DE3719398C2 - Ligands and their use - Google Patents

Description

The present invention is directed to ligands such as Antibody molecules or fragments of antibody molecules, or other ligands, such as lymphokines, which bind to a 50 kDa Bind B cell surface marker, here referred to as Bp50, in B-cell proliferation, but not in the early B-cell activation acts.

In a particular embodiment of the present invention A monoclonal antibody, G28-5, is described which defines Bp50 and has a role in proliferation of activated B cells seems to play, but none noticeable effect on the proliferation of resting B-cells exercises.  

The ligands of the present invention, such as antibodies per, lymphokines and fragments thereof may be used to the proliferation and / or differentiation of to direct and regulate human B-cells. Zusätz Lich, the ligands according to the invention by addition be modified by other compounds which Be treatment and / or detection of Bp50 antigen expressing malignant cells can be used.

Activation of resting B cells from G₀ to G₁ Phase of the cell cycle and the subsequent induction of Activated B cells are proliferating, distinct Stu fen, which require a distinct regulatory mechanism. Some agents, including murine (mice or rats) -B- Cell stimulating factor-p1 (BSF-p1) (Rabin, et al., 1985, Proc. Nat. Sci. USA 82, 2935-2939) or low doses of anti-immunoglobulin (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" factor that is, they induce B cells to enlarge, synthesize more RNA and enter the G₁ phase, alone, however, they induce DNA synthesis in B cells Not. Other "growth" factors, such as growth factor for human B-cells (BCGF) and interleukin-2 (IL-2) allow activated B cells to cycle through the cell cycle and to enter the S phase, however, resting B cells can do not cause them to start (Kehrl, et al., 1984, J. Im munol. 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 has been described by persons who are both Mu rin systems (of mice or rats) as well as human Have studied systems. These factors include B-cell Growth Factors (BCGF), of several different Sources, including T cell lines or hybridomas, B cells Cell lines or dendritic cells can be derived. Although both interleukin-1 (IL-1) and interleukin-2 (IL-2) has been found to increase B-cell growth, they are different from other specific BCGFs. For example, monoclonal antibodies (mAb) block for a murine BCGF (O'Hara, et al., 1985, Nature (London). 315: 333) or human BCGF (Ambrus, et al., 1985, J. Exp. Med. 162: 1319) BCGF activity but not IL-1 or IL-2 activity. Although distinct from IL-1 or IL-2, The BCGFs themselves appear to be heterogeneous, based on biochemical data and differential activity on ver different B-cell subunits or costimulating pro ben. For example, a 60 kilodalton (kDa), a high Molecular weight-bearing, human BCGF, BCGF (high), as different from a 12 kDa, a low one Molecular weight, BCGF (low) identified (Ambrus, et al., 1985, J. Clin. Invest. 75: 732). The cDNA, that encodes a 20 kDa murine BCGF, tentatively B cells called stimulating factor p1 (BSF-p1), has recently been reported cloned and sequenced (Noma, et al., 1986, Nature 319: 640). The recombinant lymphokine has not only BCGF activity, but can also activate resting B cells ren and the differentiation of IgG₁-producing cells induce. Thus, it differs from human BCGF (high) and BCGF (low) in both its molecular weight as well as in his activity area.

These activation and growth signals are supposed to regulate cells through interaction with specific B cell Surface structures. In addition to the antigen specifi  signal through surfaces Ig were various others Identified candidate B-cell surface peptides which in some way has a function on the activation or the growth of B-cells. Beispielswei The surface receptors for 1L-1 (Dower, et al. 1985, J. Exp. Med. 160: 501) and IL-2 (Robb, et al., 1984, J. Exp. Med. 160: 1126), and have recently been functional IL-2 receptors on B cells 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 However, activation factors still need to be completely charac terisiert. Some candidates of B cell surface chen polypeptides, which in some way has a function in activation or growth of B cells the identified. For example, subbarao and Mosier (Subbarao, et al., 1983, Immunol., Rev. 69: 81-97), that monoclonal antibodies (mAb) to the murine B-cell antigen Lyb2 B cells have been activated, and have recently become evidence due to which Lyb2 will be the receptor for BSF-p1 can (Yakura, 1985, Fed. Proc. 44: 1532). In a similar way It was found that the corresponding mAb (1F5) to a 35 kDa polypeptide, Bp35, human B cells from G₀ to G₁ acti fourth (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 to the 140 kDa C3d Receptor, Bp140, cause proliferation of B cells, 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). whether probably identifies BCGFs in both mice and humans The receptors for these factors were identified not yet isolated. Wang and co-workers (Wang, et al., 1979, J. Exp. Med. 149: 1424-1433) made a polyclonal Antiserum expressing a 54 kDa polypeptide (gps4) on human B cell len and showed that the rabbit antiserum  to gp54 Tonsillar B cells induced to divide. Recently Jung and Fu isolated (Jung, et al., 1984, J. Exp. Med. 160: 1919-1924) a mAb (AB-1) to a 55 kDa antigen, be borders on activated B-cells, which are the BCGF-dependent Proliferation blocked. However, whether anti-gp54 or AB-1 one Detecting BCGF receptor or not is not yet known.

The invention provides the ligands according to claim 1 available supply. It is a substantially pure ligand, the Bp50 binds a 50 kilodalton B cell surface antigen by the monoclonal antibody G28-5 defined by a hybrid cell line as deposited under ATCC and pertaining thereto ger identification number HB9110 is manufactured. Preferred ligands are described in the dependent claims 2 to 16. Ver Uses of the ligands of the invention are in Ansprü 17 to 21 and 23 to 24, respectively. The wording of all By making reference to the content of Be spelled out.

The present invention accordingly comprises on the one hand Ligands linked to Bp50, a 50 kDa B cell specific Surface polypeptide described below and, (b) the proliferation of activated B cells ver multiply. On the other hand, the invention includes ligands which Bp50 bind, but no biological effect or function, like the proliferation of activated B-cell proliferation len, show.

The ligands of the present invention include anti body molecules, monoclonal antibody molecules and fragments of these antibody molecules that are linked to the antigen containing combining site, or chemically modified Antibodies and fragments. Such fragments include but not limited to Fv, Fab, F (ab ') ₂, Fab' and the  same. The ligands of the invention further include Lymphokines, which are human B-cell growth factors as well chemically modified lymphokines, however not limited thereto. The ligands according to the present The present invention may be chemically modified, for Example by linking or coupling a connection the ligand. Such compounds include, without it to be restricted, cytotoxic agents, therapeutic agents  tel, chemotherapeutic agents, markers such as radiolabels, Enzymes, radioopaque compounds and the like. The Ligan The according to the present invention can be modifi in their modifi ornamented or unmodified form used to Human B-cell proliferation and / or differentiation too direct, regulate and modify.

The present invention is based on the recognition that two human B cell differentiation antigens, Bp35 and the B-cell antigen described here, Bp50, apparently a di Stuctive role as signal receptors in the B-cell Akti play. Monoclonal antibodies (mAb) to Bp35 and Bp50 both provide positive signals to B cells, the latter of which Continue to stimulate the cell cycle. mAb to Bp35 acts similar to anti-Ig antibodies in principle activating on ru B cells to enable them to enter the G₁- Phase of the cell cycle. In contrast, it works a monoclonal antibody described herein, or its F (ab ') ₂ fragment to Bp50, a 50 kDa polypeptide is expressed by all B cells, in the sense of a stimulus activation of activated B cells to cycle through the cell cycle and proliferates the proliferation of activated B cells. Monoclonal antibodies to Bp35 activate like anti-Ig-anti body tonsiliary B cells and induce low levels B-cell proliferation. The anti-Bp50 monoclonal antibody By itself, on the other hand, neither activating acts on B cells still inducing B cell proliferation len; together with anti-Bp35 or anti-Ig antibodies ver however, it does increase B-cell proliferation. In this way The effect of anti-Bp50 antibodies of the Akti is similar of B-cell growth factor (BCGF). It will be single small amounts such as 0.05 μg / ml of anti-Bp50 are needed to treat the Enhance proliferation, and anti-Bp50 is like BCGF effective even if it is 12 to 24 hours after the activity addition of B cells with anti-Ig or anti-Bp35. Without additional exogenous signals induce anti-Bp35 and  Anti-Bp50 antibodies share a strong proliferation of purified resting B cells. This result lets on it conclude that the Bp35 and Bp50 surface molecules on the regulatory control of B cell activation and pro act through the cell cycle. Because of the significance of anti-Bp35 and similar molecules on the effect and the Effect of the ligands according to the invention is based on Clark et al., 1985, Proc. Natl. Acad. Sci. (USA) 82: 1766-1770 verwie sen, and reference.

Although the activity of anti-Bp50 is that of BCGF (low) is similar because both anti-Bp50 and BCGF (low) kostimu ling by 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 differentiated from the activity of BCGF (low), since the proliferation of B cells treated with optimal amounts of anti-Bp50 and anti-Bp50 Bp35 (or anti-Ig) are further enhanced May be with BCGF (low) and both blood B cells as well certain B-cell lymphomas differ on anti-Bp50 Reply to BCGF. For optimal activity, anti- Bp50 within 12 hours after B-cell activation whereas BCGF (low) gives its optimal ac even if it is 24 hours after Activation is added. In addition, Bp50 will be out of all B cells express, whereas receptors or BCGF (low) are limited to activated B cells. Thus, anti- Bp50 and BCGF (low) coordinate B cell growth However, they obviously do so by difference light signals.

In one embodiment of the invention, the ligands, which bind to Bp50 and activate the proliferation of Multiply B cells, used to increase immune response become. For example, these ligands, which are Bp50  Bind, used as a "resource" to boost an immune response to a vaccine. On the other hand, the se ligands to increase the immune response of an immune un oppressed individual.

In another embodiment, the ligands may be after the Chemically modified so that the cells to which bind the ligands can be killed. Because all B cells express the Bp50 antigen, this approach would become a Suppression of immune response. A cytotoxic Drug linked to a ligand according to the invention can be used in vivo, for example, in vivo Immunosuppression for refraction of histocompatibility bar cause grafting in transplant patients. other On the other hand, these modified ligands can also be used for the control used by autoimmune diseases.

According to another embodiment of the invention can be evil like diseases, such as tumor cells expressing Bp50, treated using ligands of the invention linked to a chemotherapeutic agent are for the treatment of such neoplastic Erkran useful. These modified ligands can be found in vivo to be used to the chemotherapeutic agent to direct a type of malignant cells, which causes the Bp50 antigen expressed, including those cells are not B cells, but express Bp50. Using of ligands according to the invention containing the B cell Proli multiply feration, a particular advantage can be derived from it that in the treatment of malignant B cells Diseases using one attached to the ligand such a chemotherapeutic agent is used, that is especially effective for the killing of proliferating Cells is. In this way, a potentiation of the Arz can be obtained.  

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

The purified Bp50 antigen according to the invention can be converted to Bil antibodies and to form or design others Ligands are used according to the invention. The Bp50 anti gene can continue to be used for samples and tests such as diagnostic immunoassays. In addition, can Bp50 itself as a cell immune mediator in vivo or in vivo used in vitro.

definitions

The following abbreviations used here have the bene meaning:

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-area polypeptide defined by mAb G28-5
Fv = the variable region or antigen combining site of an antibody molecule. This may be any, the idiotype of the molecule of the fragment, 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 = kilodaltons
mAb = monoclonal antibody
SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis
TPA = 12-O-tetradecanoylphorbol-13-acetate.

Figure 1. Expression of Bp50 is limited to Bp35 ± B cells. A two-color cytometric flow analysis of 50,000 cells was performed as described (Clark, et al., 1985, Proc Nataz Asad Sci., USA 82: 1766-1770). The data are plotted as cell number against the logarithm of green fluorescence and the logarithm of red fluorescence, with 4-5 points approximating a duplication of fluorescence. The data are given to show autofluorescent negative cells. PE (red) anti-3p35 (1F5) against FITC (green) anti-Bp50 (G28-5) staining shows that all Bp50 + cells are also Bp35 +.

Figure 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 by autoradiography and intensifying screens.

Panel A: lane 1, anti-Bp50 (G28-5); Lane 2, anti-Bp95 (G28-8); Lane 3, only sepharose goat anti-mouse Ig. Einwir 4 days.  

Panel 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, only Sepharose goat anti-mouse Ig. An exposure time of 2 days was selected so that the bands in lanes 2 to 4 were not overdeveloped and could be clearly distinguished relative to Bp50. (One of three attempts).

Fig. 3. Two-color immunofluorescence analysis of Bp50 expression. Peripheral blood or tonsillar 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 per, including 2C3 (anti-Bp50). 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 logarithm amplifiers in both red and green dimensions. Forward and rectangular light scattering was used to demarcate monocytes. Uncolored cells were positioned at the back of the grid or plate. Red fluorescence is right and green fluorescence is left.

Fig. 4. Dose response curves for the proliferation amplification of dense tonsillar Er-B cells by anti-Bp50 antibody 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 quadruple samples was measured on day 3.

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

Figure 6. Comparison of the ability of anti-Bp35 and anti-Bp50 to induce quiescent Tonsillar B lines to exit the G₀-stage of the cell cycle. Day 3 post-treatment medicine 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 number against the logarithm of AO red fluorescence (RNA).

Figure 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 indicated days by an 18 hour pulse of 3 H-thymidine. Proliferation was measured four-fold and standard deviations are shown. (One of three experiments).

Figure 8. Times after anti-Bp35 stimulation in which anti-Bp50 (A) or BCGF (B) optimally enhance 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 indicated time points; Anti-Bp50 or BCGF alone; Anti-Bp35, added at the onset of culture, followed by the addition of anti-Bp50 or BCGF at the indicated time points; Anti-Bp50 or BCGF added at the beginning of culture followed by anti-Bp35. (One of two experiments).

Proliferation was measured four times and standard deviation tions 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 additive effect on B-cell proliferation. Dense tonsillar EB cells were stimulated with graded doses of BCGF (low) together 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 10⁶ cells: anti-Bp35, 5 μg / ml; Anti-Bp50, 0.2 μg / ml; Anti-Ig beads, 50 μg / ml.

Figure 10. Comparative 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 of3 H-thymidine during a 12 hour pulse. Proliferation was measured fourfold and standard deviations are shown.

As already mentioned, the present invention comprises ligand those which bind to Bp50, a 50 kDa B-cell specific surface polypeptide, and activate the proliferation of multiply B cells. Furthermore, the invention includes Ligands that bind to Bp50, but no biological Effect or function, such as the proliferation of proliferation of activated B cells.

The ligands of the present invention include anti body molecules, monoclonal antibody molecules and fragments of these antibody molecules that combine with the antigen contain binding to the Bp50 receptor, including chemically modified antibody and Frag mente. Such fragments include but are not be limits to Fv, Fab, F (ab ') ₂, Fab' and the like. more The ligands according to the present invention include Lymphokines that bind to the Bp50 receptor. these can the BCGFs as well as chemically modified lymphokines and the like include, but are not limited to. The ligands According to the invention, in their modified or unmodi fied form can be used for modulation and regulation immune response and for the treatment of malignant disease foci expressing the Bp50 antigen. This use training opportunities are described in more detail later.

Is the ligand a monoclonal antibody or a fragment of it, then the monoclonal antibody against Bp50 can be made using any technique used to Production of antibody molecules by continuous Cell lines in culture is used. For example, in the frame The invention applies the hybridoma technique, such as  originally by Kohler and Milstein (1975, Nature 256: 495-497), as well as other techniques, which became available only recently, like the Human B cell hybridoma technique (Kozbor et al., 1983, Imm noly Today 4:72) and the EBV hybridoma technique for the manufacture 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. at games for such fragments include without being be to be limited: the F (ab ') ₂ fragment obtained by treatment the antibody molecule can be formed with pepsin; the Fab 'fragments formed by reduction of the disulfide bridges of the F (ab ') ₂ fragment can be formed; the F (ab ') ₂-Frag ment, by treating the antibody molecule with papain can be formed; and the 2Fab or Fab fragments, the by treating the antibody molecule with papain and Re Duktionsmitteln for reducing the disulfide bridges gebil can be.

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

Depending on the intended use of the Ligand or suitable fragments of the ligand chemically modi be fied by connecting a compound from a Variety of available connections to 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-pyridyldi thio) propionate (SPDP), Schiff's base reactions, Anknüp sulfhydryl functions, the use of sodium isothiocyanate or enzymatic bonds, to name but a few call. Where a radioisotope is to be added to the ligand, This can also be done via enzymatic pathways, oxidative sub institution, chelation and the like. An overview of the chemical reagents used for the Protein modification can be used is published light by Lundblad and Noyes, Chemical Reagents for Protein Modification, Volume II, CRC Press, Inc., Boca Raton, Flori there, Ch. 5, pp. 123-139, 1984.

The chemical linkage or coupling of a compound the ligand can be directed to a site on the ligand that does not participate in binding to Bp50. This can be achieved by protecting the binding site on the Ligands before performing the coupling reaction. at For example, the ligand can first be attached to Bp50, to protect the Bp50 binding site, after which the Kupp lungsreaktion to link the desired compound available reactive sites on the ligand Bp50 complex can be carried out. Is the coupling reaction complete then the complex can be taken apart again to create a modified ligand the desired compound is linked, whereby the Bp50 binding site of the molecule minimally affected becomes. Where the ligand comprises a monoclonal antibody, like G28-5, where the Fc domain or region of the molecule not required for the ligand to make its effect (see Section 5.3.3), it may be advantageous to use the Coupling of the desired compounds to the Fc region of the To direct molecule.  

The following sections describe the 50 kDa B cell surface marker, Bp50, which is evident has an effect on B cell proliferation, as well Ligands that bind to the new 50 kDa receptor, as well their uses. As an example of the ligands of present invention, a monoclonal antibody, which defines Bp50 and its F (ab ') ₂ fragments if described, similar to BCGF, the prolifera tion of B cells increased. Unlike anti-Bp35 mAb, which resting B-cells of G₀-phase to enter the G₁ phase can induce activated anti-Bp50 mAb quiescent B cells do not. Anti-Bp35 and anti-Bp50 mAbs induce I mean a strong activation and proliferation of gerei B cells without additional exogenous signals.

The experiments described below show that anti- Bp50 activity is similar to BCGF activity, but that of anti-Bp50 is different from a BCGF, since anti-Bp50 and BCGF with low molecular weight are clearly additive and different on different B-cell subgroups or diseases Act. Bp50 can be a receptor for a distinct BCGF or be for a transmembrane signal that affects BCGF production or modulates BCGF receptor expression.

Used methods for characterization of the Bp50 receptor

Cell preparations. Mononuclear cells were normal or leukemic heparinized peripheral blood by Fi coll-hypaque gradient (Pharmacia, Piscataway, NJ) iso profiled. Mononuclear cells were obtained from tonsillar Tissue as described (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1756-1770). T cells were removed by Rosetting using AET-treated sheep erythrocytes and Ficoll-Hypaque gradient separation. at In some experiments, blood-3 cells were enriched by  Isolation of nylon wool adherent cells. monocytes were removed by incubation on plastic Petri dish len, once or twice at 37 ° C for 45 minutes, when 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. Blood B cell-enriched preparations had 60-85% sIg + cells. B-cell lymphoma cells were isolated by careful separation of lymphoma cells in Medium, followed by Ficoll-Hypaque gradient centrifugation tion.

Monoclonal antibodies. The G28-5 antibody became Bp50 produced by immunizing BALB / c mice with human E Tonsillarlymphozyten and fusion of immune spleen cells with NS-1 myeloma (Kohler, et al., 1975, Nature 256: 495-497; Ledbetter, et al., 1979, Immunol. Rev. 47: 63-82). Hy Broth cell cultures that secrete an antibody, the is reactive with tonsillar B cells and not with T cells, were identified by the use of indirect Immunofluorescence (IF) and analysis with a FACS IV cell sorter; Cultures with antibody with a histogram pattern, similar to the known mAb to Pan B cell markers (eg. Bp35), were cloned and for further study selected. The G28-5 clone produced an IgG₁ mAb that only with normal or malignant B-cells or B-cell lines rea lusted. Other mAbs used in this study are (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 (IgG₂a) anti-Bp35, HB10a (IgG₂a), anti- HLA-DR, 2C3 (IgG₁) anti-u-chain, G19-4 (IgG₁) anti-CD3, FC-2  (IgG₂a) anti-Fc receptor CD16, and 9.6 (IgG₂a) anti-CD2 (E Receptor), by Dr. med. Paul Martin (Martin, et al., 1983, J. Immunol. 131: 180). The IgG₁ mAbs were passed through Precipitation purified using 45% or 50% saturated ammonium sulfate and DEAE-Sephacryl column chromatography tography, and the IgG₂a mAbs were purified by the Use of protein A Sepharose columns. The F (ab ') ₂-Frag Elements of G28-5 were prepared by the method of Parham (Parham, et al., 1983, J. Immunol. 131: 2895), on a 2 m long Sephacryl S200 column and purified to purity examined by SDS-PAGE (Ledbetter, et al., 1985, J. Immunol. 135: 1819). The 2C3 mAb to u chains were ligated to Sepharose 4B Beads conjugated (Pharmacia Fine Chemicals, Uppsala, Schwe den), using the cyanogen bromide coupling.

Fluorescein and phycoerythrin conjugations. purified mAb was either directly conjugated with fluorescein below Use of fluorescein isothiocyanate (FITC; Molecular Probes) (green) using the Goding method (Goding, et al., 1976, J. Immunol. Meth. 13: 215-226), or conjugated R-phycoerythrin (PE) (red) using SPSP (Phar macia), with a method described in detail by Letbetter, et al., 1985, in Perspectives in Immunogenetics and Histocompa tibility, ASHI, New York, 6, 119-129. Lym Phoid cells were grown in round bottom microtiter plates for 30 minutes with a suitable dilution of green and / or red mAb incubated, washed twice and then on a FACS IV cell sorter.

Two-color immunofluorescence. Two-color studies were performed with a fluorescence activated cell sorter (FACS IV: Becton-Dickinson, Mountain View, CA) under use a 560-nm dichroic mirror for splitting the Beams and a 580 long-pass filter and a 540 shortcut Pass filters (Ditric Optics, Hudson, MA) in front of the red or green Fotomultiplierrohre. In addition, a two  color compensator (T. Nozaki, Stanford University) det, for a slight overflow of green and red signa len correct. For each two-color staining were Data collected from 40,000 cells and stored on floppy disks stores. The data are given as cell number (vertical) against the logarithm of green fluorescence against the logarithm mus red fluorescence on a 64 × 64 dot grid. Annæ 4.5 points mean a doubling of the fluorescence. Uncolored cells were at the back corner of the grid positioned; red fluorescence is right and green fluorescence Zenz is on the left. The flow cytometry system for two-color IF with fluorescein and phycoerthrin is more detailed (Ledbetter, et al., 1985, in Perspectives in Im munogenetics and histocompatibility, ASHI, New York, 6, 119-129 and 325-340).

Cell culture. Blood or tonsillar lymphoid cells were cultured at 5 - 10 × 10⁵ cells / ml fourfold in 96-well Microtiter plates containing 200 μl RPM1-1640 medium, that with 15% fetal bovine serum, antibiotics, glutamine and Pyruvate (R15) was supplemented. After 1 to 7 days, the Cells 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 placed on glass fiber filters with a cell harvested, and the radioactivity was in one Scintillation counter measured. In some experiments w the antibody or factors at different times added to the start of the cultures. Proliferation at the Experiments were measured on day 3.

Costimulating factors. Purified BCGF was obtained from Zyto kine technology (Buffalo, New York) and contained no detectable IL-1, IL-2 or interferon activity. This BCGF was produced according to the method of Maizel and coworkers (Maizel, et al., 1982, Proc. Nat. Acad. Sci. USA 79: 5998) who have shown that the major BCGF acti  is based on a 12 kDa species in this material, hereafter referred to as "BCGF (low)" (Mehta, et al., 1985, J. Immunol, 135: 3298). The cleaning stages completed preparative DEAE affinity chromatography, followed by hydroxylapatite column chromatography. IL-1 in purified to homogeneity form became generous by Dr. med. Steven Dower provided (Dower, et al., 1985, J. Exp. Med. 162: 501). Recombinant IL-2 wur This is available from Cetus Corporation posed. TPA (12-O-tetradeconoyl-phorbol-13-acetate) was sourced from Sigma.

Detection of cell activation. Changes in cell volume, which were induced by mAbs and / or factors measured using a cell sorter and a win supplied to kel light scatterers. Cell cycle changes in cellu Linear RNA and DNA levels were measured by staining Activated cells with acridine orange and measuring the relati of cellular RNA (red) and DNA (green) content with a Cell sorter according to the method of Darzynkiewicz et al. (Dar zynkiewicz, 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 by direct IF fluorescence levels with an Epics V cell sorter quantified.

Biochemical characterization of Bp50. immunoprecipitation of Bp50 from surface ¹²⁵I-labeled tonsillar cells was performed as described (Ledbetter, et al., 1985 J. Immunol. 134: 4250-4254). Isolated antigens were elek trophoresed on 10% SDS polyacrylamide gel plates without Reduction. The gels were visualized using Autoradiography at -70 ° C and Cronex illumination plus In tensifying screens or foils (Dupont).  

Characterization of the Bp50 receptor

The following sections describe the results of the Experiments using the methods described above Methods were performed.

Identification of a B-cell specific 50 kDa cell surface marker, B-50

A mAb to Bp50 was caused by immunization tion of BALB / c mice with human tonsillar lymphocytes and Fusion of immune spleen cells with NS-1 myeloma. On Clone, G28-5, produced an IgG₁ mAb that was the NS-1-light Chain not included. After examination by IF analysis, it turned out that G28-5 only with normal or ma B cells or B cell lines react. A comprehensive one Screening of normal tissues according to accepted methods (Clark, et al., 1985, Proc. Nat. Acad Sci. USA 82: 1766-1770; Ledbetter et al., 1986, Human Immunol. 15: 30-44; Led better, 1985, in Perspectives in Immunogenetics and Histo comoatibility, ASHI, Mew York, 6, pp. 325-340), that the G28-5 antibody with E rosettes negative (er) cell Blood or tonsils do not react to Ny non-adherent T cells, PHA-induced T cell blasts or with blood granulocytes, monocytes and red cells len or platelets. It reacted strongly with all seven tested B-lymphoplastoid cell lines and with three Bur putty lymphoma (Raji, Daudi, Namalwa), but not with four T-cell lines (CEM, HSB-2, JURKAT and HPB-ALL). All tested chronic lymphocytic leukemia (3/3) and 90% (9/10) of the tested B lymphomas expressed the Bp50 Markers, whereas only 28% (2/7) of non-T, not B-CALLA⁺ acute lymphocytic leukemia Bp50.  

The limited distribution of Bp50 to normal tissues 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 was restricted to Bp35 + B cells ( Figure 1) are 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 at similar levels on tonsillar B-cell subpopulations separated on Percoll gradients into floating and dense fractions. 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 do not express Bp50.

The G28-5 antibody reacted with a single polypeptide on tonsillar lymphocytes migrating at approximately 50 kDa under non-reducing conditions ( Figure 2A). This molecule is larger than previously reported B cell markers of the same molecular weight range as 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, Editor: Reinherz, et al., Springer-Verlag, Berlin, Chapter 12, Vol 2, 155-167; Slovin, et al., 1982, Proc. Nat Acad Sci., USA 79: 2649-2653, Thor ley-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 be easily 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) made available by an international working group (Clark, et al., 1986, in Leukocyte Typing II, eds. Reinherz et al., Springer Verlag, Berlin, Chapter 12, Vol. 2, 155-167) do not block the binding of fluoresceinated anti-Bp50 antibodies to B cells. Based on tissue distribution, biochemical analysis and blocking studies, the G28-5 monoclonal antibody thus recognizes a 50 kDa structure that is different from other known B cell antigens.

Expression of Bp50 is limited to B cells

Distribution studies on hematopoietic tissues and cell lines as well as detailed bi-color flow cytometric analyzes indicate 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. Nearly all Bp50 + cells in both the blood and the tonsils also expressed Bp35 and HLA-DR, but did not express the CD2 ( Fig. 1) or CD3, T-cell or IgG Fc receptors found on MK Cells are found. In addition, ConA-activated CD3 + T cell-blasts expressed I1-2 receptors, but not Bp50.

Two-color flow cytometric analysis allows the quantitative measurement of the density ratio between the surface antigens. The dense, resting B cells in the sheath zone of secondary follicles were previously shown to 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, indicating that Bp50 is expressed in vivo on both resting B cells and activated B cells ,

Propagation of B-cell proliferation with anti-Bp50 antibody

As already explained, B cells with low doses of Anti-u-chain specific antibodies are activated. It was recently found to be the B cell-specific marker Bp35 (B1), a 35 kDa polypeptide, also in the early B cell activation works: the 1F5 mAb to Bp35 activates B- Cells, such as low doses of anti-U antibodies, to Er increase in cell volume and RNA content and the emergence of Responsiveness to BCGF (Clark et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770; Gollay, et al., 1985, J. Immunol. 135: 3795-3801). Therefore it was of interest, the Effect of anti-Bp50 mAb on the proliferation of unbehan defective B cells or either with anti-Bp35 or anti-u-an antibodies (Table 1) to compare activated B cells. Anti-Bp35 in solution or anti-u antibody, attached to Sepharose beads, could be suitable for themselves under suitable Conditions stimulate some B-cell proliferation (Table 1, line 1). In contrast, anti- Bp50 antibody alone does not increase proliferation (Table 1, Line 2). However, anti-Bp50 mAb increases proliferation considerably when cultivating with anti-u beads or with anti-Bp35 is fourth. In this regard, anti-Bp50 is similar to BCGF (Ta belle 1, line 3). It was important to determine if anti-Bp50 and BCGF together may induce B-cell proliferation As explained in Table 1, line 4, anti- Bp5a and BCGF together did not proliferate, ever increasing  but the proliferation of either with anti-u or -Bp35 activated cells just over one of the two alone stimulated. BCGF has, if it is with anti-Bp50 without other sig nale, no effect over a 3-log area on the proliferation of dense B-cells, even if An ti-Bp50 at doses ranging from 0.1 to 10 μg / ml becomes.

Table 1

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

Anti-Bp50 mAb only proliferates proliferation after the B cells are incubated with anti-Bp35 or anti-U Antibodies are activated

The results in Table 1 indicate that anti-Bp50 mAb can not induce proliferation by itself. As shown in Figure 4, doses of anti-Bp50 in the range of 0.05 μg to 2.0 μg / ml have no effect on ³H-thymidine uptake. However, in the presence of optimal levels of anti-Bp35 mAb, as small as 0.1 to 0.5 μg / ml of anti-Bp50 antibodies significantly increased proliferation. There are even 50,000 to 70,000 cpm detectable at the optimal proliferation time when highly purified B cells are cultured only with anti-Bp35 plus anti-Bp50. Similar 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 act upon the activation of B cells by other signals. The data in Fig. 5 show that this is indeed the case. When B cells are first activated with anti-Bp35, anti-Bp50 can still be added at as late as 24-48 hours later and still proliferate on day 4. If, conversely, the cells are first treated with anti-Bp50 anti-Bp35 was effective only when added within a few hours of 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, starting from G₀, but induces activated B cells to pass through run the cell cycle

Anti-Bp35, such as anti-U antibodies, has been found to induce dormant G-phase Tonsillar B cells to increase (Clark, et al., 1986, Leukocyte Typing II, Editor: Reinherz, et al., Springer Verlag, Berlin, Vol 2, 455-462) and to enter the G₁ phase of the cell cycle (Gollay, et al., 1985, J. Immunol., 135: 3795-3801). Thus, it was of interest to compare the ability of anti-Bp50 mAb to anti-Bp35 mAb for its efficacy on B cell activation. As shown in Fig. 6A, unstimulated dense tonsillar B cells had a uniform RNA profile characteristic of cells in G₀ even after 3 to 4 days in culture (Darzynkiewicz, 1980, Proc Nat Natl Acad Sci USA 77 : 6697-6702). However, about 15-30% of anti-Bp35 or anti-u stimulated cells had an increased RNA content, which is an indication of G₁-phase entry. In contrast, neither anti-Bp50 ( Figure 6B) nor NCGF ( Figure 6C) alone induced significant numbers of B cells to enter G₁. For example, anti-Bp35 and anti-Ig mAb, respectively, induced 13.5% and 20.9% of tonsillar cells to enter G₁ two days after activation, whereas cells treated with anti-Bp50 alone (2.7%) ) or BCGF (3.2%) remained at the media control level (2.2%). However, when anti-Bp50 or BCGF were added along with anti-Bp35 or anti-u antibodies, the proportion of cells in phase G₁ dramatically increased. Similarly, anti-Bp50 and BCGF alone did not induce B cells to enter S phase (Table 2), however, along with either anti-Bp35 or anti-u, the number of S-phase cells increased to the second. up to triple.

Table 2

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

Optimum conditions for propagation of the B-cell len proliferation with anti-Bp50 antibodies

Antibodies to Bp50 by themselves have little or no no noticeable effect on dense, round B cells (Tabel le 3). However, in the presence of agents that activate B cells can increase, such as anti-Ig, anti-Bp35 and TPA increased Anti-Bp50 mAb proliferation clearly. Anti-Bp50 has no costimulatory effect with different interleuki including purified IL-1, recombinant IL-2 and  BCGF (low). A comparison of the effects of anti-Bp50 with those of BCGF (low) showed that the same means, the costimulating with anti-Bp50 were as great stimu ling with BCGF (low) (Table 3). From Of particular interest was finding that BCGF and anti-Bp50 are not costimulating for dormant cells.

Table 3

Proliferation of B-cell proliferation with anti-Bp50 antibodies or B-cell growth factor

The kinetics of anti-Bp50 enhanced proliferation is shown in FIG . 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 kinetic processes of proliferation when propagated by BCGF or by anti-Bp50 were similar.

Even small amounts such as 0.05 μg of anti-Bp50 antibody ver increased proliferation. An optimal dose of 0.3 μg / ml was used in subsequent studies. A Observation was made that when using whole anti body 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 ug / ml.

Human B cells are extremely sensitive to inhibitor effects caused by Fc receptors of antibodies which bind to surfaces Ig (Parker, 1980, Immunol. Rev. 52: 115; Bÿsterbosch, et. al., 1985, J. Exp. Med. 162: 1825). So it was important to check the effectiveness of the ge total anti-Bp50 mAb compared with that of anti-Bp50 F (ab ') ₂ fragments. Over a 100-fold dose range were F (ab ') ₂ fragments clearly as effective as or still more effective than the whole antibody in multiplying the B- Cell proliferation (Table 4). So the Fc domain or Region of anti-Bp50 mAb not needed at anti-Bp50 order to exert its effect and, if at all effective, be inhibitory. In other words, anti-Bp50 can be like BCGF obviously act as a soluble mediator, without the Help the Fc receptor mediated access function to the cell le.  

Table 4

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

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 indicates that anti-Bp50 and the BCGF used in this study apparently work on different signals. First, unlike BCGF (low) receptors (Bsterbosch, et al., 1985, J. Exp. Med. 162: 1825), Bp50 molecules are expressed on quiescent blood B cells ( Figure 3). Although both anti-Bp50 and BCGF (low) are most effective when added to anti-Bp35 or anti-Ig, the second least anti-3p50 is clearly optimally effective when added 12 hours after the onset of culture ( Figure 8A ). In contrast, BCGF (low) could be added 24 hours after initiation of culture and still proliferate optimally ( Figure 8B). These kinetic experiments, modeled after the proposals of Howard and Paul (1983, Ann., Rev. Immunol., 1: 307), indicate that a Bp50-dependent signal can normally exert its action before BCGF.

Both anti-Bp50 and BCGF (low) increase the proliferation of anti-Bp35 or anti-Ig activated B cells (Table 3). The effects of anti-Bp50 and BCGF (low) were additive in many experiments ( Figure 7). Fig. 9 shows a titration of BCGF (low) in an experiment using anti-Bp50 at its optimal concentration (0.2 μg / ml). BCGF (low) could further increase the proliferation of quiescent 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 ( Figure 10), was therefore necessary to allow the blood cells to respond to anti-Bp50. While dense tonsillar B cells were generally unreacted to either BCGF or anti-Bp50, floating B cells reacted (Table 5). B-cell disease sites were also different in their response to anti-Bp50 compared to BCGF. For example, some B-cell lymphomas responded to TPA plus BCGF (low), but not to 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).

Use of anti-Bp50 ligands and Bp50

The ligands of the present invention may be in vivo or in vitro in their unmodified or modified Forms used to modulate immune responses. For example, the ligands themselves can be used as an "adjunct" to increase an immune response a vaccine or to increase the immune response of an immune oppressed individual. When cytotoxins or anti-proli ferative agents are coupled to the ligands, these can modified ligands on the other hand can be used to to reduce an immune response, for example in the car immune disorders or in transplant patients to the Avoid prosthesis or plantat rejection. This modifi Decorated ligands can also be used for treatment malignant disease centers that include cells or tumors, which express the Bp50 antigen, regardless of whether the disease is the source of B-cells or not.

The ligands according to the invention and / or the Bp50 itself, can be used in vitro. Such applications conclude in vitro tests, such as immunoassays to find cells that express Bp50 antigen, and / or for locating in body fluids, for example hand-segregated Bp50 antigen. In these cases can the ligand or Bp50 be labeled with a radiolabel, Fluorine, enzyme, enzyme substrate, dye and the like. to In addition, the ligands can be used to those expressing, separating and / or adding the Bp50 antigen identify, in which case the ligand to a immovable carrier can be coupled or attached to a Fluorine which is expressed in a FACS (fluorescence-activated cell sorter) can be used.  

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

Bp50 receptor and uses of ligands, such as Anti-Bp50, for proliferation of B-cell proliferation

Early research suggested that the factors which in the induction of B cells from the G₀- to the G₁- Phase of the cell cycle are different, are different from the factors or requirements for the transition to S phase. This model is mainly based on studies, this show that agents, such as low doses of anti-Ig B cells Activation factors or anti-Bp35 alone, a small one or have no effect on B-cell proliferation. However, the same agents can B cells to a point drive the cell activation, in which they are compared to wax factors are susceptible. In contrast, have Growth factors such as BCGF or IL-2 alone have no effect resting D cells, however, increase the growth of ak tivated B cells.

Although the present invention is not limited should bring to any theory or interpretation, so the Here presented results but an additional support for a model of distinct regulation of B-cell assets tion and growth stages. It was shown that the Ak activation and proliferation signals in human B cells transmitted through distinct cell surface structures can. Although anti-Bp35 mAb B cells to enter the Activated G₁ phase of the cell cycle, it induces alone kei ne or only a small proliferation. Has anti-Bp50 mAb the opposite effect: it can not activate B cells, However, if it is even as late as 12 to 24 hours after Activation is added, it may be B-cell growth induce.  

The Bp50 molecule can function normally as a receptor for a ligand, such as a soluble growth factor or a signal transmitted through cell-to-cell contact (i.e., a ligand that is on the surface of another cell Is found). Previous studies identified different T cell-derived BCGFs similar to anti-Bp50, the Proliferate B cell proliferation. Both a high and a low molecular weight forms of B-cell len 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, are the receptors on B cells for growth signals yet not identified. The mAb AB-1 reacts with a B- Cell marker that expresses only on activated B cells is blocked and BCGF-dependent proliferation and thus to recognize the BCGF receptor or a related structure can. Bp50 appears different from AB-1 markers 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 react (Jung, et al., 1984, J. Exp. Med. 160: 1919-1924). Bp50 is on all B cells, which, based on Absorption analysis and direct binding tests, at BCGF Receptors do not seem to be the case. The current ones Data show that Bp50 and the receptor for BCGF with low according to molecular weight have different structures. Under Use of a rabbit heteroantiserum showed Wang and Employees (Wang, 1979, J. Exp. Med. 149: 1424-1433) already a 54 kDa glycoprotein, gp54, which like Bp50 at all B cells expressed but at lower levels  Blood B cells as on tonsillar B cells. It is possible, but unlikely that the rabbit heteroantiserum and Anti-Bp50 recognize the same or related structures: im Contrary to anti-Bp50 mAb is the rabbit antiserum too gp54 alone is able to stain B-cell proliferation formulate.

Anti-Bp35 alone, unlike Bp50, can activate B cells from the G₀ to the G₁ stage and thus can be referred to as an "activation" signal. Whether or not Bp35 acts only in early B-cell activation is not yet clear, as anti-Bp35 antibody can stimulate some B-cells to share (Clark, et al., 1985, Proc. Nat. Acad. Sci USA 82: 1766-1770). Similarly, Bp50 may not strictly work only as a "growth" signal: anti-Bp50 antibodies not only proliferate proliferation along with activation signals (anti-Bp35 or anti-u), but also increase the total number of G₁s the B cells (Table 2). In other words, Bp50 acts as a costimulant to promote the progression of both the Akti vierungs- (G₀ to G₁) and the 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 on growth regulation of fibroblasts. How B cells respond to anti-Bp35 or anti-Bp50 clearly depends on their stage of differentiation or activation.

It is shown here that two mAbs, anti-Bp35 (a "compe tenz- "signal) and anti-Bp50 (a" progression "signal) together a considerable proliferation of highly purified B cells in the absence of antigens or other known Can induce factors. The natural ligands for these structures are not yet known. However, because mAb too  suitable epitopes as well as soluble factors Mimic signals transmitted to cell and cell interaction it is possible to use suitable combinations of mAbs for Conducting and regulating Huamn B cell proliferation and to use differentiation. This will in turn be helpful be for the development of strategies in vivo for the control of human diseases, such as malignant B-cell diseases, immunodeficiencies and certain autoimmune diseases Diseases.

The monoclonal antibody G28-5, which is linked to a single-chain Polypeptide of approximately 50 kDa on the surface is expressed by human B cells a particular embodiment of the ligands of the present invention Invention showing the profile homing of activated B cells can multiply. Since the human B cell profile in Similarly, by T cell-derived BCGFs, including Low and high molecular weight BCGF, The activity of anti-Bp50 was increased G28-5 compared with that of a BCGF preparation, the predominant Low molecular weight BCGF. Anti-BP50 G28-5 and BCGF (low) were very similar in their costimulatory Effect with the same activating agents (Anti-Ig, anti-Bp35 and TPA), however, were not costimulating with each other or with IL-1 or IL-2. In addition, it was the activity of anti-Bp50 G28-5 does not depend on its Fc domain or region, since F (ab ') ₂ fragments of G28-5 functional were active. This indicates that soluble Anti-Bp50, such as soluble BCGF Fc receptor-bearing carrier cells not required to exercise an effect. Farther Both anti-Bp50 and BCGF are only effective in the presence an activation stimulus. In other words, anti- Bp50 and BCGF do not promote "competence" factors but promote them rather the "progression" of B cells through the cell cycle.  

While it is possible that Bp50 may serve as a receptor for a ligand, such as a B-cell growth factor, several results indicate that Bp50 is at least not the receptor for the BCGF (low) used in this study it is expressed on blood B cells, while BCGF (low) receptors are apparently not expressed. Candidate structures for the BCGF (low) receptor, unlike Bp50, are also expressed only on activated B cells. In addition, normal and malignant B cell populations are different in their responsiveness to anti-Bp50 compared to BCGF (low) (Table 5 and Figure 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 indicates that Bp50 can function as a receptor for BCGF (high).

Although Bp50 may be a receptor for a soluble ligand, Bp50 may also act as a receptor for a cell to cell-signaling signal that regulates BCGF receptor levels and / or autocrine production. The model for differentiation antigens that serve as enhancers of an auto-krin receptor pathway comes from studies with T cells. MAb to the lp220 common leukocyte antigen proliferates proliferation by increasing IL- ² receptor expression on activated T cells (Ledbetter et al., 1985, J. Immunol. 135: 1819). An analogous mechanism may occur with anti-Bp50 and the expression of certain BCGF receptors. Bp50 and BCGF (low) are apparently under well-coordinated control because, as with IL-1 and IL-2 receptors, BCGF increases the expression of Bp50 on certain leukemic cells. The Bp50 molecules also share similarities with the Tp44 molecule, which acts to influence IL-2 production. The 9.3 anti-Tp44 antibody has been shown to increase the proliferation of T cells 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 activated with anti-Bp35 or TPA. The Tp44 signal works by stimulating IL-2 production rather than by stimulating T-cell growth. The Bp50 signal may possibly function in an analogous manner by stimulating B cell autocrine production (Gordon, et al., 1984, Nature, London 319: 145).

For immunosuppression or treatment of nasty modified diseases used ligands

According to this embodiment, the ligands according to the Invention by adding an antiproliferative agent be modified so that the resulting molecule kills of cells expressing Bp50 antigen can. Such modified ligands may be used in the treat autoimmune disease to suppress the proli feration of B cells while suppressing the car immune response can be used. These modified ligands can also help to immunopatiorate a transplant patient used to reject the graft to avoid. Similarly, cytotoxic Agent used to suppress immune response be added to the ligands of the invention. When ligands are used, the proliferation of  Increase B cells, an increased effect can be achieved who as the drug directs to the proliferating B cells becomes.

In another embodiment, ligands may be added after Invention by the addition of an antiproliferati The agents are modified for the treatment of Erkran kung herds are used in which tumors or cells express the Bp50 antigen The addition of this chemo therapeutic agent to the ligands of the invention should lead to greater specificity of the drug against the ma lead ligaments cells. In addition, a specific Benefit obtained when a B-cell disease with a ligand which is linked to a cytotoxin pelt, which has a stronger killing effect than pro liferierenden cells as compared to non-proliferating Has cells. Treatment with such a ligand may be too potentiate the action of the cytotoxin.

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

Table 6

Chemotherapeutic agents coupled to anti-Bp50 ligands

Any known method can be used to test the ligand to the chemotherapeutic or antiproliferative agent to pair. Examples of such methods are already above listed (see section 5).

Other uses of ligands and Bp50

In addition to the therapeutic applications, the Li and Bp50 itself have other applications in in vitro as well as in vivo diagnostic studies, Sepa ration schemes, etc.  

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

In addition to therapeutic treatment and diagnostic Examination can also use the ligands according to the invention used to identify or separate from Cells expressing Bp50 antigen. In addition, the Ligand, if a suitable radio marker or a radio opaque compound is attached to the ligand, also for in vivo imaging of tumors expressing Bp50 antigen be used. Other applications erge for the skilled person from the previous description environment.

Deposit of cell lines

The following hybridoma was deposited on the American Type Culture Collection, Rockville, MD, and has the specified ID number received:

hybridoma ATCC identification number G28-5 HB9110.

The present invention is not be in scope restricts to the deposited hybridoma, since the deposited Embodiment is determined as a single illustration one aspect of the invention and each cell line that functions is equivalent, within the scope of the invention lies. In fact, numerous modifications of the inventions in addition to those shown and described  visibly to the person skilled in the art from the preceding description and the attached figures or drawings. Such Modifications are intended to be included within the scope of the claims fall.

Claims (24)

1. Essentially pure ligand that binds to Bp50 50 kilodaltons (kDa) B-cell surface antigen, defi niert produced by monoclonal antibody G28-5 by a hybrid cell line as deposited under ATCC and associated identification number. HB9110. 2. Ligand according to claim 1, characterized in that it an antibody molecule or an Fv, Fab, F (ab ') ₂ or Fab 'portion of the antibody molecule. 3. ligand according to claim 2, characterized in that the Antibody molecule is a monoclonal antibody molecule or a Fv, Fab, F (ab ') ₂ or Fab'-part of the mono clonal antibody molecule. 4. Ligand according to claim 1, characterized in that it includes a lymphokine. 5. Ligand according to claim 4, characterized in that it a B-cell growth factor.   6. ligand according to one of claims 1 to 5, characterized gekenn records that it has a verb coupled to the ligand tion. 7. ligand according to claim 6, characterized in that the ligand coupled compound an antiprolifera active agent, an alkylating agent, an antimetabo lit, an antibiotic, a vinca alkaloid, an enzyme, a platinum-coordinated complex, a radioisotope or a fluorescent compound. 8. Substantially pure ligand according to claim 1, at Binding to activated B cells the activated B cells stimulated to go through the cell cycle, so that the Proliferation of B cells is increased. 9. Ligand according to claim 8, characterized in that it an antibody molecule or an Fv, Fab, F (ab ') ₂ or Fab 'portion of the antibody molecule. 10. Ligand according to claim 9, characterized in that the Antibody molecule is a monoclonal antibody molecule or a Fv, Fab, F (ab ') ₂ or Fab'-part of the mono clonal antibody molecule. 11. Ligand according to claim 10, characterized in that it G28-5, produced by a hybrid cell line, as filed under ATCC and associated identification no. HB9110, or a mutant, recombinant or a gene table manipulated derivative thereof.   12. Ligand according to claim 8, characterized in that it includes a lymphokine. 13. Ligand according to claim 12, characterized in that it a B-cell growth factor. 14. Ligand according to claim 12, characterized in that the lymphokine is on the surface of a cell located. 15. ligand according to any one of claims 8 to 14, characterized ge indicates that it is coupled to the ligand Compound includes. 16. Ligand according to claim 15, characterized in that the ligand-coupled compound is an antiprolife active agent, an alkylating agent, an antimeta bolit, an antibiotic, a vinca alkaloid, an enzyme, a platinum-coordinated complex, a radioisotope or a fluorescent compound. 17. Use of a ligand according to one of claims 8 to 14 in an effective dose to increase the Pro Liferation of B cells in vitro by treatment of activated B cells, so that the activated B cells Cells go through the cell cycle and proliferation is increased. 18. Use according to claim 17, wherein the B cells by Treatment of an effective dose of a second ligand which binds to Bp35, a 35 kDa B- Cell surface antigen, so that the B cells from the G₀- to G₁ stage or phase of the cell cycle continued stride.   19. Use according to claim 18, characterized in that the second ligand comprises an antibody molecule that binds to Bp35 is able to bind. 20. Use according to claim 19, characterized in that the Bp35 binding antibody is monoclonal Antibody comprises. 21. Use according to claim 20, characterized in that the activated B cells within 12 hours after activation by the second binding to Bp35 Ligands treated with the binding to Bp50 ligand become. 22. Use of a ligand according to one of claims 1 to 7, coupled with an antiproliferative agent is, in an effective dose to suppress the Proliferation of cells expressing Bp50, a 50 kDa B-cell surface antigen, defined by the monoclonal antibody G28-5; made by one Hybrid cell line as deposited under ATCC and affiliation ger identification no. HB9110, in vitro by the treatment of Bp50 expressing cells. 23. Use according to claim 22, characterized in that the Bp50 expressing cells B cells or malignant Cells include. 24. Use according to claim 22 or 23, characterized records that the ligand is a monoclonal antibody molecule comprising G28-5, manufactured by one Hybrid cell line as deposited under ATCC with the label No. HB9110 or a mutant, recombinant or gene tampered derivatives thereof, or a Fv, Fab, F (ab ') ₂ or Fab'-part thereof.