresced anti-Bp50 antibodies against B cells. Accordingly, the monoclonal antibody G28-5 recognizes the 50 Kd structure in detail from other B cell antigens based on the tissue distribution of biochemical analyzes and blocking studies.

The expression of Bp50 is restricted to B cells

Hematopoietic tissues such as cell line distribution studies and extensive two-color cytometric flow analyzes showed that Bp50 is only expressed on B lymphocytes. As illustrated in Figure 3, Bp50 is expressed on a small subset of the blood lymphocytes and on a large population of tonsillar lymphocytes. Essentially all Bp50-t cells expressed both in blood and tonsils also Bp35 and HLA-DR, but did not express CD2 (Fig. 1) or CD3, T cell molecules or the IgG-Fc receptors which responded the NK cells can be found. ConA-activated CD3 + T cell blasts further expressed 11-2 receptors, but do not express Bp50.

The two-color cytometric flow analysis allows the quantitative measurement of the density relationship between two surface antigens. It was previously shown that the dense resting B-cells in the secondary follicle mantezone express IgM and small amounts of Bp35, while the floating activated B-cells in the germinal center are IgM-negative and express increased amounts of Bp35 (Ledbetter, et al ., Human Immunol. 15:30). Figure 3 shows that both IgM positive and IgM negative B cell subsets expressed Bp50 in equal amounts, showing that bp50 is expressed in both resting B cells and in vivo activated B cells.

Improvement of B-cell orifification with anti-Bp50 antibodies

As previously explained, B cells can be activated with low doses of anti-u chain specific antibodies. It has recently been found that the B cell-specific marker bp35 (Bl) a 35 kDa polypeptide can also function in early B cell activation. The 1F5 mAb against Bp35, like low doses of anti-u antibodies, activated B cells by reducing the cell volume and the

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Increase RNA content and become reactive against BCGF (Ciark, et al., 1985, Proc. Nat. Acad, Sei. USA 82: 1766-1770; Gollay, et al., 1985, J. Immunol, 135: 3795-3801 ). Accordingly, it was of interest to compare the effect of anti-Bp50 mAb on the proliferation of untreated B cells or from B cells activated with either anti-Bp35 or anti-u antibodies (Table 1). Anti-Bp35 in solution or anti-u antibodies bound to Sepharose beads under appropriate conditions alone could somewhat stimulate B cell proliferation (Table 1, line 1); however, the anti-BpSO alone could not stimulate proliferation (Table 1, line 2).

Anti-Bp50 mAb, however, significantly improved proliferation when cultured with anti-u beads or with anti-Bp35. In this regard, anti-Bp50 was similar to BCGF (Table 1, line 3). As a result, it was important to determine whether anti-Bp50 and BCGF together could induce B cell proliferation. As illustrated in Table 1, line 4, anti-Bp50 and BCGF together did not induce proliferation, but they increased the proliferation of anti-u or anti-Bp35 activated cells slightly more than when stimulated alone. BCGF over a three-hole range, when used with anti-BpSO without other signals, had no effect on the proliferation of dense B cells, even when anti-Bp50 was used in the dose range 0.1 to 10 fig / ml.

Table 1

Improvement of anti-Ig or anti-Bp35-induced B cell proliferation with anti-Bp50 antibodies Medium proliferation + S.E, cultivated by B cells with:

line

Costimulant

medium

Anti-u beads

Anti-Bp35

1

none

1.212 ± 547

10.219 ± 462

5.539 ± 308

2nd

Anti-Bp5Q

7194718

38.792 ± 1.329

25.465 ± 616

3rd

BCGF

456 ± 217

14.217 ± 445

9.443 ± 343

4th

Anti-Bp50 + BCGF

1.456 ± 126

54.393 ± 2.537

46.488 ± 3.387

The proliferation of dense Er tonsillary B cells (95% surface IgM + cells) was measured on day 3 as described. Briefly described, 2 x 105 cells / 200 nl / well were fourfold for 48 hours, with RPMI 1640 medium, which contains 15% fetal bovine serum plus additives without antibody, or with 2C3 monoclonal antibody against u-chains, which were attached to Sepharose beads was bound ("anti-u-beads" 50 μg / ml) or free 1F5 anti-Bp35 antibody (5 (ig / ml). The cultures which contained the medium "anti-u-beads" or anti-Bp35 were used alone or with BCGF (5% final concentration, Cytokine Technology, Buffalo, New York, with no detectable IL-1 or IL-2 activity) with anti-BP50 (1: 1000 dilution of the ascites) as a dietary supplement After 40 hours, the cells were pulsed with 3H-thymidine and the incorporated counts were measured after 18 hours.

Anti-BP50 mAb only improves proliferation after the B cells have been activated by anti-Bp35 or anti-u antibodies

The results of Table 1 lead to the conclusion that anti-BpSO-mAb could not induce proliferation itself. As shown in Table 4, doses from 0.05 g to 2.0 ng / ml of anti-BpSO had no effect on 3H-thymidine uptake. In the presence of optimal amounts of anti-Bp35 mAb, only 0.1 to 0.5 ng / ml anti-BpSO antibodies significantly increased the proliferation. Amounts of 50,000 to 70,000 cpm were detectable with an optimal proliferation time when highly purified B cells were only cultured anti-Bp35 plus anti-Bp50. A consistent observation showed that higher doses of anti-Bp50 (greater than 2 to 5 fig / ml) were less effective than doses in the range of 100 to 200 ng.

These results lead to the conclusion that anti-Bp50 only worked after the B cells were activated by other signals. The data shown in Fig. 5 suggest that this is indeed the case. If B cells were first activated with anti-Bp35, the anti-BpSO could be added up to 24 to 48 hours later, with day 4 proliferation still improving. If, on the other hand, the cells were first treated with anti-Bp50, anti-Bp35 was only effective if it was added within a few hours after the start of the cultures. Similar results were found when anti-u was used instead of anti-Bp35.

Anti-Bp50 mAb does not activate B cells from Gn. however, it induces activated B cells to tune through the cell cycle

It has previously been found that anti-Bp35, like lower doses of anti-u antibodies, stimulates resting tonsillar B cells in Go to enlarge (Clark, et al., 1986, Leukocvte Typing II. Editor Reinherz, et al., Springer Verlag, Berlin, Volume 2, 455-462) and in the Gi phase of the cell cycle

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(Gollay, et al., 1985, J. Immunol. 135: 3795-3801). It was therefore of interest to compare the ability of anti-Bp50 mAb with anti-Bp35 mAb for their effects related to B cell activation. As shown in Fig. 6A, non-stimulated, dense tonsillar B cells even after 3 to 4 days in culture had a uniform RNA profile which is characteristic of cells in Go (Darzynkiewicz, 1980, Proc. Nat. Acad. Sci. USA 77: 6697-6702). However, approximately 15-30% of the cells that were stimulated with anti-Bp35 or anti-u had an increased RNA content, which indicated the entry into Gì. In contrast, neither anti-Bp50 (Fig. 6B) nor BCGF (Fig. 6C) alone induced a significant number of B cells to transition to Gì. 2 days after the activation, for example, anti-Bp35 or anti-Ig mAb induced 13.5% or 20.9% of the tonsillar cells to cross into Gi, while cells which only had anti-Bp50 (2.7%) or treated with BCGF (3.2%) remained at the control medium level (2.2%). However, when both anti-Bp50 and BCGF were added together with anti-Bp35 or anti-u antibodies, the number of cells that passed into Gi increased dramatically. Similarly, anti-Bp50 and BCGF alone showed no induction of B cells to transition to the S phase (Table 2) together, however, with either anti-Bp35 or anti-u the number of S-phase cells increased two - up to three times.

Table 2 _____

Effect of anti-Bp50 and BCGF on the progress of the cell cycle in tonsillary lymphocytes Competence-signal progression-signal% cells

Go Gt S / G2 / M

Medium none

89.9

7.1

2.5

Anti-Bp35

none

80.4

14.5

3.7

Anti-lg none

65.6

27.6

5.7

medium

Anti-Bp50

83.6

12.0

3.3

Anti-Bp35

Anti-Bp50

54.1

35.5

9.7

Anti-lg

Anti-Bp50

43.6

36.2

16.2

medium

BCGF

85.4

11.7

2.2

Anti-Bp35

BCGF

56.6

32.6

11.6

Anti-lg

BCGF

48.4

36.1

14.1

Percentage of cells in Go, Gi or S and G2 determined using the acridine orange staining method (Darzynkiewicz et al., 1980, Proc. Nat. Acad. Sci. USA 77: 6697-6702); 1 x 106 dense tonsillary lymphocytes with anti-Bp35 (5 jig / ml), anti-u on Kug oak (50 ng / ml), anti-Bp50 (0.4 ng / ml), BCGF (5%) or combinations as indicated .

Optimal conditions for the improvement of B cell proliferation with anti-Bp50 antibodies

Antibodies against Bp50 itself have no or no detectable effect on dense resting B cells (Table 3). In the presence of agents that can activate B cells, for example anti-Ig, anti-Bp35 and TPA, anti-Bp50 mAb, the proliferation was clearly improved. Anti-Bp50 did not costimulate with some interleukins, including purified IL-1, recombinant IL-2, and BCGF (low). A comparison of the effects of anti-BpSO with those of BCGF (low) showed that some drugs that were costimular with anti-Bp50 also showed costimulation with BCGF (low) (Table 3). Of particular interest was the finding that BCGF and anti-Bp50 together showed no costimulation for resting cells.

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Table 3

Improvement of B-Zi sllproliferata'on with j

^ nti-Bp50 antibodies or B-cell growth factors

Mean proliferation ± S.E. cultivated by B cells with:

Kostimuians

medium

Anti-Bp50

BCGF

(200 ng / ml)

(5%)

none

96 ± 1

267 ± 15

285 ± 74

Anti-lg

5.833 ± 391

41.634 ± 2.103

25.094 ± 61

Anti-Bp35 (5ng / ml)

457 ± 45

8.143 ± 280

1.733 ± 32

TPA (2ng / ml)

7.361 ± 537

21.163 ± 871

13.064 ± 1.030

IL-1 (10 U / ml)

264 ± 2

308 ± 23

221 + 8

IL-2 (100 (U / ml)

204 ± 34

350 ± 7

220 ± 11

BCGF (5%)

220 ± 7

851 ± 28

270 + 18

Dense Er tonsillary B cells (more than 95% slgM + cells) were cultured with 2 x 105 cells / well for 48 hours and then pulsed with 3H-thymidine for 24 hours before counting.

The kinetics of proliferation, which was improved by Antl-Bp50, is shown in FIG. 7. The peak of proliferation occurred on day 4 and then decreased regardless of whether the cells were activated with anti-Bp35 or other activators such as anti-Ig or TPA. The kinetics of proliferation, which were improved by BCGF or by anti-Bp50, were similar.

Anti-Bp50 antibodies were sufficient to improve proliferation. The optimal dose used in the subsequent studies was 0.3 ng / ml. Continuous observation showed that when using whole antibody molecules higher Doses of anti-Bp50 (greater than 2-5 ng / ml) were less effective than doses in the range 0.1-0.5 ng / ml.

Human B cells were excellently sensitive to inhibitory effects mediated by Fc receptors of antibodies bound to the surface ig (Parker, 1980, Immunoi. Rev. 52: 115; Bijsterbosch, et al., 1985, J. Exp. Med. 162: 1825). It was therefore important to compare the effectiveness of the whole anti-Bp50 mAb with that of anti-Bp50-F (ab ') 2 fragments. Over a 100-fold dose range, F (ab ') a fragments were clearly as effective or even more effective than whole antibodies at improving B cell proliferation (Table 4). As a result, the Fc range of the anti-Bp50 mAb is not required for anti-Bp50 to exert its effect, and if something can be inhibitory. In other words, anti-Bp50, like BCGF, can obviously act as a soluble mediator without the help of the Fc-receptor-mediated target cell function,

Table 4

The Fc range of anti-Bp50 antibodies is not necessary for the improvement of B cell proliferation

Medium proliferation of B cells cultivated with:

Anti-Bp50 Dose Medium Anti-Bp35

(ug / mt)

none

-

295 ± 16

269 ± 27

whole ab

0.125

278 ± 32

5.140 ± 20

1.25

275 ± 24

4,686 ± 342

12.5

163 ± 15

3.852 ± 203

F (from02

0.125

594 ± 21

10.635 ± 449

1.25

531 ± 3

10.893 ± 575

12.5

179 ± 8

9.411 ± 870

The cell culture conditions were the same as described in Table 3.

Differences between anti-Bp50 and BCGF fniederi activity

Anti-Bp50 and BCGF (low) had a similar effect on B cells and showed costimulation with the same active ingredients (Table 3). Certain evidence, however, shows that anti-Bp50 and BCGF, which were used in this study, apparently operate by different signals. First, in contrast to BCGF (lower), the Bp50 molecules (Bijsterbosch, et al., 1985, J.

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Exp. Med. 162: 1825) expressed on resting B-lines from blood (FIG. 3). Second, although both anti-Bp50 and BCGF (low) work most effectively when added after anti-Bp35 or anti-Ig, the anti-Bp50 was clearly the most effective when added 12 hours after culture started ( 8A). BCGF (lower), however, could be added up to 24 hours after the start of the culture and still improved the proliferation optimally (FIG. 8B). These kinetic experiments, which were shaped according to Howard and Paul (1983, Ann. Rev. Immunol. 1: 307), lead to the conclusion that a Bp-50-dependent signal normally exerts its effect before BCGF.

Both anti-Bp50 and BCGF (low) improved the proliferation of B-lines which were activated with anti-Bp35 or anti-Ig (Table 3). However, the effects of Anti-Bp 50 and BCGF (low) were additive in many experiments (Fig. 7). Figure 9 shows a titration of BCGF (low) in an experiment using anti-Bp50 in its optimal concentration (0.2 ng / ml). BCGF (lower) was able to further improve the proliferation of resting B cells in the presence of anti-Bp50 after activation by anti-Ig or anti-Bp35. Optimal concentrations of BCGF (low) were 5-20%, while 25% were inhibitory. Accordingly, when anti-Bp50 and BCGF (low) were both used at their optimal concentrations, they still showed additive effects on B cell proliferation.

Finally, both normal and malignant B cell subunits differed in their response to anti-Bp50 and BCGF (low). For example, some blood B cells responded to BCGF (low) but not to anti-Bp50 (Table 5). An additional activation signal such as anti-Bp35 (Table 5) or TPA (Fig. 10) was consistently required for the blood B cells to respond to anti-Bp50. While dense tonsillary B-lines generally did not respond to either BCGF or anti-Bp50, the floating B-lines responded (Table 5). The malignant B cells also differ in their response to anti-Bp50 against BCGF. For example, some B cell lymphomas responded to TPA plus BCGF (low) but not TPA plus G28-5 anti-BP50 (Figures 10B and D). In contrast, dense tonsillary B cells and peripheral blood B cells responded to TPA plus both BCGF (lower) and anti-Bp50 (Figures 10A and C).

Table 5

B cell subunits differ in their response to anti-Bp50 or BCGF mean proliferation (± S.E.M.) from lymphocytes:

Stimulation blood tonsils (tonsils)

Exp 1 Exp 2 Exp 3 tightly floating none

527 ± 70

650 ± 133

906 ± 106

385 ± 43

387 ± 12

BCGF (5%)

13.918 ± 1.082

37.594 ± 2.023

3,347 ± 174

332 ± 33

1.451 ± 57

Anti-Bp50 (0.5 ng / ml)

519 ± 28

1.013 ± 81

1.151 ± 28

472 ± 8

1.543 ± 20

Anti-Bp35 (5ng / ml)

554 ± 90

645 ± 89

665 ± 115

2.415 ± 80

1.129 ± 68

Anti-BpSO + Anti-Bp35

-

12.274 ± 546

15.667 ± 333

36.589 ± 1.335

4,843 ± 136

Anti-Bp50 + BCGF

-

-

3,943 ± 115

1.342 ± 19

2,899 ± 91

The cell culture conditions correspond to those of Table 1. Blood lymphocytes (B cells plus monocytes) adhering to nylon wool were freed from the monocytes by incubating them on plastic dividers overnight before stimulation (Exp 1 + 2). In Exp 3, the blood E-lymphocytes were freed from the monocytes by incubation on plastic dividers for 1 hour before stimulation. The tonsillary Er lymphocytes were dosed using a Percoll gradient into dense (pellet) or floating (fraction 1) subsets (32).

Use of anti-BD50 Lioanden and Bo50

The Ugands according to the invention can be used in vivo and in vitro in their unmodified or modified form in order to modulate immune responses. For example, the Ugands themselves can be used as an "adjuvant" to increase an immune response to a vaccine or to increase the immune response to an individual with immunosuppression. Alternatively, when cytotoxins or anti-proliferative agents are coupled to the ligands, they can be used to reduce an immune response, e.g. in autoimmune disorders or in transplant patients to help prevent graft rejection. These modified ligands could also be used to treat malignancies, which include cells or tumors that express the Bp50 antigen, regardless of whether the malignancy originally comes from a B-line.

Both the ligands according to the invention and / or Bp50 itself can be used in vitro. Such applications include in vitro tests such as immunoassays for the detection of cells expressing the Bp59 antigen and / or for the detection of the release of the Bp50 antigen in the body13

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liquids, if available. In these cases, the ligand or BpSO could be labeled with a radio label, fluorine, an enzyme, an enzyme substrate, a dye, etc. In addition, the ligands can be used to separate and / or identify cells that express the Bp50 antigen, in which case the ligand can be coupled to an immobile support or can be bound to fluorine used in a FACS can (Fiuorescen activated cell sorter).

The numerous uses of the ligands and Bp5Q according to the invention are discussed in detail below.

The BoSO receptor and the use of lioands. such as anti-Bp50 to improve B cell proliferation

Previous studies concluded that the factors involved in inducing B cells from the go to the Gt phase of the cell cycle are different from the actuators or the requirements in the S phase. This model is based in principle on studies which show that agents such as low doses of anti-Ig-B cell activation factors or anti-Bp35 alone have no or only minor effects on B cell proliferation. However, the same agents can direct the B cell lines to a point in cell activation where they are susceptible to growth factors. In contrast, growth factors such as BGGF or IL-2 alone have no effect on resting B cells, but improve the growth of activated B cells.

While the present invention is not intended to be limited to theory or explanation, the results presented here provide additional support for a model of specific regulation of B cell activation and growth levels. It has been shown here that the activation and proliferation signals in human B cells can be transmitted via certain cell surface structures. Although anti-Bp35 mAb activated B cells to enter the Gi phase of the cell cycle, it induced little or no proliferation. Anti-BpSO-rnAb had the opposite effect. He couldn't activate the B cells; however, if it was added 12 to 24 hours after activation, it could induce B cell growth.

The BpSO molecule may presumably be normal, either as a Ugand receptor, e.g. function as a soluble growth factor or as a signal mediated by cell-cell contact (i.e. a ligand is found on the surface of another cell). Previous studies have identified various T cell-derived BCGFs that, like anti-Bp50, improve B cell proliferation. The low and high molecular weight B cell growth factors have been identified and various types have shown additive effects (Kendet, et al., 1984, Immunol. Rev. 18: 75-96; Kishimoto, 1985, Ann. Rev. Immunol. 3: 133 -157; Swaïn, et al., 1983, J. Exp. Med. 158: 822-835; Howard, et al., 1984, Immunol. Rev. 78: 185-210; Ambrus, et al., J. Clin. Invest. 75: 732-739; Ambrus, 1985, J. Exp. Med. 162: 1319-1335). As a result, Bp50 could be a receptor for one of these factors.

With the exception of IL-2 receptors and the C3D receptor, the receptors on the B cells for growth signals have not yet been identified. The mAb-AB-1 reacts with a B cell marker, which is only expressed on activated B cells, and blocks BCGF-dependent proliferation and could therefore recognize the BCGF receptor or a related structure. Bp50 appears to be different from the AB-1 marker since the AB-1 mAb does not block the binding of the G28-5 anti-Bp50 antibody and, in contrast to the G28-5 mAb, only reacts with activated B cells ( Jung, et al., 1984, J. Exp, Med. 160: 1919-1924). Bp50 is on all B cells, which does not appear to be the case based on absorption analyzes and direct binding tests for BCGF receptors. The present data show that Bp5Ö and the receptor for the low molecular weight BCGF are different structures. Using a rabbit heteroantiserum, Wang and co-workers (Wang, 1979, J. Exp. Med. 149: 1424-1433) described a glycoprotein with a molecular weight of 54 kDa gp54, which, like Bp50, is expressed on all B cells, however in smaller amounts on blood B cells than on tonsillary B tents. The rabbit hetero-roantiserum and anti-Bp50 are likely, but unlikely, to have the same or related structures: in contrast to the anti-Bp50 mAb, the rabbit antiserum against gp54 alone was sufficient to stimulate B cell proliferation .

Anti-Bp35 alone, in contrast to Anti-Bp50, can activate B cells from Go to Gi and can therefore be referred to as an “activation” signal. Whether Bp35 only works in early B cell activation is not yet clear that anti-Bp35 antibodies can stimulate some B cells to divide (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770). Similarly, Bp50, strictly speaking, can not only function as a "growth" signal: anti-Bp50 antibodies together with activation signals (anti-Bp35 or anti-u) not only improve proliferation but also increase the total number of B cells, which occur in Gi (Table 2). In other words, anti-Bp59 reacts as a costimulant to promote the progression of activation (Go in Gi) and growth phases (G to S) of the cell cycle. The BCGF used in these studies also had a similar activity (Figure 6C). As a result, Anti-Bp35 and Anti-BpSO (or BCGF) seem to be the greatest analogy to the “competence” and “progression” factors that were used in the studies on

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growth regulation. How the B cells respond to anti-Bp35 or anti-Bp50 clearly depends on their state of differentiation or activation.

Here, it was shown that two mAb, anti-Bp35 (a "competence" signal) and anti-Bp50 (a "progression" signal) together represent a substantial proliferation of highly purified B cells in the absence of antigens or other known factors can induce. The natural Ugands for these structures are not yet known. However, since mAb against appropriate epitopes can mimic both soluble factors and signals mediated by cell-cell interactions, it is possible to use an appropriate combination of mAb to control and regulate human B cell proliferation or differentiation. This will support the development of in vivo strategies for the control of human ailments, such as B-cell malignancies from immune deficiencies and certain autoimmune disorders. The new monoclonal antibody G28-5, which is expressed with a single chain polypeptide of approximately 50 Kd, which is expressed on the surface of human B cells, is only a special embodiment of Ugandans of the present invention which the proliferation of activated B cells can improve. Since human B cell proliferation can be improved in a similar manner by T cell-derived BCGF including low and high molecular weight BCGF, the activity of anti-Bp50 G28-5 was compared with that of BCGF preparations, which are predominantly low molecular weight BCGF included, compared. Anti-Bp50 G28-5 and BCGF (lower) were very similar in that they were costimulatory with the same activators (Anti-Ig, Anti-Bp35 and TPA), but not costimulatory with others such as IL-1 or IL -2 were. Furthermore, the activity of anti-Bp50 G28-5 was not dependent on its Fc region, since the F (ab ') 2 fragments of G28-5 were functionally active. This leads to the conclusion that soluble anti-Bp50, such as soluble BCGF, does not need Fc receptor-carrying additional cells to exert an effect. Furthermore, both Antl-Bp50 and BCGF are only effective in the presence of an activation stimulus. In other words, anti-Bp50 and BCGF are not “competence” factors, but promote the “progression” of B cells through the cell cycle.

While it is possible that Bp50 may function as a Ugand receptor, such as a B cell growth factor, some results suggest that Bp50 is not the receptor at least for the BCGF (used) in the present study, represents: it is expressed on blood B cells, whereas BCGF (low) receptors obviously do not. Possible structures for the BCGF (lower) receptor in contrast to Bp50 are also expressed on activated B cells. Furthermore, the normal and malignant B cell populations differ in their sensitivity to anti-Bp50 to BCGF (low) (Table 5 and Fig. 10). For example, some B lymphomas proliferate in response to BCGF (low) but not in response to anti-Bp50. Finally, in a number of experiments, optimal concentrations of anti-Bp50 and BCGF together induced more proliferation than either alone. Anti-Bp50 mimics the activity of other BCGF, such as BCGF (high), which is costimulatory with anti-IgM (Ambrus, et al., 1985, J. Exp. Med. 162: 1319; Ambrus, et al., 1985, J. Clin. Invest. 75: 732). This leads to the conclusion that Bp50 could act as a receptor for BCGF (high).

Although Bp50 could be a receptor for a soluble ligand, Bp50 can alternatively serve as a receptor for a cell-cell mediated signal that regulates the amount of BCGF receptor and / or autocrine production. The precedent for the differentiation of antigens, which serve as enhancers for an autocrine receptor pathway, comes from studies with T cells. mAb against the common leucocyte antigen, Lp 220, improves proliferation by increasing IL-2 receptor expression on activated T cells (Ledbetter, et al., 1985, J. Immunol. 135: 1819). An analogous mechanism can operate with anti-Bp50 and the expression of certain BCGF receptors. Bp50 and BCGF (low) are obviously under some coordinated control, like the IL-1 and IL-2 receptors, BCGF improves the expression of Bp50 on certain leukemic cells. The Bp50 molecule also shares similarities with the Tp44 molecule, which works by influencing IL-2 production. The inventors and others have shown that the 9.3 anti-Tp44 antibody improves the proliferation of T cells activated by anti-CD3 or TPA (Ledbetter, et al "1985, J. Immunol. 135: 2331; Hara , et al., 1985, J. Exp. Med. 161: 1513). Similarly, anti-Bp50 improves the proliferation of B cells activated by anti-Bp35 or TPA. The Tp44 signal works by inhibiting IL-2 production and less by stimulating T cell growth. The Bp50 signal could presumably function in an analogous manner through B-cell autocrine production (Gordon, et al., 1984, Nature, London, 310: 145).

Modified Liaanden. which are used for immunosuppression or treatment of malignancies

For this purpose, the ligand according to the invention can be modified by binding an anti-proliferative agent, so that the resulting molecule can be used to kill cells which express the Bp50 antigen. Ligands modified in this way can be used in the treatment of autoimmune disorders to suppress the proliferation of B cells and thereby suppress the autoimmune response. These modified Ugands can also be used in the immunosuppression of a transplant patient to reject the graft site

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to prevent. Accordingly, cytotoxic agents which are used for the suppression of immune responses can be bound to the ligand according to the invention. If ligands which increase the proliferation of B cells are used, an improved effect should be brought about since the agent is directed against the proliferating B cells.

Furthermore, ligands according to the invention which are modified by the binding of an anti-proliferative agent can be used for the treatment of malignancies which comprise tumors or cells which express the Bp50 antigen. The attachment of these chemotherapeutic agents to ligands according to the invention should bring about a greater specificity of the agent against the malignant cells. Furthermore, a particular advantage should be obtained when treating B cell malignancy with a ligand to which a cytotoxin is coupled which is more effective in killing proliferative cells than in non-proliferative cells. Treatment with such a ligand should potentiate the action of the cytotoxin.

Accordingly, the chemotherapeutic agents or the anti-proliferative agents which can be coupled to the Ugands according to the invention include the agents which are listed in Table 6 below, and which are from Goodman and Gilman, the Pharmacological Basis of Therapy, Sixth Edition , MacMillan Publishing Co., Inc., New York, pages 1249-1313, 1980. However, the possible active ingredients are not limited to those specified below.

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Table 6

Chemotherapeutic agents that can be coupled to anti-Bp50 ligands are class type agents

Alkylating agent

N-Lost

Mechlorethamine

Cyclophosphamide

Melphalan

Uracil lost

Chlorambucii

Ethyleneimine derivatives

Thiotepa

Alkyl sulfonates

Busulfan

Nitrosourea

Carmustln Lomustin Semustin Streptozocin

Triazine derivatives

Dacarbazine

Antimetabolite

Foic acid analogs

Methotrexate

Pyrimidine analogs

Fluorouracil

Cytarabine

Azaribine

Purine analogue

Mercaptopurine thioguanine

Natural products

Vinca alkaloid

Vinblastin Vincristìn

Antibiotics

Dactinomycin

Daunorubicin

Doxorubicin

Bleomycin

Mithramycin

Mitomycin

Enzymes

L-asparaginase

Different means

Platinum coordination complexes

Cisplatin

Substituted ureas

Hydroxyurea

methyl

Hydrazine

Derivatives

Procarbazine

Adrenocorticai suppressant

Mitotan

Hormones and antagonists

Adrenocorticosteroids

Prednisone

Pragestine

Hydroxyprogesterone caproate

Medroprogesterone acetate

Megestrol acetate

Estrogens

Diethylstilbesotot Ethinylestradiol

Antiestrogen

Tamoxifen

Androgen

Testosterone propionate fluoxymesterone

Radioactive isotopes

Phosphorus derivatives

Sodium phosphate 32p

iodine

Sodium iodide 1311

All methods of the prior art can be used to couple the Ugands with the chemotherapeutic or anti-proliferative agents. Examples of such methods are mentioned in the present description.

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Other uses of Liaanden and Bp50

In addition to the therapeutic uses of the Ugands and Bp50 itself, there are other uses, both in vitro and in vivo diaonosis tests. Separation process, etc. The Bp50 receptor can be used in the manufacture and / or design of the Ugands according to the invention. Bp50 can also be used with the ligands according to the invention in in vitro tests which serve as a reference for the detected Bp50 in the sample. Finally, Bp50 itself can be useful as a soluble factor in mediating immunity, e.g. a lymphokine.

In addition to the therapeutic treatment and diagnostic tests, the ligands according to the invention can also be used for the identification or separation of cells which express the Bp50 antigen. Appropriate radio labels or radio-opaque compounds can also be bound to the ligand, the ligand being able to be used for in vivo imaging of tumors which express the Bp50 antigen. Other uses are apparent to those skilled in the art from the present description.

Deposit of cell lines

The present hybridoma was deposited on May 22, 1986 with the American Type Culture Collection, Rock-ville, MD, and has the following deposit number:

Hybridoma

ATCC deposit no.

G28-5

HB9110

The present invention is not intended to be limited by the scope of the hybridoma deposited, since the deposited embodiment serves only as a single illustration and is an aspect of the invention, and other cell lines that function equivalent are within the definition of the invention. Numerous modifications of the invention will be apparent to those skilled in the art when the present description and accompanying drawings are available.

Claims (21)

Claims 1. Ligand which binds Bp50, a B cell surface antigen with a molecular weight of 50 ku (kilodalton), which is defined by the monoclonal antibody G28-5. 2. Ligand according to claim 1, which a) binds Bp5Q and b) after binding to an activated B cell that stimulates the activated B cell, traverses the cell cycle so that the proliferation of the B cell is increased. 3. Ligand according to claim 1 or 2, characterized in that it comprises an antibody molecule or an Fv, Fab, F (ab ') 2 or Fab' part of the antibody molecule. 4. Ligand according to claim 1, 2 or 3, characterized in that the antibody molecule consists of a monoclonal antibody molecule or an Fv, Fab, F (ab ') 2 or Fab' part of the monoclonal antibody. 5. Ligand according to claim 4, characterized in that the monoclonal antibody molecule is the monoclonal antibody G28-5 or an Fv, Fab, F (ab ') 2 or Fab' part thereof. 6. Ligand according to claim 1 or 2, characterized in that it contains a lymphokine, in particular a lymphokine, which is a B cell growth factor. 7. Ligand according to claim 6, characterized in that the lymphokine is on the cell surface. 8. Ligand according to one of claims 1-3 and 6, characterized in that it is coupled with a compound. 9. Ligand according to claim 8, characterized in that the compound is an anti-proliferative agent, an alkylating agent, an antimetabolite, an antibiotic, a vinca alkaloid, an enzyme, a platinum coordination complex, a radioisotope or a fluorescent compound. 10. The method for producing the ligand according to claim 5 in the form of the monoclonal antibody G28-5, characterized in that the hybridoma cell line with the deposit no. ATCC HB 9110 or a mutant thereof is grown and the monoclonal antibody is obtained, 11. B cell surface antigen with a molecular weight of 50 ku (kilodalton), which is defined by the monoclonal antibody G28-5 according to claim 5. 12. B-cell surface antigen according to claim 11, characterized in that it comprises a polypeptide. 13. A method for improving the in vitro proliferation of B cells, characterized in that B cells are treated with an effective dose of a ligand according to claim 1 in such a way that the activated B cells traverser the cell cycle and proliferation is improved. 18th 5 10th 15 20th 25th 30th 35 40 45 50 55 60 CH 676 600 A5 14. The method according to claim 13, characterized in that the B cells are activated by treating them with an effective dose of a second ligand which binds Bp35, a B cell surface antigen with a molecular weight of 35 ku (kilodaltons) Way that the B cell progresses from the Go to the Gi stage of the cell cycle. 15. The method according to claim 13 or 14, characterized in that a ligand according to one of claims 3-9 is used. 16. The method according to claim 14, characterized in that the second ligand contains an antibody molecule which binds Bp35. 17. The method according to claim 16, characterized in that the antibody which binds Bp35 is a monoclonal antibody. 18. The method according to claim 14, characterized in that the activated B-lines are treated with the ligand which binds Bp50 within 12 hours after activation by the second ligand which binds Bp35. 19. A method for suppressing the in vitro proliferation of cells which express Bp50, a B cell surface antigen with a molecular weight of 50 ku (kilodalton), which is defined by the monoclonal antibody G28-5, characterized in that the Cells expressing Bp50 are treated with an effective dose of a ligand according to claim 1, the ligand being coupled with an anti-proliferative agent. 20. The method according to claim 19, characterized in that the cells which express Bp50 contain B cells or malignant cells. 21. The method according to claim 19, characterized in that a ligand according to one of claims 3 to 9 is used. 19th