DE3719398A1 - LIGANDS AND METHOD FOR PROPAGING B-CELL PROLIFERATION - Google Patents

Abstract

The B-cell receptor, Bp50, is a 50 kilodalton polypeptide, that functions in B-cell proliferation. Ligands, such as lymphokines, antibody molecules or the Fv fragments of antibody molecules that bind to Bp50, augment the proliferation of activated B-cells and can be used to regulate B-cell proliferation of differentiation. Toxic substances can be bound to the ligand to suppress proliferation. The ligands can be used in conjunction with a second ligand that binds Bp35, a 35 kd B-cell surface antigen which is involved in progression from G0 to G1 of the cell cycle.

Description

1. Introduction

The present invention is directed to ligands such as Antibody molecules or fragments of antibody molecules, or other ligands, such as lymphokines, attached to a 50 kDa Bind B cell surface markers, referred to here as Bp50, that in B cell proliferation, but not in early B cell activation works. The present invention also targets the Bp50 B cell antigen itself. In a special embodiment of the present invention describes a monoclonal antibody, G28-5, which Bp50 defines and plays a role in the proliferation of activated B cells seem to play, but not noticeable Effect on the proliferation of resting B cells exercises.  

The ligands according to the present invention, such as antibodies, Lymphokines and fragments thereof can be used the proliferation and / or differentiation of direct and regulate human B cells. In addition the ligands according to the invention can be added by modified by other compounds for treatment and / or detection of Bp50 antigen expressing malignant cells can be used.

2. Background of the Invention

The activation of resting B cells from the G ₀ to G ₁ phase of the cell cycle and the subsequent induction of activated B cells to proliferate are distinct stages that require a distinct regulatory mechanism. Some agents, including Murin (mouse or rat) -B cell stimulating factor-pl (BSF-pl) (Rabin, et al., 1985, Proc. Nat. Sci. USA 82, 2935-2939) or low doses of Antiimmunoglobulin (anti-Ig) (DeFranco, et al., 1985, J. Immunol. 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. That is, they induce B cells to enlarge, synthesize more RNA, and enter the G ₁ phase, but they alone do not induce DNA synthesis in B cells. Other "growth" factors, such as growth factor for human B cells (BCGF) and interleukin-2 (IL-2), cause activated B cells to go through the cell cycle and to enter the S phase, but may be quiescent B cells do not initiate (Kehrl, et al., 1984, J. Immunol. Rev. 18: 75-96; Muraguchi, et al., 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 cells have been described by individuals who have studied both murine (mouse or rat) systems and human systems. These factors include B cell growth factors (BCGF), which can be derived from several different sources, including T cell lines or hybridomas, B cell lines or dendritic cells. Although both interleukin-1 (IL-1) and interleukin-2 (IL-2) have been found to increase B cell growth, they are different from other particular BCGFs. For example, monoclonal antibodies (mAb) block a murine BCGF (O'Hara, et al., 1985, Nature (Lond.) 315: 333) or human BCGF (Ambrus, et al., 1985, J. Exp. Med. 162 : 1319) BCGF activity, but not IL-1 or IL-2 activity. For their part, although different from IL-1 or IL-2, the BCGFs appear to be heterogeneous based on biochemical data and differential activity on different B cell subunits or costimulatory samples. For example, a 60 kilodalton (kDa), high molecular weight, human BCGF, BCGF (high), has been identified as different from a 12 kDa, low molecular weight, BCGF (low) (Ambrus, et al., 1985, J. Clin Invest 75: 732). The cDNA encoding a 20 kDa murine BCGF, tentatively called B-cell stimulating factor pl (BSF-pl), has recently been cloned and its sequence elucidated (Noma, et al., 1986, Nature 319: 640). The recombinant lymphokine not only has BCGF activity, but can also activate resting B cells and induce the differentiation of IgG ₁ -producing cells. Thus, 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 cells by interacting with specific B-cell surface structures. In addition to the antigen-specific signal by surface Ig, various other candidates of B-cell surface peptides have been identified which can to some extent function on the activation or growth of B-cells. For example, the surface receptors for IL-1 (Dower, et al., 1985, J. Exp. Med. 160: 501) and IL-2 (Robb, et al., 1984, J. Exp. Med. 160: 1126) and recently, functional IL-2 receptors on B cells have been 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). Receptors for B cell growth and activation factors still need to be fully characterized. Some candidates of B cell surface polypeptides that somehow function in the activation or growth of B cells have been identified. For example, Subbarao and Mosier (Subbarao, et al., 1983, Immunol. Rev. 69: 81-97) found that monoclonal antibodies (mAb) activated Lyb2 B-cells to the murine B-cell antigen, and evidence recently appeared on the basis of which Lyb2 can be the receptor for BSF-pl (Yakura, 1985, Fed. Proc. 44: 1532). Similarly, the associated mAb (1F5) to a 35 kDa polypeptide, Bp35, was found to activate human B cells from G ₀ to G ₁ (Clark, et al., 1985, Proc. Nat. Acad. Sci USA 82: 1766-1770; Gollay, et al., 1985, J. Immunol. 135: 3795-3801). Aggregated C3d or antibodies to the 140 kDa C3d receptor, Bp140, induce the proliferation of B cells that 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 BCGFs have 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) made a polyclonal antiserum that identified a 54 kDa polypeptide (gp54) on human B cells and showed that the rabbit antiserum to gp54 induced tonsillar B cells for division. Recently, Jung and Fu (Jung, et al., 1984, J. Exp. Med. 160: 1919-1924) isolated an mAb (AB-1) to a 55 kDa antigen, limited to activated B cells, which the BCGF -dependent proliferation blocked. However, it is not yet known whether Anti-gp54 or AB-1 recognize a BCGF receptor or not.

3. Description of the invention

The present invention is directed to ligands which (a) on Bp50, a 50 kDa B cell specific surface polypeptide, described below, and (b) increase the proliferation of activated B cells. The Invention is also directed to the Bp50 antigen itself, which is defined by monoclonal antibody G28-5 and acts on the proliferation of activated B cells. Furthermore, the invention is directed to ligands which Bp50 bind, but no biological effect or function, like the proliferation of activated B cells, demonstrate.

The ligands of the present invention include antibody molecules, monoclonal antibody molecules and fragments of these antibody molecules containing the site combining with the antigen, or chemically modified antibodies and fragments. Such fragments include, but are not limited to, Fv, Fab, F (ab ′) ₂ , Fab ′, and the like. The ligands of the invention further include lymphokines, which may include, but are not limited to, human B cell growth factors as well as chemically modified lymphokines. The ligands according to the present invention can be chemically modified, for example by linking or coupling a compound to the ligand. Such compounds include, but are not limited to, cytotoxic agents, therapeutic agents, chemotherapeutic agents, markers such as radio markers, enzymes, radio opaque compounds, and the like. The ligands according to the present invention can be used in their modified or unmodified form to direct, regulate and modify 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 antigen described here, Bp50, apparently play a distinctive role as signal receptors in B cell activation. Monoclonal antibodies (mAb) to Bp35 and Bp50 both provide positive signals to B cells that stimulate their progression through the cell cycle. Similar to anti-Ig antibodies, mAb to Bp36 has an activating effect on resting B cells in order to enable them to enter the G ₁ phase of the cell cycle. In contrast, a monoclonal antibody described here, or its F (ab ′) ₂ fragment to Bp50, a 50 kDa polypeptide that is expressed by all B cells, acts in the sense of stimulating activated B cells to go through the cell cycle and increases the proliferation of the activated B cells. Monoclonal antibodies to Bp35, like anti-Ig antibodies, activate tonsillar B cells and induce low levels of B cell proliferation. In contrast, the anti-Bp50 monoclonal antibody by itself has neither an activating effect on B cells nor an inducing effect on the proliferation of B cells; however, together with anti-Bp35 or anti-Ig antibodies, it increases B cell proliferation. In this regard, the action of anti-Bp50 antibody equals the activity of B cell growth factor (BCGF). Only small amounts such as 0.05 µg / ml anti-Bp50 are required to increase proliferation, and anti-Bp50 is effective like BCGF, even if it is 12 to 24 hours after activating the B cells with anti-Ig or anti-Bp35 is added. Without additional exogenous signals, anti-Bp35 and anti-Bp50 antibodies together induce strong proliferation of purified resting B cells. This result suggests that the Bp35 and Bp50 surface molecules act on the regulatory control of B cell activation and progression through the cell cycle. Because of the significance of anti-Bp35 and similar molecules on the effect and action of the ligands according to the invention, Clark et al., 1985, Proc. Natl. Acad. Sci. (USA) 82: 1766-1770.

Although the activity of anti-Bp50 is that of BCGF (low) resembles that both anti-Bp50 and BCGF (low) costimulatory with the same means, but not with each other and both anti-Bp50 and BCGF (low) only activated Affect B cells and work in a soluble form, the activity of anti-Bp50 can be distinguished from the activity of BCGF (low) because the proliferation of B cells that contain optimal amounts of anti-Bp50 and anti Bp35 (or anti-Ig) are stimulated to be further enhanced can with BCGF (low) and both blood B cells as well certain B cell lymphomas differed on anti-Bp50 Answer BCGF. For optimal activity, anti Bp50 within 12 hours after B cell activation added, whereas BCGF (low) its optimal activity even if it stays 24 hours after the Activation is added. In addition, Bp50 is used by everyone B cells expressed whereas receptors or BCGF (low) are restricted to activated B cells. Thus, anti Bp50 and BCGF (low) coordinate B cell growth in a coordinated manner, but obviously do this through different Signals.

In one embodiment of the invention, the ligands, that bind to Bp50 and the proliferation of activated B-cell proliferation is used to increase immune response will. For example, these ligands, which are Bp50  bind, used as "aids" to increase an immune response to a vaccine. On the other hand, these can Ligands to increase the immune response of an immune-suppressed Be used individually.

In another embodiment, the ligands can be after Invention can be chemically modified so that the cells adhere that bind the ligands are killed. Because all B cells expressing the Bp50 antigen, this approach would become one Suppress immune response. A cytotoxic Drug linked to a ligand according to the invention can be used, for example, in vivo to produce a Immunosuppression to break histocompatibility barriers in graft patients. On the other hand can also use these modified ligands for control of autoimmune diseases.

According to another embodiment of the invention, malignant Diseases such as tumor cells that express Bp50 treated using ligands according to the invention be linked to a chemotherapeutic agent are that for the treatment of such neoplastic diseases is useful. These modified ligands can be found in used in vivo to make the chemotherapeutic agent to direct a type of malignant cell that does Bp50 antigen, including those cells expressed are not B cells but express Bp50. Using of ligands according to the invention which promote B cell proliferation multiply, a special advantage can be drawn from it that when treating malignant B-cells Diseases using a ligand attached chemotherapeutic agent such is used which is particularly effective for killing proliferating Cells is. This can potentiate the drug effect be preserved.  

Alternatively, the ligands according to the invention can also be used in used in vitro for identification or separation of cells expressing the Bp50 antigen and / or for Examination of body fluids for their presence of Bp50 antigen, which may or may not be common Not. In addition, the ligands according to the invention in used in vivo to target cells or tumors Express Bp50 antigen.

The purified Bp50 antigen according to the invention can be used for formation of antibodies and for the formation or design of others Ligands according to the invention can be used. The Bp50 antigen can still be used for samples and tests like diagnostic immunoassays. Furthermore, can Bp50 itself as a mediator of cell immunity in vivo or in used in vitro.

3.1 Definitions

The following abbreviations used here have the given ones Importance:

AO = acridine orange BCGF = B cell growth factor BCGF (high) = a 60 kDa human BCGF BCGF (low) = a 12 kDa human BCGF Bp35 = a 35 kDa B cell specific surface polypeptide (CD20 ) defined by mAb 1F5 Bp50 = a 50 kDa B cell-specific surface polypeptide defined by mAb G28-5 Fv = the variable region or antigen-combining site of an antibody molecule. This can be any fragment containing the idiotype of the molecule, including but not limited to Fab, F (ab ′) ₂ , Fab ′ and the like. IF = immunofluorescence Ig = immunoglobulin IL-1 = interleukin 1 IL-2 = interleukin 2 kDa = kilodalton mAb = monoclonal antibody SDS-PAGE = sodium dodecyl sulphate-polyacrylamide gel electrophoresis TPA = 12-O-tetradecanoylphorbol-13-acetate.

4. Description of the figures

Fig. 1. Expression of Bp50 is restricted to Bp35 ± -B cells. Two color flow cytometric analysis of 50,000 cells was performed as described (Clark, et al., 1985, Proc. Nat. Asad. Sci. USA 82: 1766-1770). The data are plotted as the number of cells against the logarithm of green fluorescence and the logarithm of red fluorescence, with 4-5 points approximately meaning a doubling of the fluorescence. The data are given to show autofluorescent negative cells. PE (red) -Anti-Bp35 (1F5) against FITC (green) -Anti-Bp50 (G28-5) staining shows that all Bp50 + cells are also Bp35 +.

Fig. 2. Biochemical comparison of Bp50 polypeptide with other B cell surface antigens. Immunoprecipitation of Bp50 from surface ¹²⁵ I-labeled tonsillar cells was performed as described. Isolated antigens were electrophoresed on 10% SDS polyacrylamide gel plates without reduction. The gels were visualized using autoradiography and intensifying screens. Plate A: lane 1, anti-Bp50 (G28-5); Lane 2, anti-Bp95 (G28-8); Lane 3, Sepharose goat anti-mouse Ig only. Exposure time: 4 days. Plate B: lane 1, anti-Bp50 (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 only. An exposure time of 2 days was chosen so that the bands in lanes 2 to 4 were not overdeveloped and could be clearly distinguished relative to Bp50. (One of three attempts).

Figure 3. 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 antibody, 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 equipped with four decade log amplifiers in both red and green dimensions. Forward and right angle light scattering was used to demarcate monocytes. Unstained cells were positioned at the back of the grid or plate. Red fluorescence is on the right and green fluorescence is on the left.

Figure 4. Dose response curves for the proliferation of dense tonsillar Er-B cells by anti-Bp50 antibodies, as indicated: media alone; Anti-Bp50 alone; Anti-Bp35 (5 µg / ml) alone; BCGF alone; Anti-Bp35 plus BCGF; Anti-Bp35 plus divided doses of Anti-Bp50. Mean proliferation ± standard deviation of four-fold samples were measured on day 3.

Figure 5. Anti-Bp50 mAb are most effective in increasing proliferation when added following a B cell activation signal. Dense tonsillar Er-B cells are only incubated with medium for 4 days, anti-Bp50 (0.5 µg / ml) is added at different times after the incubation, anti-Bp35 (5 µg / ml) is added at different times after the Incubation added; Anti-Bp50 is kept constant, at which time Anti-Bp35 is added at different times; Anti-Bp35 is kept constant at which anti-Bp50 is added to cultures at different times. ³ H-thymidine is added during the last 10 hours and its incorporation is measured.

Figure 6. Comparison of the ability of anti-Bp35 and anti-Bp50 to induce resting tonsillar B cells to exit the G ₀ stage of the cell cycle. Day 3 after treatment medium alone (     ), Anti-Bp35 alone (-----); and Ig alone (.....), A, no additional additives; B, anti-Bp50 (0.5 µg / ml) added to each group; C, 5% BCGF added to each group. The data are plotted as the relative cell count against the logatrithm of AO red fluorescence (RNA).

Fig. 7. Kinetics of B cell proliferation after stimulation with anti-Bp50 against BCGF. Dense tonsillar EB cells were stimulated with medium alone; 10% BCGF alone; Anti-Bp35 alone; Anti-Bp50 alone; Anti-Bp35 + 10% BCGF; Anti-Bp35 + anti-Bp50; and Anti-Bp35 + Anti-Bp50 + 10% BCGF. Proliferation was measured on the specified days by an 18 hour pulse of ³ H-thymidine. Proliferation was measured four times and standard deviations are shown. (One of three experiments).

Fig. 8. Show after anti-Bp35 stimulation, in which anti-Bp50 (A) or BCGF (B) optimally increase the proliferation. Dense tonsillar EB cells were stimulated as shown and proliferation was measured by an 18 hour pulse of ³ H-thymidine on day 3. Media; Anti-Bp35 alone, added at the times indicated; Anti-Bp50 or BCGF alone; Anti-Bp35 added at the beginning of the culture, followed by the addition of Anti-Bp50 or BCGF at the indicated times; Anti-Bp50 or BCGF added at the beginning of the culture, followed by Anti-Bp35, (one of two experiments). Proliferation was measured four times and standard deviations are shown. Doses used: Anti-Bp35, 5 µg / ml; Anti-Bp50, 0.2 µg / ml; BCGF (low) 5%. Concentrations were applied as follows: Anti-Bp35, 5 µg / ml; Anti-Bp50, 0.2 µg / ml; BCGF, 5%.

Figure 9. Anti-Bp50 and BCGF have an additive effect on B cell proliferation. Dense tonsillar EB cells were stimulated with graded doses of BCGF (low) along with anti-Bp50 alone; Anti-Bp35 alone, anti-Ig beads alone; Anti-Bp35 + anti-Bp50; or Anti-Bp50 + Anti-Ig. Proliferation was measured on day 3 after stimulation with an 18 hour pulse of ³ H-thymidine. Proliferation was measured four times and standard deviations are shown. (One of four experiments). Doses used 10 ⁶ cells: Anti-Bp35, 5 µg / ml; Anti-Bp50, 0.2 µg / ml; Anti-Ig beads, 50 µg / ml.

Fig. 10. Comparison test of anti-Bp50 and BCGF on normal and malignant B cells. Peripheral blood EB cells (A) or dense tonsillar EB cells (C) were stimulated with or without TPA (75 ng / ml) in the presence of 10% BCGF or 1 µg / 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 incorporation of ³ H-thymidine during a 12 hour pulse. The proliferation was measured four times and standard deviations are shown.

5. Detailed description of the invention

The present invention is directed to ligands which (a) on Bp50, a 50 kDa B cell specific surface polypeptide, bind and (b) the proliferation of activated Multiply B cells. The invention is also directed to the Bp50 antigen itself, which is defined by mAb G28-5 and acts on B cell proliferation. Continue judging the invention relates to ligands that bind to Bp50, however, no biological effect or function like reproduction the proliferation of activated B cells.

The ligands of the present invention include antibody molecules, monoclonal antibody molecules, and fragments of these antibody molecules that contain the antigen combining site that binds to the Bp50 receptor, including chemically modified antibodies and fragments. Such fragments include, but are not limited to, Fv, Fab, F (ab ') ₂ , Fab', and the like. Furthermore, the ligands according to the present invention include lymphokines that bind to the Bp50 receptor. These may include, but are not limited to, the BCGFs and chemically modified lymphokines and the like. The ligands of the invention, in their modified or unmodified form, can be used to modulate and regulate immune response and to treat malignant lesions that express the Bp50 antigen. These uses are described in more detail in section 5.4.

The ligand is a monoclonal antibody or fragment of this, then the monoclonal antibody against Bp50 can be produced are made using every technique used to produce of antibody molecules through continuous Serves cell lines in culture. For example, in the frame The invention uses the hybridoma technique, such as  originally from Kohler and Milstein (1975, Nature 256: 495-497), as well as other techniques, which have only recently become available, like the Human B cell hybridoma technique (Kozbor et al., 1983, Immonoly Today 4:72) and the EBV hybridoma manufacturing technique human monoclonal antibody (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) and the like.

Antibody fragments containing the idiotype of the molecule can be created by known techniques. Examples of such fragments include, but are not limited to: the F (ab ′) ₂ fragment, which can be formed by treating the antibody molecule with pepsin; the Fab ′ fragments, which can be formed by reducing the disulfide bridges of the F (ab ′) ₂ fragment; the F (ab ′) ₂ fragment, which can be formed by treating the antibody molecule with papain; and the 2Fab or Fab fragments that can be formed by treating the antibody molecule with papain and reducing agents to reduce the disulfide bridges.

If the ligand that binds Bp50 is a lymphokine, then it can the lymphokine is obtained from natural sources or if its amino acid sequence is known or derived, then the lymphokine can be synthesized through chemical methods be synthesized. If the gene sequence of the lymphokine known recombinant DNA techniques can be applied to clone the gene into an expression vector, which is for the transcription and translation of the gene sequence in a suitable host cell.

Depending on the intended use, the Ligand or suitable fragments of the ligand chemically modified are made by connecting a Variety of connections available at the  Ligands using known coupling techniques. Such techniques include, but are not limited to, the use of carbodiimide, cyanogen bromide, bifunctional Reagents such as glutaraldehyde, N-succinimidyl-3- (2-pyridyldithio) - propionate (SPDP), Schiff base reactions, connection of sulfhydryl functions, the use of sodium isothiocyanate or enzymatic bonds, just a few call. Where to add a radioisotope to the ligand this can also be done via enzymatic routes, oxidative substitution, Chelation and the like can be made. An overview of the chemical reagents needed for the Protein modification can be used is published at Lundblad and Noyes, Chemical Reagents for Protein Modification, Volume II, CRC Press, Inc., Boca Raton, Florida, Ch. 5, pp. 123-139, 1984.

The chemical linkage or coupling of a compound the ligand can be directed to a location on the ligand that does not participate in the binding to Bp50. This can be achieved by protecting the binding site on the Ligands before performing the coupling reaction. For example the ligand can be bound to Bp50 first, to protect the Bp50 binding site, after which the coupling reaction to link the desired connection available reactive sites on the ligand Bp50 complex can be carried out. When the coupling reaction is complete, then the complex can be dismantled again to create a modified ligand which the desired connection is made, whereby the Bp50 binding site of the molecule minimally affected becomes. Where the ligand comprises a monoclonal antibody like G28-5, in which the Fc domain or region of the molecule is not required for the ligand to exert its effect (see section 5.3.3), it can be advantageous to Coupling of the desired compounds to the Fc region of the To direct the molecule.  

The following sections describe the new 50 kDa B cell surface marker, Bp50, which obviously has an effect on B cell proliferation, as well as ligands that bind to the new 50 kDa receptor, and their uses. As an example of the ligands of the present invention, a monoclonal antibody which defines Bp50 and its F (ab ′) ₂ fragments are also described which, like BCGF, increase the proliferation of B cells. In contrast to anti-Bp35 mAb, which can induce resting B cells of the G ₀ phase to enter the G ₁ phase, anti-Bp50 mAb does not activate resting B cells. Anti-Bp35 and Anti-Bp50 mAb together induce strong activation and proliferation of purified B cells without additional exogenous signals.

The experiments described below show that anti Bp50 activity is similar to BCGF activity, but anti-Bp50 is different from a BCGF, because Anti-Bp50 and BCGF also low molecular weight are clearly additive and different to different B cell subsets or diseases Act. Bp50 can be a receptor for a distinct BCGF or for a transmembrane signal that is the BCGF production or modulates BCGF receptor expression.

5.1 Characterization methods used of the Bp50 receptor Cell preparations.

Mononuclear cells were made from normal or leukemic heparinized peripheral blood from Ficoll Hypaque gradients (Pharmacia, Piscataway, NJ) isolated. Mononuclear cells were obtained from tonsillar Tissue as described (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770). T cells were removed by Rosetting using AET-treated sheep erythrocytes and Ficoll-Hypaque gradient separation. At In some experiments, blood B cells were enriched by  Isolation of cells adhering to nylon wool. Monocytes were removed by incubation on plastic petri dishes, once or twice at 37 ° C for 45 minutes if not stated otherwise. Floating or dense Tonsillar B cell fractions were isolated by Percoll Step gradients as described (Clark et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770). Dense tonsillar B cell Accordingly, preparations had more than 95% sIg + Bp35 + cells. Preparations enriched with blood B cells had 60-85% sIg + cells. B cell lymphoma cells were found isolated by careful separation of lymphoma cells in Medium followed by Ficoll-Hypaque gradient centrifugation.

Monoclonal antibodies.

The G28-5 antibody to Bp50 was generated by immunizing BALB / c mice with human E tonsillar lymphocytes and fusing immune spleen cells with NS-1 myeloma (Kohler, et al., 1975, Nature 256: 495- 497; Ledbetter, et al., 1979, Immunol. Rev. 47: 63-82). Hybrid cell cultures that secreted an antibody that is reactive with tonsillar B cells and not with T cells were identified using indirect immunofluorescence (IF) and analysis with a FACS IV cell sorter; Cultures with antibodies with a histogram pattern similar to the known mAb to Pan B cell markers (e.g. Bp35) were cloned and selected for further studies. The G28-5 clone produced an IgG ₁ mAb that only reacted with normal or malignant B cells or B cell lines. Other mAbs used in this study are described in detail (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 Perspectives in Immunogenetics and Histocompatibility, ASHI, New York, 6, pp. 325-340). These include 1F5 (IgG _2a ) anti-Bp35, HB10a (IgG _2a ), anti-HLA-DR, 2C3 (IgG ₁ ) anti-u chain, G19-4 (IgG ₁ ) anti-CD3, FC-2 (IgG _2a ) Anti-Fc Receptor CD16, and 9.6 (IgG _2a ) Anti-CD2 (E Receptor), by Dr. Paul Martin provided (Martin, et al., 1983, J. Immunol. 131: 180). The IgG ₁ mAbs were purified by precipitation using 45% or 50% saturated ammonium sulfate and DEAE-Sephacryl column chromatography, and the IgG _2a mAbs were purified by using Protein A-Sepharose columns. The F (ab ′) ₂ fragments of G28-5 were prepared by the method of Parham (Parham, et al., 1983, J. Immunol. 131: 2895), purified on a 2 m long Sephacryl S200 column Purity checked by SDS-PAGE (Ledbetter, et al., 1985, J. Immunol. 135: 1819). The 2C3 mAb to u chains were conjugated to Sepharose 4B beads (Pharmacia Fine Chemicals, Uppsala, Sweden) using the cyanogen bromide coupling.

Fluorescein and phycoerythrin conjugations.

Cleaned mAb was either directly conjugated with fluorescein Use of fluorescein isothiocyanate (FITC; Molecular Probes) (green) using the method of Goding (Goding, et al., 1976, J. Immunol. Meth. 13: 215-226), or conjugated to R-phycoerythrin (PE) (red) using SPSP (Pharmacia), using a method detailed by Letbetter, et al., 1985, in Perspectives in Immunogenetics and Histocompatibility, ASHI, New York, 6, 119-129. Lymphoid Cells were placed in round-bottomed microtiter plates for 30 minutes with a suitable dilution of green and / or red mAb incubated, washed twice and then on analyzed using a FACS IV cell sorter.

Two-color immunofluorescence.

Two-color studies were done performed with a fluorescence activated cell sorter (FACS IV: Becton-Dickinson, Mountain View, CA) using of a 560 nm dichroic mirror to split the Rays and a 580 long pass filter and a 540 short Pass filters (Ditric Optics, Hudson, MA) in front of the red one or green photomultiplier tubes. In addition, a two-color  Compensator (T. Nozaki, Stanford University) used for a slight overflow of green and red signals to correct. For every two-tone coloring Data from 40,000 cells collected and stored on floppy disks. The data are given as cell number (vertical) against the logarithm of green fluorescence against the logarithm red fluorescence on a 64 × 64 dot grid. Nearly 4.5 points mean a doubling of the fluorescence. Unstained cells were at the back corner of the grid positioned; red fluorescence is on the right and green fluorescence is on the left. The flow cytometry system for two colors IF with fluorescein and phycoerthrin is described in more detail (Ledbetter, et al., 1985, in Perspectives in Immunogenetics and Histocompatibility, ASHI, New York, 6, 119-129 and 325-340).

Cell culture.

Blood or tonsillar lymphoid cells were cultured fourfold at 5-10 × 10 ⁵ cells / ml in 96-well microtiter plates containing 200 μl RPMl-1640 medium containing 15% fetal bovine serum, antibiotics, glutamine and pyruvate ( R15) was added. After 1 to 7 days, the cells were pulsed with 0.5 μCi of ³ H-thymidine per well (New England Nuclear, 6.7 Ci / mmol; 1 Ci = 37) for 18 hours. The cells were then collected on glass fiber filters with a cell harvester and the radioactivity was measured in a scintillation counter. In some experiments, antibodies or factors were added at different times after the start of the cultures. The proliferation in these experiments was measured on day 3.

Cost-stimulating factors.

Purified BCGF was from cytokines Technology (Buffalo, New York) related and contained no detectable IL-1, IL-2 or interferon activity. This BCGF was made using the Maizel method and co-workers (Maizel, et al., 1982, Proc. Nat. Acad. Sci. USA 79: 5998), which have shown to be the main BCGF activity  based on a 12 kDa species in this material, hereinafter referred to as "BCGF (low)" (Mehta, et al., 1985, J. Immunol. 135: 3298). The cleaning levels concluded a preparative DEAE affinity chromatography, followed by hydroxyapatite column chromatography. IL-1 in a form purified to homogeneity was generously from Dr. Steven Dower provided (Dower, et al., 1985, J. Exp. Med. 162: 501). Recombinant IL-2 was made courtesy of Cetus Corporation posed. TPA (12-O-tetradeconoyl-phorbol-13-acetate) was sourced from Sigma.

Detection of cell activation.

Changes in cell volume, induced by mAb and / or factors measured using a cell sorter and an angular Light control fed. Cell cycle changes in cellular RNA and DNA levels were measured by staining activated cells with acridine orange and measure the relative cellular RNA (red) and DNA (green) content with a Cell sorters using the method of Darzynkiewicz et al. (Darzynkiewicz, et al., 1980, Proc. Nat. Acad. Sco. United States 77: 6697-6702). Changes in the relative levels of the cell surface Antigens were monitored using mAb directly conjugated to fluorescein, and then through direct IF fluorescence mirrors with an Epics V cell sorter quantified.

Biochemical characterization of Bp50.

Immunoprecipitation of Bp50 from surface ¹²⁵ I-labeled tonsillar cells was performed as described (Ledbetter, et al., 1985 J. Immunol. 134: 4250-4254). Isolated antigens were electrophoresed on 10% SDS polyacrylamide gel plates without reduction. The gels were visualized using autoradiography at -70 ° C and Cronex lighting plus intensifying screens or foils (Dupont).

5.2 Characterization of the Bp50 receptor

The following sections describe the results of the Experiments using the ones described above Methods were carried out.

5.2.1 Identification of a B cell specific 50 kDa cell surface marker, Bp50.

A mAb against Bp50 was elicited by immunization of BALB / c mice with human tonsillar lymphocytes and fusion of immune spleen cells with NS-1 myeloma. One clone, G28-5, produced an IgG ₁ mAb that did not contain the NS-1 light chain. After analysis by IF analysis, it was found that G28-5 only reacts with normal or malignant B cells or B cell lines. A comprehensive screening of normal tissues using recognized methods (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770; Ledbetter et al., 1986, Human Immunol. 15: 30-44; Ledbetter , 1985, in Perspectives in Immunogenetics and Histocompatibility, ASHI, New York, 6, pages 325-340), found that the G28-5 antibody with E-rosettes does not react with negative (Er) cells from blood or tonsils on non-adherent nylon wool T cells, PHA-induced T cell blasts or with blood granulocytes, monocytes and red cells or platelets. It reacted strongly with all seven B-lymphoplastoid cell lines tested and with three Burkitt's lymphoma lines (Raji, Daudi, Namalwa), but not with four T cell lines (CEM, HSB-2, JURKAT and HPB-ALL). All chronic lymphocytic leukemias (3/3) and 90% (9/10) of the B-lymphomas tested expressed the Bp50 marker, whereas only 28% (2/7) of non-T-, not B-CALLA⁺ acute lymphocytic Leukemia expressed Bp50.

The restricted distribution of Bp50 to normal tissue was further confirmed by quantitative two-color immunofluorescence (two color IF) analysis. Using an R-phycoerythrin (PE) -conjugated antibody (red) to the Pan-B cell antigen Bp35 (B1, CD20) and fluorescein-conjugated anti-Bp50 antibody (green), it was found that Bp50 only on Bp35 + B Cells ( Fig. 1) is expressed in blood or tonsils. Blood B cells consistently expressed slightly lower levels of Bp50 than tonsillar B cells. This is similar to HLA-DR expression (Ledbetter et al., 1986, Human Immunol. 13: 30-44) and gp54 expression (Wang, et al., 1979, J. Exp. Med. 149: 1424- 1433), which are also lower on blood cells. Bp50 was expressed with similar levels on tonsillar B cell subpopulations that were separated into floating and dense fractions on Percoll gradients. Using the PE-conjugated mAb to 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 expressing high levels of IL-2 receptors did not express Bp50.

The G28-5 antibody reacted with a single polypeptide to tonsillar lymphocytes, which migrated at approximately 50 kDa under non-reducing conditions ( Fig. 2A). This molecule is larger than previously reported B cell markers from the same molecular weight range as, for example, Bp39 or Bp45 (Zipf, et al. 1983, J. Immunol. 131: 3064-3072; Kitner, et al, 1981, Nature 294, 458-460 ; Clark, et al., 1986, in Leukocyte Typing II, publisher: Reinherz, et al., Springer-Verlag, Berlin, chapter 12, volume 2, 155-167; Slovin, et al., 1982, Proc. Nat Acad. Sci. 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. The Bp39 marker, unlike Bp50, is expressed on granulocytes, and Bp45, unlike Bp50, is restricted to B cell blasts. Antibodies to Bp39 (41-H16) and Bp45 (MNM6, Blast-1, Blast-2), which were made accessible by an international working group (Clark, et al., 1986, in Leukocyte Typing II, publisher: Reinherz et al. , Springer Verlag, Berlin, chapter 12, volume 2, 155-167) do not block the binding of fluoresceinized anti-Bp50 antibodies to B cells. Based on tissue distribution, biochemical analysis and blocking studies, the G28-5 monoclonal antibody recognizes a 50 kDa structure that is different from other known B cell antigens.

5.2.2 Expression of Bp50 is restricted to B cells

The distribution studies in hematopoietic tissues and cell lines and also detailed two-color flow cytometric analyzes show that Bp50 is only expressed on B lymphocytes. As illustrated in Figure 3, Bp50 is expressed on a small subunit of blood lymphocytes and on a large population of tonsillar lymphocytes. Almost all Bp50 + cells in both the blood and tonsils also expressed Bp35 and HLA-DR, but did not express the CD2 ( Fig. 1) or CD3, T cell molecules or the IgG Fc receptors that target NK- Cells are found. In addition, ConA-activated CD3 + T cell blasts expressed Il-2 receptors, but not Bp50.

Two-color flow cytometric analysis allows the quantitative measurement of the density ratio between the surface antigens. It has previously been shown that the dense, dormant B cells in the envelope of secondary follicles express IgM and low levels of Bp35, whereas the floating, activated B cells in the germinal center are IgM negative and express elevated levels of Bp35 (Ledbetter , et al., Human Immunol. 15:30). Figure 3 shows that both IgM positive and IgM negative B cell subunits expressed Bp50 in equal amounts, suggesting that Bp50 is expressed on both resting B cells and activated B cells in vivo.

5.3 Increase in B cell proliferation with anti-Bp50 antibody

As explained earlier, B cells can take low doses of Anti-u chain specific antibodies are activated. It It has recently been found that the B cell specific marker Bp35 (B1), a 35 kDa polypeptide, also in early B cell activation works: the 1F5 mAb to Bp35 activates B- Cells, such as low doses of anti-u antibody, to increase in cell volume and RNA content and the emergence of Responsibility to BCGF (Clark et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770; Gollay, et al., 1985, J. Immunol. 135: 3795-3801). So it was of interest that Effect of anti-Bp50 mAb on the proliferation of untreated B cells or with either anti-Bp35 or anti-u antibodies (Table 1) to compare activated B cells. Anti-Bp35 in solution or anti-u antibody bound to Sepharose pearls, by themselves, could be used under suitable ones Conditions stimulate some B cell proliferation (Table 1, line 1). In contrast, anti- Bp50 antibodies alone do not proliferate (Table 1, Line 2). Anti-Bp50 mAb, however, increases proliferation considerable when cultivated with anti-u pearls or with anti-Bp35 becomes. In this regard, Anti-Bp50 is similar to BCGF (Table 1, line 3). It was important to determine whether anti-Bp50 and BCGF together could induce B cell proliferation. As explained in Table 1, line 4, anti Bp50 and BCGF together did not proliferate, but increased  the proliferation of either with anti-u or -Bp35 activated cells a little more than either one alone stimulates. BCGF has when using Anti-Bp50 with no other signals no effect is used over a 3-log area on the proliferation of dense B cells, even if anti Bp50 used in doses ranging from 0.1 to 10 µg / ml becomes.

Table 1

Multiplication of anti-Ig or anti-Bp35-induced B cell proliferation with anti-Bp50 antibodies

5.3.1 Anti-Bp50 mAb only increases proliferation after the B cells with anti-Bp35 or anti u antibodies are activated

The results in Table 1 show that anti-Bp50 mAb cannot induce proliferation by itself. As shown in Fig. 4, doses of anti-Bp50 in the range of 0.05 µg to 2.0 µg / ml had no effect on ³ H-thymidine uptake. However, in the presence of optimal levels of anti-Bp35 mAb, amounts as small as 0.1 to 0.5 µg / ml of anti-Bp50 antibodies increased proliferation significantly. Even 50,000 to 70,000 cpm can be determined with the optimal proliferation time if highly purified B cells are only cultivated with anti-Bp35 plus anti-Bp50. A corresponding observation shows that high doses of anti-Bp50 (greater than 2-5 µg / ml) are less effective than doses in the range of 100-200 ng.

These results show that anti-Bp50 can only work after activation of B cells by other signals. The data in Figure 5 show that this is indeed the case. If B cells are activated with anti-Bp35 first, anti-Bp50 can be added as late as 24-48 hours later and still increase proliferation on day 4. Conversely, if the cells are 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.

5.3.2 Anti-Bp50 mAb does not activate B cells, starting from G ₀ , but induces activated B cells to go through the cell cycle

It has already been found that anti-Bp35, like low doses of anti-u antibodies, induce resting tonsillar B cells of the G ₀ phase for enlargement (Clark, et al., 1986, Leukocyte Typing II, editor: Reinherz, et al., Springer Verlag, Berlin, Volume 2, 455-462) and to enter the G ₁ phase of the cell cycle (Gollay, et al. 1985, J. Immunol. 135: 3795-3801). So it was of interest to compare the ability of anti-Bp50 mAb versus anti-Bp35 mAb to compare its effectiveness on B cell activation. As shown in Fig. 6A, had unstimulated dense tonsillar B cells even after 3 to 4 days in culture, a uniform RNA profile characteristic of cells in G ₀ (Darzynkiewicz, 1980, Proc. Nat. Acad. Sci. USA 77: 6697-6702). However, approximately 15-30% of cells stimulated with anti-Bp35 or anti-u had an increased RNA content, which is an indication of the entry into the G ₁ phase. In contrast, neither anti-Bp50 ( FIG. 6B) nor NCGF ( FIG. 6C) alone induced significant numbers of B cells to enter G ₁ . For example, 2 days after activation, anti-Bp35 and anti-Ig mAb induced 13.5% and 20.9% of tonsillar cells to enter G ₁ , whereas cells that only had anti-Bp50 (2.7% ) or BCGF (3.2%) remained at the media control level (2.2%). However, when anti-Bp50 or BCGF was added along with anti-Bp35 or anti-u antibodies, the proportion of cells entered in phase G ₁ increased dramatically. Similarly, anti-Bp50 and BCGF alone did not induce B cells to enter the S phase (Table 2), but along with either anti-Bp35 or anti-u, the number of S-phase cells increased to two. up to three times.

Table 2

Effect of anti-Bp50 and BCGF on cell cycle progression in tonsillar lymphocytes

5.3.3 Optimal conditions for the multiplication of the B-cell Proliferation with anti-Bp50 antibodies

Antibodies to Bp50 alone have little or no noticeable effect on dense, round B cells (table 3). However, in the presence of agents that activate B cells can, such as anti-Ig, anti-Bp35 and TPA multiplies Anti-Bp50 mAb proliferation clearly. Has anti-Bp50 no costimulatory effects with different interleukins, including purified IL-1, recombinant IL-2 and  BCGF (low). A comparison of the effects of anti-Bp50 with those from BCGF (low) showed that the same means, which were costimulatory with anti-Bp50, equally stimulating with BCGF (low) (Table 3). From of particular interest was to find that BCGF and Anti-Bp50 are not costimulatory for resting cells.

Table 3

Increasing B cell proliferation with anti-Bp50 antibodies or B cell growth factor

The kinetics of anti-Bp50 enhanced proliferation is shown in Figure 7. The proliferation peak 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 kinetic processes of proliferation when augmented by BCGF or by anti-Bp50 were similar.

Even small amounts such as 0.05 µg anti-Bp50 antibodies increased the proliferation. An optimal dose of 0.3 µg / ml was used in the subsequent investigations. A It has been observed that when using whole antibody molecules higher doses of anti-Bp50 (greater than 2- 5 µg / ml), were less effective than doses in the range of 0.1 -0.5 µg / ml.

Human B cells are extremely sensitive to inhibitory effects mediated by FC receptors of antibodies that bind to surfaces Ig (Parker, 1980, Immunol. Rev. 52: 115, Bÿsterbosch, et al., 1985, J. Exp. Med. 162: 1825). So it was important to compare the effectiveness of the entire Anti-Bp50 mAb with that of Anti-Bp50 F (ab ′) ₂ fragments. Over a 100-fold dose range, F (ab ′) ₂ fragments were clearly as effective as or even more effective than the entire antibody in increasing B cell proliferation (Table 4). Thus, the Fc domain or region of anti-Bp50 mAb is not required for anti-Bp50 to exert its effect and can, if effective, be inhibitory. In other words, like BCGF, anti-Bp50 can obviously act as a soluble mediator without the help of the Fc receptor mediated cell access function.

Table 4

The Fc region of anti-Bp50 antibodies is not necessary to increase B cell proliferation

5.3.4 Differences between the Anti-Bp50 BCGF (low) activity

Anti-Bp50 and BCGF (low) have a similar effect on B cells and were costimulatory with the same agents (Table 3). However, some evidence shows that anti-Bp50 and the BCGF used in this study obviously work on different signals. First, Bp50 molecules, unlike BCGF (low) receptors (Bÿsterbosch, et. Al., 1985, J. Exp. Med. 162: 1825), are expressed on resting blood B cells ( Fig. 3). Second, although both anti-Bp50 and BCGF (low) work most effectively when added after anti-Bp35 or anti-Ig, anti-Bp50 is clearly optimally effective when added 12 hours after the start of culture ( Fig. 8A) . In contrast, BCGF (low) could still be added 24 hours after the start of cultivation and is still increasing the proliferation optimally ( FIG. 8B). These kinetic experiments, modeled on the suggestions of Howard and Paul (1983, Ann. Rev. Immunol. 1: 307), indicate that a Bp50-dependent signal can normally exert its effect before BCGF.

Both anti-Bp50 and BCGF (low) increase the proliferation of B cells activated with anti-Bp35 or anti-Ig (Table 3). The effects of anti-Bp50 and BCGF (low) were additive in many experiments ( Fig. 7). Figure 9 shows a titration of BCGF (low) in an experiment using anti-Bp50 at its optimal concentration (0.2 µg / ml). BCGF (low) increased proliferation of resting cells in the presence of anti-Bp50 after activation by either anti-Ig or anti-Bp35. Optimal concentrations of BCGF (low) were 5-10%, while 25% were inhibitory. Thus, when anti-Bp50 and BCGF (low) are both used at their optimal concentrations, they still show additive effects on B cell proliferation.

Ultimately, both normal and malignant B cell subunits differed in their response to anti-Bp50 and BCGF (low). For example, some blood B cells respond 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 therefore necessary in order to make the blood cells respond to Anti-Bp50. While dense tonsillar B cells generally did not respond to either BCGF or anti-Bp50, floating B cells responded (Table 5). B cell foci were also different in their response behavior to anti-Bp50 compared to BCGF. For example, some B cell lymphomas responded to TPA plus BCGF (low) but not TPA plus G28-5 AntiBp50 ( Figures 10B and D). In contrast, dense tonsillar B cells and peripheral blood B cells respond to TPA plus either BCGF (low) or anti-Bp50 ( Figures 10A and C).

Table 5

B-cell subunits differ in their responsiveness to anti-Bp50 or BCGF

Cell culture conditions as described in Table 1. Blood lymphocytes (B cells plus monocytes) adhering to nylon wool were freed from monocytes by incubation on plastic dishes overnight before stimulation (Exp. 1 and Exp. 2). In Exp. 3, blood E-lymphocytes were freed from monocytes by incubation on plastic dishes for one hour before stimulation. Tonsilar e _r lymphocytes were fractionated using Percoll gradient in density (pellet) or floating (fraction 1) subunits (32).

5.4 Use of anti-Bp50 ligands and Bp50

The ligands according to the present invention can be used in vivo or in vitro in their unmodified or modified Forms for modulating immune responses can be used. For example, the ligands themselves can be used as an "aid" to increasing an immune response a vaccine or to increase the immune response of an immune suppressed Individual. If cytotoxins or anti-proliferative Agents coupled to the ligands can do so modified ligands on the other hand can be used to reduce an immune response, for example in autoimmune Diseases or in transplant patients to the Avoid prosthesis or implant rejection. This modified Ligands can also be used for treatment malignant lesions that include cells or tumors, which express the Bp50 antigen regardless of whether the focus of the disease is in the origin of B cells or not.

The ligands according to the invention and / or the Bp50 itself, can be used in vitro. Such applications close a: in vitro tests, such as immunoassays to find cells that express Bp50 antigen and / or to find anything present in body fluids secreted Bp50 antigen. In these cases you can the ligand or Bp50 are marked with a radio marker, Fluorine, enzyme, enzyme substrate, dye and the like. In addition the ligands can be used to target cells that express, separate and / or express the Bp50 antigen identify, in which case the ligand to a immobile carrier can be coupled or to a Fluorine, which is in a FACS (fluorescence activated cell sorter) can be used.  

The different uses and uses of the ligands and Bp50 according to the present invention described in detail below.

5.4.1. Bp50 receptor and uses of ligands such as Anti-Bp50, to increase B cell proliferation

Early studies indicated that the factors involved in inducing B cells from the G ₀ to the G ₁ phase of the cell cycle are different from the factors or requirements for the transition to the S phase. This model is primarily based on studies showing that agents such as low doses of anti-Ig-B cell activation factors or anti-Bp35 alone have little or no effect on B cell proliferation. However, the same agents can drive B cells to a point in cell activation where they are susceptible to growth factors. In contrast, growth factors such as BCGF or IL-2 alone have no effect on resting B cells, but increase the growth of activated B cells.

While the present invention is not intended to be limited to any theory or interpretation, the results presented here provide additional support for a model of distinct regulation of B cell activation and growth levels. It has been shown that the activation and proliferation signals in human B cells can be transmitted through distinct cell surface structures. Although anti-Bp35 mAb activates B cells to enter the G ₁ phase of the cell cycle, it alone induces little or no proliferation. Anti-Bp50 mAb has the opposite effect: it cannot activate B cells, but if it is added as late as 12 to 24 hours after activation, it can induce B cell growth.

The Bp50 molecule can function normally as a receptor for a ligand, such as a soluble growth factor or for a signal that is transmitted through cell-to-cell contact (i.e. a ligand that is on the surface of another cell Is found). Previous studies identified several BCGFs derived from T cells, which are similar to anti-Bp50, which Increase B cell proliferation. Both a high as well low molecular weight forms of B cell Growth factors have been identified and it has been shown that different types have additive effects (Kehrl, et al., 1984, Immunol. Rev. 18: 75-96; Kishimoto, 1985, Ann. Rev. Immunol. 3: 133-157; Swain, 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). Thus Bp50 be a receptor for one of these factors.

With the exception of IL-2 receptors and the C3d receptor, the receptors on B cells for growth signals are still not identified. The mAb AB-1 reacts with a B- Cell marker that only expresses on activated B cells is blocked and thus BCGF-dependent proliferation recognize the BCGF receptor or a related structure can Bp50 appears different from AB-1 marker because of the AB-1 mAb does not bind the G28-5 anti-Bp50 antibody blocked and, unlike the G28-5 mAb, only with activated B cells respond (Jung, et al., 1984, J. Exp. Med. 160: 1919-1924). Bp50 is what, based on all B cells Absorption analysis and direct binding tests, with BCGF- Receptors do not appear to be the case. The current one Data show that Bp50 and the BCGF receptor are low Molecular weight have different structures. Under Wang and showed use of a rabbit heteroantiserum Coworkers (Wang, 1979, J. Exp. Med. 149: 1424-1433) yes a 54 kDa glycoprotein, gp54, which like Bp50 on all B cells are expressed, but with lower levels  Blood B cells than on tonsillar B cells. It is possible, but unlikely that the rabbit heteroantiserum and Anti-Bp50 recognize the same or related structures: in In contrast to anti-Bp50 mAb, the rabbit antiserum is too gp54 alone is able to stimulate B cell proliferation.

Anti-Bp35 alone, unlike Bp50, can activate B cells from the G ₀ to G ₁ stage and can thus be referred to as an "activation" signal. It is not yet clear whether Bp35 only works in early B cell activation or not because anti-Bp35 antibodies can stimulate some B cells to share (Clark, et al., 1985, Proc. Nat. Acad. Sci USA 82: 1766-1770). Similarly, Bp50 may not function strictly as a "growth" signal: anti-Bp50 antibodies not only increase proliferation together with activation signals (anti-Bp35 or anti-u), but also increase the total number of G ₁ reaching B cells (Table 2). In other words, Bp50 acts as a costimulant to promote the progression of both the activation (G ₀ to G ₁ ) and growth (G to S) phases of the cell cycle. The BCGF used in these studies also had similar activity ( Figure 6C). Thus, anti-Bp35 and anti-Bp50 (or BCGF) appear to be highly analogous to the "competence" and "progression" factors described in studies of growth regulation of fibroblasts. How B cells react to anti-Bp35 or anti-Bp50 clearly depends on their stage of differentiation or activation.

It is shown here that two mAb, anti-Bp35 (a "competence" - Signal) and anti-Bp50 (a "progression" signal) together a significant proliferation of highly purified B cells in the absence of antigens or other known ones Factors can induce. The natural ligands for these structures are not yet known. However, since mAb too  suitable epitopes both by soluble factors and by Imitate cell-to-cell interaction transmitted signals suitable combinations of mAb to Direction and regulation of human B cell proliferation and differentiation. Again, this will be helpful be for in vivo strategy development for control of human diseases, such as malignant ones B-cell disorders, immune deficiencies and certain autoimmune Diseases.

The new monoclonal antibody G28-5, which reacts with an approximately 50 kDa single-chain polypeptide expressed on the surface of human B cells, is a particular embodiment of the ligands of the present invention which inhibit the proliferation of activated B- Can multiply cells. Since human B cell proliferation can be similarly increased by BCGFs derived from T cells, including low and high molecular weight BCGF, the activity of anti-Bp50 G28-5 was compared to that of a BCGF preparation, which primarily contained low molecular weight BCGF. Anti-Bp50 G28-5 and BCGF (low) were very similar in their costimulatory effects with the same activators (anti-Ig, anti-Bp35 and TPA) but were not costimulatory with each other or with IL-1 or IL-2. In addition, the activity of anti-Bp50 G28-5 was not dependent on its Fc domain or region, since F (ab ′) ₂ fragments of G28-5 were functionally active. This suggests that soluble anti-Bp50, like soluble BCGF, does not require Fc receptor-bearing carrier cells to have an effect. Furthermore, both anti-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 rather promote the "progression" of B cells through the cell cycle.

While it is possible that Bp50 can serve as a receptor for a ligand, such as a B cell growth factor, various results suggest that Bp50 is at least not the receptor for the BCGF (low) used in this study: it will expressed on blood B cells, while BCGF (low) receptors are apparently not expressed. Unlike Bp50, candidate structures for the BCGF (low) receptor are also only expressed on activated B cells. In addition, normal and malignant B cell populations differ in their responsiveness to anti-Bp50 compared 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. Ultimately, in a number of experiments, optimal concentrations of anti-Bp50 and BCGF together induced greater proliferation than either alone. Anti-Bp50 mimics the activity of other BCGFs, such as BCGF (high), which are costimulatory with anti-IgM (Ambrus, et al., 1985, J. Exp. Med. 162: 1319; Ambrus, et al. , 1985, J. Clin. Invest. 75: 732). This suggests that Bp50 can function as a receptor for BCGF (high).

Although Bp50 can be a receptor for a soluble ligand Bp50 can also act as a receptor for one from cell to cell transmitted signal act, the BCGF receptor level and / or autocrine production regulated. The role model for Differentiation antigens that act as enhancers of an autocrine Receptor pathways come from studies with T cells. MAb to the Lp220 common leukocyte antigen increases the Proliferation by increasing IL-2 receptor expression on activated T cells (Ledbetter et al., 1985, J. Immunol. 135: 1819). An analogous mechanism may work with Anti-Bp50 and the expression of certain BCGF receptors. Bp50 and BCGF (low) are obviously below a certain level coordinated control, as with IL-1 and IL-  2 receptors, BCGF the expression of Bp50 on certain leukemic cells increased. The Bp50 molecules also share Similarities with the Tp44 molecule that is influencing of IL-2 production works. It was shown that the 9.3- Anti-Tp44 antibody increases the proliferation of T cells which are activated with Anti-CD3 or TPA (Ledbetter, et al., 1985, J. Immunol. 135: 2331; Hara, et al., 1985, J. Exp. Med. 161: 1513). Similarly, anti-Bp50 increases the proliferation of B cells with anti-Bp35 or TPA are activated. The Tp44 signal works by stimulation of IL-2 production rather than by stimulating the T cell growth. The Bp50 signal may be in analogous work by producing B cell autocrine is stimulated (Gordon, et al., 1984, Nature, Lond. 319: 145).

5.4.2. For immunosuppression or treatment of malignant Diseases used modified Ligands

According to this embodiment, the ligands after Invention by adding an antiproliferative agent be modified so that the molecule obtained for killing of cells expressing Bp50 antigen can. Such modified ligands can be used in the treatment of autoimmune diseases to suppress proliferation of B cells and thereby suppressing the autoimmune response be used. These modified ligands can also be used to suppress a graft patient's immune system used to reject the graft to avoid. Similarly, cytotoxic Agent used to suppress immune response are added to the ligands according to the invention. If ligands are used that increase the proliferation of  Increase B cells, an increased effect can be achieved because the drug directs to the proliferating B cells becomes.

In another embodiment, ligands according to the Invention by the addition of an antiproliferative Modified for the treatment of disease centers used where tumors or cells express the Bp50 antigen. The addition of this chemotherapy Agents for the ligands according to the invention should increase the specificity of the drug against malignancy Lead cells. In addition, a specific Benefit can be obtained when having a B cell disorder a ligand that is coupled to a cytotoxin which is a stronger killing effect than proliferating Cells as opposed to non-proliferating Has cells. Treatment with such a ligand can be too potentiate the effect of the cytotoxin.

The chemotherapeutic or antiproliferative Agents coupled to the ligands according to the invention may include, but are not limited to, Agents according to the list in Table 6 below, which by Goodman and Gilman, The Pharmacological Basis of Therapeutics, Sixth Edition, MacMillan Publishing Co., Inc, New York, pages 1249-1313, 1980, which are incorporated herein by reference is derived.  

Table 6

Chemotherapeutic agents coupled to anti-Bp50 ligands

Any known method can be used to make the ligand to the chemotherapy drug or the antiproliferative agent to couple. Examples of such methods are already given above enumerated (see section 5).

5.4.3. Other uses of ligands and Bp50

In addition to therapeutic uses, the ligands and Bp50 itself other applications in vitro as well as in vivo diagnostic investigations, separation schemes etc.  

The Bp50 receptor can be used to make and / or the structure of the ligands according to the invention. BP50 can can also be used with the ligands according to the invention in Examination methods in vitro, which require a standard to quantify the amount of Bp50 in a Sample was found. Ultimately, Bp50 itself can be used its as a soluble factor that transmits immunity, for example a lymphokine.

In addition to therapeutic treatment and diagnostic The ligands according to the invention can also be examined are used to identify or separate Cells expressing Bp50 antigen. In addition, the Ligand if a suitable radio marker or a radio opaque Link is attached to the ligand, also for in vivo imaging of tumors expressing Bp50 antigen be used. There are further possible uses himself for the expert from the previous description.

6. Deposit of cell lines

The following hybridoma was deposited with the American Type Culture Collection, Rockville, MD, and has the specified Receive identification number:

HybridomaATCC identification number: G28-5HB9110.

The present invention is not limited in scope on the deposited hybridoma, since the deposited one Embodiment is intended as a single illustration an aspect of the invention and any cell line that is functional is equivalent within the scope of the invention lies. Indeed, there are numerous modifications to the invention in addition to those shown and described obviously  to the expert from the preceding description and the attached figures or drawings. Such Modifications are intended to be within the scope of the claims fall.

Claims (67)

1. Substantially pure ligand that binds (a) to Bp50, a 50 kilodalton (kDa) B cell surface antigen, the is defined by the monoclonal antibody G28-5, and (b) those that bind to activated B cells Stimulates B cells to go through the cell cycle, so that the proliferation of B cells increases becomes. 2. Ligand according to claim 1, characterized in that it comprises an antibody molecule or an Fv, Fab, F (from ') ₂ or Fab' part of the antibody molecule. 3. Ligand according to claim 2, characterized in that the antibody molecule comprises a monoclonal antibody molecule or an Fv, Fab, F (ab ') ₂ or Fab' part of the monoclonal antibody molecule. 4. Monoclonal antibody molecule according to claim 3, characterized in that it comprises G28-5 or an Fv, Fab, F (from ') ₂ or Fab'-part thereof. 5. Monoclonal antibody molecule according to claim 3 through a hybrid cell line as deposited below ATCC and associated code no. HB9110, or a mutant, Recombinant or a genetically engineered derivative from that. 6. Ligand according to claim 1, characterized in that it includes a lymphokine. 7. Lymphokine according to claim 6, characterized in that it comprises a B cell growth factor. 8. Ligand according to claim 6, characterized in that itself the lymphokine is on the surface of a cell. 9. Ligand according to one of claims 1, 2 or 6, characterized characterized in that a connection with the ligand is coupled. 10. Ligand according to claim 9, characterized in that the an antiproliferative compound coupled to the ligand Means included. 11. Ligand according to claim 9, characterized in that the compound coupled to the ligand is an alkylating agent includes. 12. Ligand according to claim 9, characterized in that the an antimetabolite coupled to the ligand includes.   13. Ligand according to claim 9, characterized in that the an antibiotic coupled to the ligand includes. 14. Ligand according to claim 9, characterized in that the a Vinca alkaloid linked to the ligand includes. 15. Ligand according to claim 9, characterized in that the compound coupled to the ligand comprises an enzyme. 16. Ligand according to claim 9, characterized in that the a platinum-coordinated compound coupled to the ligand Complex includes. 17. Ligand according to claim 9, characterized in that the a radioisotope coupled to the ligand includes. 18. Ligand according to claim 9, characterized in that the fluorescent compound coupled to the ligand Connection includes. 19. Essentially pure ligand that bind to Bp50 capable of defining a 50 kDa B cell surface antigen by monoclonal antibody G28-5. 20. Ligand according to claim 19, characterized in that it comprises an antibody molecule or an Fv, Fab, F (ab ') ₂ or Fab' part of the antibody molecule. 21. Ligand according to claim 20, characterized in that the antibody molecule comprises a monoclonal antibody molecule or an Fv, Fab, F (ab ') ₂ or Fab' part of the monoclonal antibody molecule. 22. Ligand according to claim 19, characterized in that it includes a lymphokine. 23. Lymphokine according to claim 22, characterized in that it includes a B cell growth factor. 24. Ligand according to claim 19, 20 or 22, characterized in that that it is a compound coupled to the ligand includes. 25. Ligand according to claim 24, characterized in that the an antiproliferative compound coupled to the ligand Means included. 26. Ligand according to claim 24, characterized in that the compound coupled to the ligand is an alkylating agent includes. 27. Ligand according to claim 24, characterized in that the an antimetabolite coupled to the ligand includes. 28. Ligand according to claim 24, characterized in that the an antibiotic coupled to the ligand includes. 29. Ligand according to claim 24, characterized in that the a Vinca alkaloid linked to the ligand includes. 30. Ligand according to claim 24, characterized in that the compound coupled to the ligand comprises an enzyme. 31. Ligand according to claim 24, characterized in that the a platinum-coordinated compound coupled to the ligand Complex includes.   32. Ligand according to claim 24, characterized in that the a radioisotope coupled to the ligand includes. 33. Ligand according to claim 24, characterized in that the fluorescent compound coupled to the ligand Connection includes. 34. Essentially pure 50 kDa B cell surface antigen, which is defined by monoclonal antibody G28-5 is. 35. Substantially pure 50 kDa antigen according to claim 34, characterized in that it comprises a polypeptide. 36. method for increasing the proliferation of B cells, characterized in that it is the treatment of activated B cells with an effective dose of a ligand which is able to bind to Bp50, a 50 kDa B- Cell surface antigen defined by monoclonal Antibody G28-5, so that the activated B cells the Go through the cell cycle and proliferation increases is. 37. The method of claim 36, wherein the B cells are activated by treating an effective dose of a second ligand capable of binding to Bp35, a 35 kDa B cell surface antigen so that the B cells are off the G ₀ - advance to the G ₁ stage or phase of the cell cycle. 38. The method according to claim 36 or 37, characterized in that that it is carried out in vivo. 39. The method according to claim 36 or 37, characterized in that that it is carried out in vitro.   40. The method according to claim 36 or 37, characterized in that the ligand comprises an antibody molecule which is capable of binding to Bp50, or an Fv, Fab, F (ab ′) ₂ or Fab′-part of the antibody molecule with binding ability to Bp50. 41. The method according to claim 36 or 37, characterized in that the ligand is a monoclonal antibody molecule that is able to bind to Bp50, or an Fv, Fab, F (ab ′) ₂ or Fab′-part of a monoclonal antibody molecule with binding ability to Bp50 includes. 42. The method according to claim 41, characterized in that the monoclonal antibody molecule comprises G28-5 or an Fv, Fab, F (ab′2) ₂ or Fab′-part thereof. 43. The method according to claim 41, characterized in that the monoclonal antibody through a hybrid cell line, as stored under ATCC and the associated identification number. HB9110, or a mutant, recombinant or genetic manipulated derivatives of which is produced. 44. The method according to claim 36 or 37, characterized in that that the ligand comprises a lymphokine. 45. The method according to claim 44, characterized in that the lymphokine comprises a B cell growth factor. 46. The method according to claim 44, characterized in that the lymphokine is present on the surface of a cell. 47. The method according to claim 37, characterized in that the second ligand comprises an antibody molecule attached to Bp35 can bind.   48. The method of claim 47, characterized in that the antibodies binding to Bp35 a monoclonal antibody includes. 49. The method according to claim 37, characterized in that the activated B cells within 12 hours activation by the second ligand binding to Bp35 are treated with the ligand binding to Bp50. 50. method of suppressing cell proliferation, expressing Bp50, a 50 kDa B cell surface antigen, by the monoclonal antibody G28-5 is defined, characterized in that the Bp50 express Cells with an effective dose of one Bp50 binding ligand treated with a antiproliferative agent is coupled. 51. The method according to claim 50, characterized in that the cells expressing Bp50 include B cells. 52. The method according to claim 50, characterized in that the cells expressing Bp50 include malignant cells. 53. The method according to any one of claims 50 to 52, characterized characterized in that it is carried out in vivo. 54. The method according to any one of claims 50 to 52, characterized characterized that it is carried out in vitro. 55. The method according to any one of claims 50 to 54, characterized in that the ligand comprises an antibody molecule or an Fv, Fab, F (ab ′) ₂ or Fab ′ part of the antibody molecule. 56. The method according to claim 55, characterized in that the antibody molecule comprises a monoclonal antibody molecule or an Fv, Fab, F (ab ′) ₂ or Fab ′ part of the monoclonal antibody molecule. 57. The method according to claim 56, characterized in that the monoclonal antibody molecule comprises G28-5 or an Fv, Fab, F (ab ′) ₂ or Fab′-part thereof. 58. The method according to claim 56, characterized in that the monoclonal antibody through hybrid cell lines like deposited under ATCC with the identification no. HB9110 or one Mutant, recombinant or genetically engineered derivatives of which is produced. 59. The method according to claim 50, characterized in that the ligand comprises a lymphokine. 60. The method according to claim 59, characterized in that the lymphokine comprises a B cell growth factor. 61. The method according to claim 50, 55 or 59, characterized in that that the antiproliferative agent is an alkylating agent includes. 62. The method according to claim 50, 55 or 59, characterized in that that the antiproliferative agent is an antimetabolite includes. 63. The method according to claim 50, 55 or 59, characterized in that that the antiproliferative agent is an antibiotic includes. 64. The method according to claim 50, 55 or 59, characterized in that that the antiproliferative agent is a vinca alkaloid includes.   65. The method according to claim 50, 55 or 59, characterized in that that the antiproliferative agent is an enzyme includes. 66. The method according to claim 50, 55 or 59, characterized in that that the antiproliferative agent is a platinum coordinated complex. 67. The method according to claim 50, 55 or 59, characterized in that that the antiproliferative agent is a radioisotope includes.