DK173940B1 - Ligands and Methods for Enhancing B Cell Proliferation and B Cell Surface Antigen Bp50 - Google Patents

Description

DK 173940 B1

The present invention relates to ligands such as antibody molecules or fragments of antibody molecules or other ligands such as lymphokines which bind to a 50kDa B cell surface marker, herein referred to as Bp50, which functions at B cell proliferation but does not at early B cell activation. The present invention also relates to the Bp50 B cell antigen itself. In a particular embodiment of the present invention, a monoclonal antibody, G28-5, is defined that defines Bp50 and appears to play a role in the proliferation of activated B cells, but has no detectable effect on the proliferation of quiescent ΒΙΟ cells.

The ligands, such as antibodies, lymphokines and fragments thereof according to the invention, can be used to control and regulate human B cell proliferation and / or differentiation. Furthermore, the ligands of the present invention can be modified by attaching other compounds thereto which can be used for the treatment and / or detection of malignant cells expressing the Bp50 antigen.

The activation of dormant B cells from GQ to Gj phase 1 cell cycle and subsequent induction of activated B cells to proliferate are distinct steps that require distinct regulatory mechanisms.

Some agents, including murine B cell stimulating factor-β1 (BSF-β1) (Rabin, et al., 1985, Proc. Nat. Acad. Sci. USA 82, 2935-2939) or low doses of anti-immunoglobulin ( 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, 0. Immunol. 132: 176-180) are "activation or competence" factors, that is, they induce B cells to to grow, synthesize more RNA and enter Gj, but alone do not induce DNA synthesis in B cells. Other "growth" factors such as human B cell growth factor (BCGF) and 1terleukin-2 (IL-2) get activated B cells to undergo the cell cycle and enter S phase, but do not activate resting B cells (Kehrl, et al., 1984, 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 now been described by researchers in both murine and human systems. These include B-cell growth factors (BCGF) derived from a variety of sources, including T-cell lines or hybridomas, B-cell lines or dendritic cells. Although both interleukin-1 (IL-1) and 1terleukin-2 2 have been shown to increase B-cell growth, they appear to be different from certain BCGFs. For example, monoclonal antibodies (mAb) against a murine BCGF (O'Hara, et al., 1985, Nature (Lond.) 315: 333) or human BCGF (Ambrus, et al., 1985, J. Exp. Med. 162: 1319) block BCGF-5 activity but not IL-1 or IL-2 activity. Although the BCGFs are different from IL-1 or IL-2, they themselves appear to be heterogeneous based on biochemical data and differential activity on different B cell subgroups or in costimulation experiments. For example, 60-kilodalton (kDa) high molecular weight human BCGF, BCGF 10 (high), which is different from a 12-kDa low molecular weight form, BCGF (low) has been identified (Ambrus, et al., 1985, J. Cl in. Invest. 75: 732). The cDNA encoding a 20-kDa murine BCGF, experimentally called B-cell stimulating factor p1 (BSF-p1), has recently been cloned and sequenced (Noma et al., 1986, Nature 319: 640). The recombinant lymphokine not only has BCGF-15 activity, but can also activate resting B cells and induce differentiation of IgGj-producing cells. Thus, it differs from human BCGF (high) and BCGF (low) both in terms of molecular weight and in terms of activity range.

These activation and growth signals presumably regulate cells by interacting with specific B-cell surface structures. In addition to the antigen-specific signal through surface Ig, several other possible B-cell surface polypeptides have been identified that may somehow function in the activation or growth of B cells. Eg. are the cell surface receptors for IL-1 (Dower, et al., 1985, 0.

Exp. With. 160: 501) and IL-2 (Robb et al., 1984, J. Exp. Med. 160: 1126) have been characterized, and more recently, functional IL-2 receptors have been identified on B cells (Zubler, et al. , 1984, J. Exp. Med. 160: 1170; Jung, et al., 1984, J. Exp. Med. 160: 1597; Muraguchi, et al., 1985, J. Exp. Med. 161: 181). However, receptors for B-cell growth and activation factors are yet to be fully characterized. Several possible B cell surface polypeptides have been identified that may somehow act upon the activation or growth of B cells. Eg. found Subbarao and Hosier (Subbarao, et al., 1983, Immunol. Rev. 69: 81-97) that monoclonal antibodies (mAb) against the murine 35 B cell antigen Lyb2 activate B cells, and evidence has recently been provided for that Lyb2 should be the receptor for BSF-β1 (Yakura, 1985, Fed. Proc. 44: 1532). Similarly, the present inventors have found that appropriate mAb (1F5) against a 35 kDa polypeptide, Bp35, activates human B cells from Gq to Gj (Clark, et al., 1985, Proc. Nat. Acad.

Sci. USA 82: 1766-1770; Gollay, et al., 1985, J. Immunol. 135: 3,795 to 3,801). Aggregated C3d or antibodies against the 140 kDa C3d receptor, Bp140, cause proliferation of B cells that are T cell dependent (Melchers, 5 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) prepared a polyclonal antiserum that identified a 54-kDa polypeptide (gp54) on human B cells and showed that rabbit antiserum against gp54 induced tonsillar B cells for division, recently isolated Jung and Fu (Jung, et al., 1984, J. Exp. Med. 160: 1919-1924), a mAb (AB-1) against a 55-kDa antigen restricted to activated B cells that block BCGF-dependent and proliferation. However, whether an anti-gp54 or AB-1 recognizes a BCGF receptor remains unknown.

The present invention relates to ligands that (a) bind to Bp50, a 50kDa B cell specific surface polypeptide described herein, and (b) enhance the proliferation of activated B cells. The invention also relates to the Bp50 antigen itself, which is defined by monoclonal antibody G28-5 and acts by proliferation of activated B cells. Furthermore, the invention relates to ligands that bind to Bp50 but do not exhibit a biological effect or function, such as enhancing the proliferation of activated B cells.

The ligands of the present invention comprise antibody molecules, monoclonal antibody molecules and fragments of these antibody molecules containing the antigen-combining site or chemically modified antibodies and fragments which include, but are not limited to Fv, Fab, F (ab ') 2, Fab' and the like. Furthermore, the ligands of the present invention include lymphokines which may include, but are not limited to, human B cell growth factors as well as chemically modified lymphokines. The ligands of the invention may be chemically modified, e.g. by binding or linking a compound to the ligand. Such compounds include, but are not limited to, cytotoxic agents, therapeutics, chemotherapeutic agents, labels such as radioactive labels, dyes, enzymes, radioopaque compounds and the like. The ligands of the present invention can be used in their modified or unmodified form to control, 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-5 cell antigen described here, Bp50, apparently play distinct roles as signal receptors in B cell activation. Monoclonal antibodies (mAb) to Bp35 and Bp50 both give positive signals to B cells, which simulate their transformation through the cell cycle. mAb against Bp35 functions, like ant1-Ig antibodies, in principle, by activating quiescent B cells to become competent to enter the Gj phase of the cell cycle. In contrast, a monoclonal antibody described herein or its F (ab ') 2 fragment functions against Bp50, a 50-kDa polypeptide expressed on all B cells, so that activated B cells are stimulated to undergo cell cycle and increase proliferation of activated B cells. Monoclonal needle antibodies to Bp35 activate, as do anti-Ig antibodies, tone sillary B cells and induce low-level B cell proliferation. In contrast, anti-Bp50 monoclonal antibody alone does not activate B cells nor induce B cells to proliferate, but, along with anti-Bp35 or anti-Ig antibodies, increases B cell proliferation. In this respect, the effect of the anti-Bp50 anti-tof is similar to the activity of B-cell growth factors (BCGF). As little as 0.05 µg / ml anti-Bp50 is required to enhance proliferation, and like BCGF, anti-Bp50 is effective even when added 12 to 24 hours after B cells have been activated with anti-Ig or anti-Bp35. Without further exogenous signals, anti-Bp35 and anti-Bp50 antibodies together induce strong proliferation of purified dormant B cells. These results suggest that the Bp35 and Bp50 surface molecules function in the regulatory control of B cell activation and progression throughout the cell cycle. Because of the importance anti-Bp35 and similar molecules have on the effect and effect of the ligands of the present invention, Clark et al., 1985, Proc. Natl.

Acad. Sci. (USA) 82: 1766-1770 as part of the present disclosure.

Although the activity of anti-Bp50 is similar to that of BCGF (low), since both anti-Bp50 and BCGF (low) are costimulatory with the same agents but not with each other, and both anti-Bp50 and BCGF (low) only affect activated B cells and acting in a dissolved form, the activity of anti-Bp50 can be distinguished from the activity of BCGF (low), since the proliferation of B cells stimulated with optimal amounts of anti-Bp50 and anti-Bp50 -Bp35 (or anti-Ig), can be further increased with BCGF (low), and both blood B cells and certain B cell lymphomas respond differently to anti-Bp50 and BCGF. For optimal activity, anti-Bp50 should be added within 5 within 12 hours of B cell activation, whereas BCGF (low) retains optimal activity even when added 24 hours after activation. Furthermore, Bp50 is expressed on all B cells, whereas receptors or BCGF (low) are restricted to activated B cells. Thus, anti-Bp50 and BCGF (low) coordinate can regulate B-cell growth, but apparently this will occur through distinct signals.

In one embodiment of the present invention, the ligands that bind to Bp50 and enhance the proliferation of activated B cells can be used to enhance an immune response. Eg. For example, these ligands that bind Bp50 can be used as an "adjuvant" to enhance an immune response to a vaccine. Alternatively, these ligands can be used to enhance the immune response of an immunosuppressive individual.

In another embodiment, the ligands of the invention can be chemically modified so that the cells to which the ligands bind are killed.

Since all B cells express the Bp50 antigen, this variant will result in suppression of the immune response. Eg. For example, a cytotoxic drug bound to a ligand of the present invention can be used in vivo to induce immunosuppression to cross histocompatibility barriers in transplant patients. Alternatively, these modified ligands can be used to fight autoimmune diseases.

In another embodiment of the present invention, malignancies such as tumor cells expressing Bp50 can be treated by using a ligand of the invention bound to a chemotherapeutic agent useful in the treatment of such neoplastic diseases. These modified ligands can be used in vivo to direct the chemotherapeutic agent against any type of malignant cell expressing the Bp50 antigen, including non-B cells but expressing Bp50. When using the ligands of the invention that enhance B cell proliferation, a particular advantage should be obtained when treating B cell malignancies, wherein the ligand-bound chemotherapeutic agent comprises one that is more effective in killing proliferation. in fermenting cells. In this case, an enhancement of the drug effect could be achieved.

Alternatively, the ligands of the invention can be used in vitro to identify or separate cells expressing the Bp50 antigen 6 and / or to examine body fluids for the presence of the Bp50 antigen that may be spread. Furthermore, the ligands of the invention can be used in vivo to capture cells or tumors expressing the Bp50 antigen.

The purified Bp50 antigen of the present invention can be used to prepare antibodies and to prepare or design other ligands of the invention. Furthermore, the Bp50 antigen should be used for studies such as diagnostic immunoassays. Furthermore, Bp50 itself can be used as a mediator of cell immunity 10 in vivo or in vitro.

DEFINITIONS

As used herein, the following abbreviations will have the following meanings: 15 AO * acridine-orange BC6F 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 20 (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 combiner they place in an antibody molecule. This may be any fragment containing the idiotype of the molecule, including but not limited to Fab, FJab ', Fab' and the like.

IF - Immunofluorescence

Immunoglobulin 30 µl-I = interleukin 1 IL-2 * interleukin 2 kDa = kilodalton mAb - monoclonal antibody SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis TPA-12-O-tetradecanoylphorbol-13-acetate 7 DK 173940

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 5 (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770). The results are plotted as cell counts versus log of green fluorescence and log of red fluorescence, with 4-5 spots representing approximately a fluorescence doubling. The results are presented to show autofluorescent negative cells. PE (red) anti-Bp35 (1F5) versus FITC (green) -10 anti-Bp50 (G28-5) staining shows that all Bp50 + cells are also Bp35 +.

FIG. 2. Biochemical comparison of Bp50 polypeptide with other B cell 125 surface antigens. Immunoprecipitation of Bp50 from surface I-labeled tonsillar cells was performed as described. Isolated antigens were electrophoresed on 10% SDS-polyacrylamide plate gels without reduction.

15 Gels were visualized with autoradiography and intensifying screens. Plate A: lane 1, anti-Bp50 (G28); lane 2, anti-Bp95 (G28-8); lane 3, sepha-rose-goat anti-mouse Ig alone. Exposure time: 4 days. Plate B: lane 1, ant1-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 alone.

An exposure time of 2 days was chosen so that the bands in tracks 2 to 4 were not overexposed and clearly distinguishable from Bp50. One in 3 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 fluorescine (green) -conjugated reference antibodies, including 2C3 (anti-IgM ); 1F5 (anti-Bp35); HB1Oa (anti-DR); and 9.6 (anti-CD2, E receptor). Cells were analyzed with a FACS IV equipped with four decade log amplifiers 1 in both red and green dimensions.

Front and right light scattering was used to capture monocytes. Spotless cells are at the back of the grid, red fluorescence is to the right, and green fluorescence is to the left.

Fig.4. Dose response curves for increasing proliferation of dense tonsillar Er-B cells by anti-Bp50 antibodies, as shown: Medium alone, anti-Bp50 alone, anti-Bp35 (5 µg / ml) alone, BCGF alone , anti-Bp35 plus BCGF, anti-Bp35 plus graduated doses of anti-Bp50. Mean proliferation ± standard deviation of quadroplicat samples was measured on day 3.

8 DK 173940 B1

FIG. 5. Ant1-Bp50 mAb is most effective in enhancing proliferation if addition occurs after a B cell activation signal. Dense tonsillar Er-B cells Incubated for 4 days with medium alone, anti-Bp50 (0.5 µg / ml) added at different times after incubation, anti-5 Bp35 (5 µg / ml) added at different times after Incubation, anti-Bp50 kept constant, to which anti-Bp35 was added later at different times, anti-Bp35 kept constant, to which anti-Bp50 was added to cultures at different times. Over the last 10 hours ten 1-H-thymidine was added and its incorporation measured.

FIG. 6. Comparison of the ability of anti-Bp35 and anti-Bp50 to induce dormant tonsillar B cells to exit the Goo stage of the cell cycle. Day 3 after treatment media alone (_), anti-Bp35 alone (--------); and Ig alone (.........), A, no further additives; B, anti-Bp50 (0.5 µg / ml) added to each group; C, 5% BCGF added every 15 groups. The results are plotted as relative cell count versus log to A0 red fluorescence (RNA).

FIG. 7. Kinetics of B-cell proliferation after stimulation with anti-Bp50 versus BCGF. Dense tonsillar E-B 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-Bp50 + anti-Bp50 + 10% BCGF. Proliferation was measured on the indicated days with an 18 hour pulse of 3 H-thymidine. Proliferation was measured in quadroplicate and standard deviations are shown. One in three attempts.

FIG. 8. Times after anti-Bp35 stimulation, where anti-Bp50 (A) or 25 BCGF (B) optimally proliferates. Dense tonsillar E-B cells were stimulated as shown, and proliferation was measured by an 18 hour o pulse of H-thymidine on day 3. Medium, anti-Bp35 alone added at the indicated times, anti-Bp50 or BCGF alone, anti -Bp35 added at the onset of culture followed by addition of anti-Bp50 or BCGF to the 30 times indicated, anti-Bp50 or BCGF added at the onset of culture followed by anti-Bp35. One out of two attempts. Proliferation was measured in quadroplicate and standard deviations are shown. Doses used: anti-Bp35, 5 µg / ml, anti-Bp50, 0.2 µg / nl, BCGF (low) 5%. Concentrations used were as follows: anti-Bp35, 5 µg / ml, anti-Bp50, 0.2 Mg / ml, BCGF, 5%.

FIG. 9. Anti-Bp50 and BCGF have additive effects on B cell proliferation. Dense tonsillar E-B cells were stimulated with graduated 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. The proliferation was measured on day 3 after stimulation with an 18 hour pulse of H-thymidine. Proliferation was measured in quadroplicate and standard deviations are

C

shown. One in four attempts. Doses used for 10 cells: anti-Bp35, 5 µg / ml, anti-Bp50, 0.2 µg / ml, anti-Ig beads, 50 µg / ml.

FIG. 10. Comparative effects of anti-Bp50 and BCGF on normal and malignant B cells. Peripheral blood E-B cells (A) or dense tonsillar E-B 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 similarly stimulated. Proliferation was measured on day 3 3 10 by incorporation of H thymidine over a 12 hour pulse. Proliferation was measured in quadroplicate and standard deviations are shown.

DETAILED DESCRIPTION

The present invention relates to ligands that (a) bind to Bp50, a 50kDa B cell specific surface polypeptide, and (b) increase the proliferation of activated B cells. The invention also relates to the Bp50 antigen itself, which is defined by mAb G28-5 and acts on B cell proliferation. Furthermore, the invention relates to ligands that bind to Bp50 but do not exhibit a biological effect or function, such as enhancing proliferation of activated B cells.

The ligands of the present invention comprise antibody molecules, monoclonal antibody molecules, and fragments of these antibody molecules which contain the antigen-combining site that binds to the Bp50 receptor, including chemically modified antibodies and fragments which include, but are not limited to, Fv, Fab, 25 F (ab ') 2, Fab' and the like. Furthermore, the ligands of the present invention include lymphokines which bind to the Bp50 receptor. These may include, but are not limited to, BCGFs as well as chemically modified lymphokines and the like. The ligands of the invention can be used in their modified or unmodified forms for modulating and regulating immune responses and in the therapy of malignancies expressing the Bp50 antigen. These uses are discussed in more detail in Section 4 below.

When the ligand is a monoclonal antibody or fragment thereof, the Bp50 monoclonal antibody can be prepared using any technique that produces antibody molecules by continuous cell lines in culture. Eg. For example, the hybridoma technique originally developed by Kohier and Milstein (1975, Nature 256: 495-497) as well as other more recently available techniques, such as the human B-cell hybridoma technique (Kozbor et al. , 1983, Immunology Today 4:72) and the EBV hybridoma technique for producing human monoclonal antibodies (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., p. 77-96). and the like, all of which fall within the scope of the invention.

Antibody fragments containing the idiotype of the molecule are believed to be capable of being generated by known techniques. Such fragments include, for example, but are not limited to: the F (ab ') 2 fragment which can be generated by treating the antibody molecule with pepsin, the Fab' fragments which can be generated by reducing disulfide bridges in the F (ab ') 2 fragment, the F (ab') 2 * fragment which can be generated by treating the antibody molecule with papain and the 2Fab or Fab fragments which can be generated by treating the antibody. the papain molecule and a reducing agent for reducing the disulfide bridges.

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

Depending on the intended use, the ligand or appropriate fragments of the ligand can be chemically modified by attaching any of a variety of compounds to the ligand using known coupling techniques known per se. Such techniques may 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, attachment to sulfhydryl moieties. , use of sodium isothiocyanate or enzymatic binding to name just a few. When a radioisotope is to be attached to the ligand, this can also be done by enzymatic agents, oxidative substitution, chelating, etc. For a review of the chemical reagents that can be used for protein modification, see Lundblad and Noyes, Chemical Reagents for Protein Modification, Volume II , CRC Press, Inc., 35 Boca Raton, Florida, Chapter 5, pp. 123-139, 1984.

The chemical binding or coupling of a compound to the ligand could be directed to a site on the ligand which does not participate in binding to Bp50. This could be done by protecting the binding site of the ligand before conducting the coupling reaction. Eg. For example, the ligand can first be bound to Bp50 to protect the Bp50 binding site, after which the coupling reaction can be performed to bind the desired compound to available reactive sites on the Igand-Bp50 complex. Once the coupling reaction is complete, the complex can be cleared, thereby forming a modified ligand to which the desired compound is attached, so that the Bp50 binding site of the molecule is minimally affected. When the ligand comprises a monoclonal antibody, such as G28-5, wherein the Fc region of the molecule is not required for the ligand to exert its effect (see Section 3.3 below), it may be advantageous to control the coupling of desired compounds to the Fc region of the molecule. .

The following subsections describe the new 50-kDa B-cell surface marker, Bp50, which appears to act on B-cell proliferation, as well as ligands that bind to the new 50kDa receptor and uses thereof. As an example of the ligands of the present invention, there is also disclosed a monoclonal antibody which defines Bp50 and its Fiab'Jg fragments and which, like BCGF, enhances B cell proliferation.

Unlike anti-Bp35 mAb, which can induce dormant B cells in G0 to enter Gj, anti-Bp50 mAb does not activate dormant B cells.

Together, with no additional exogenous signals, anti-Bp35 and anti-Bp50 mAb together induce strong activation and proliferation of purified B cells.

The experiments described below also show that anti-Bp50 activity is similar to BCGF activity, but that anti-bp50 differs from one BCGF, since anti-Bp50 and low molecular weight BCGF are clearly additive and differ on different B cells. subgroups or malignancies. Bp50 may be a receptor for a distinct BCGF or for a transmembrane signal that modulates BCGF product or BCGF receptor expression.

1. METHODS OF CHARACTERIZING THE Bp50 RECEPTOR

Cell preparations. Nononuclear cells were isolated from normal or 30 leukemically heparanized peripheral blood by Ficoll-Hypague gradients (Pharmacia, Piscataway, NJ). Hononuclear cells were obtained from tonsillar tissue as described (Clark, et al., 1985, Proc. Nat. Acad. Sci.

USA 82: 1766-1770). T cells were diluted with AET-treated sheep erythrocyte rosetting and Ficoll-Hypague gradient separation. In some experiments 35 B cells from blood were enriched by Isolating Nylon Wool Adherent Cells. Monocytes were removed by incubation on plastic petri dishes once or twice at 37 ° C for 45 minutes unless otherwise noted. Liquid or dense tonsillar B cell fractions were isolated by Percoll 12 step 17 gradients as described (Clark, et al., 1985, Proc. Nat. Acad.

Sci. USA 82: 1766-1770). Dense tonsillar B cell preparations constantly had more than 95% SLG + Bp35 + cells. Blood B cell enriched preparations had 60 to 85% SLG + cells. B-cell lymphoma cells Isolated by mild influence of lymphoma cells in medium followed by Ficoll-Hypague gradient centrifugation.

Monoclonal antibodies. The G28-5 antibody to Bp50 was developed by immunizing BALB / c mice with human E-tonsillar lymphocytes and fusing immune spleen cells with an 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 antibody reactive with tonsillar B cells and not with T cells were identified using Indirect Immunofluorescence (IF) and assay with a FACS IV cell sorting apparatus. Cultures with antibody giving histogram patterns corresponding to 15 known mAbs against pan B cell markers (eg, Bp35) were cloned and selected for further study. The G28-5 clone formed an IgGj mAb that reacted only with normal or malignant B cells or B cell lines. Other mAbs used in this study have been described in detail (Clark, et al., 1985, Proc. Nat. Acad. Sc1. 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, 1n Perspectives in Immunogenetics and Histocompatibility. ASHI, New York, 6, pp. 325-340) . These include 1F5 (IgG2a) anti-Bp35, HB1Oa (IgG2a), anti-HLA-DR, 2C3 (IgGj) anti-u chain, G19-4 (IgGj) anti-CD3, FC-2 (IgG2a) anti-Fc receptor CD16 25 and 9.6 (IgG2a) anti-CD2 (E receptor) provided by Dr. Paul Martin (Martin, et al., 1983, J. Immunol. 131: 180). The IgG2 mAbs were purified by precipitation using 45% or 50% saturated ammonium sulfate and DEAE separaic column chromatography, and the IgG2a mAbs were purified using protein A sepharose columns. The F (ab '- fragments of G28-5 were prepared by Parham's method (Parham, et al., 1983, J. Immunol. 131: 2895) was purified on a 2 meter long sephacryl S200 column and examined for purity by SDS-PAGE (Ledbetter, et al., 1985, J. Immunol. 135: 1819). 2C3 mAb against u chains was conjugated to Sepharose 4B beads (Pharmacia Fine Chemicals, Uppsala, Sweden) using 35 anogen bromide coupling.

Fluorescence and phoneconvulsions. Purified mAb was either directly conjugated with fluorescence using fluorescine-1sothiocyan at (FITC, Molecular Probes) (green) by Goding's method 13 Goding etc al., 1976, J. Immunol. Meth. 13: 215- 226) or conjugated to R-phycoerythrin (PE) (red) using SPDP (Pharmacia) by a method described in detail 1 Ledbetter, et al., 1985, Perspectives in Immunogenetics and .Histocompatibility, ASHI, New York, 6, 119-5 129. Lymphoid cells were incubated in round bottom microtiter plates for 30 minutes with a suitable dilution of green and / or red mAb, washed twice, and then analyzed on a FACS IV cell sorting apparatus.

Two-color immunofluorescence. Two-color studies were performed with a fluorescence-activated cell sorting apparatus (FACS IV: Becton-Dickinson, 10 Mountain View, CA) using a 560-nm dichroic mirror for cleavage of the beam and a 580 long-pass filter and a 540 short-pass filter (Ditric Optics, Hudson, MA) in front of the red and green photomultiplier tube, respectively. In addition, a two-color compensator (T. Noza-ki, Stanford University) was used to correct for small error scatterings of 15 green and red signals. For each two-color stain, data were collected from 40,000 cells and stored on floppy disks. Results are presented as cell counts (vertical) versus log green fluorescence versus log red fluorescence on a 64 x 64 spot grid. About 4.5 spots represent a fluorescence doubling. Spotless cells are in the rear 20 corner of the grid, red fluorescence is to the right, and green fluorescence is to the left. The two-color IF cytometry system used with fluorescence and phycoerythrin 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 in a c-volume of 5-10 x 10 ml in quadroplicate in 96-well microtiter plates containing 200 μΐ RPM1-1640 medium supplemented with 15% fetal bovine serum, antibiotics, glutamine and pyruvate (R15) . After 1 to 7 days, cells were pulsed with 0.5 µCi of H-thymidine per ml. well (New England Nuclear, 6.7 Ci / mmol; 30 L Cl = 37) for 18 hours. Next, cells were harvested on fiberglass filters with a cell harvester, and radioactivity was measured in a scintillation counter. In some experiments, antibodies or factors were added at different times after starting cultures, and proliferation in these experiments was measured on day 3.

35 Costimulating factors. Purified BCGF was purchased from the Cytokine Techno logy (Buffalo, New York) and contained no detectable IL-1, IL-2 or interferon activity. This BCGF was prepared by the method of Maizel and co-workers (Maizel, et al., 1982, Proc. Nat.

DK 173940 B1 14

Acad. Sc1. USA 79: 5998), which has shown that most of the BCGF activity in this material resides in a 12-kDa compound, hereinafter referred to as "BCGF (low)" (Mehta, et al., 1985, J. Immunol. 135: 3298). The purification steps included preparative scale 5 DEAE affinity chromatography followed by hydroxylapatite column chromatography. IL-1, which was purified for homogeneity, was a generous gift from Dr. Steven Dower (Dower, et al., 1985, J. Exp. Med. 162: 501). Recombinant IL-2 was provided by Cetus Corporation. TPA (12-O-tetradeconyl phorbol 13-acetate) was purchased from Sigma.

10 Detection of cell activation. Changes in cell volume induced by mAb and / or factors were measured using a cell sorting apparatus and a light scatterer with a front light incident. Cell cycle changes in cellular RNA and DNA content were measured by staining activated cells with acridine orange and measuring relative cellular RNA (red) and DNA content (green) with a cell sorting apparatus by the method of Darzynkiewicz et al. (Darzynkiewicz, et al., 1980, Proc. Nat. Acad. Sci. USA 77: 6697-6702). Changes in relative cell surface antigen content were measured using a mAb directly conjugated with fluorescence and then quantified by direct IF fluorescence amounts by an Epics V cell sorting apparatus.

Biochemical characterization of Bp50. Immunoprecipitation of Bp50 from surface 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 plate gels without reduction.

25 Gels were visualized using autoradiography at -70 ° C and Cronex illumination plus intensifying screens (Dupont).

2. CHARACTERIZATION OF THE Bp50 RECEPTOR

The following subsections describe the results of the experiments performed using the methods described above.

2.1. IDENTIFICATION OF A B-CELL SPECIFIC 50 kDa CELL

SURFACE MARKER, Bp50

An mAb against Bp50 was generated by immunizing BALB / c mice with human tonsillar lymphocytes and fusion of immune spleen cells with the NS-1 myeloma. One clone, G28-5, formed an IgGj mAb which did not contain the NS-1 light chain. Upon closer examination by IF analysis, G28-5 was found to respond only with normal or malignant B cells or B cell lines. Extensive screening of normal tissue by established methods (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770; DK 173940 B1

Ledbetter et al., 1986, Human Immunol. 15: 30-44; Ledbetter, 1985, in Perspectives in Immunogenetics and Histocompatibility. ASHI, New York, 6, pp. 325-340) showed that the G28-5 antibody reacts with E-rosified negative (Er-) cells from blood or tonsils, but not with nylon wool nonadherent 5 T cells, PHA-induced T cells. cell blasts or with blood granulocytes, monocytes, red blood cells or platelets. It reacted strongly with all 7 examined B lymphoblastoid cell lines and with 3 Burkitt's lymphoma lines (Raji, Daudi, Namalwa), but not with 4 T cell lines (CEM, HSB-2, JURKAT, and HPB-ALL). All examined chronic lymphocytic 10 leukemias (3/3) and 90% (9/10) of examined B lymphomas expressed the Bp50 marker, while only 28% (2/7) of non-T, non-B CALLA + acute lymphocytic leukemia are expressed Bp50.

The limited distribution of Bp50 on normal tissue was further confirmed by quantitative two-color immunofluorescence (two-color IF) assays.

Using an R-phycoerythr1n (PE) -conjugated antibody (red) against pan B cell antigen Bp35 (B1, CD20) and fluorescence-conjugated anti-Bp50 antibody (green), it was found that Bp50 was expressed only on Bp35 + B cells (Fig. 1) in blood or tonsils. Blood B cells consistently expressed somewhat lower levels of Bp50 than tonsillar B cells, which corresponds to HLA-DR expression (Ledbetter et al., 1986, Human Immunol. 15: 30-44) and gp54 expression (Wang, et al., 1979, J. Exp. Med. 149: 1424-1433) which are also lower on blood B cells. Bp50 was expressed at similar levels on tonsillar B-cell subpopulations separated on Percoll gradients 1 liquid and dense fractions. Using the present PE-conjugated mAb against the T-cell marker, CD3 (T3), and NK cell-associated marker, CD16 (Fc receptor) (Ledbetter, et al., 1979, Immunol. Rev. 47:63 -82), it was found that Bp50 is not expressed on T cells or NK cells. Furthermore, using two-color IF, it was found that CD3 + PHA blasts expressing high levels of IL-2 receptors did not express Bp50.

The G28-5 antibody responded with a single polypeptide to tonsillar lymphocytes migrating at ca. 50 Kd under non-reducing conditions (Fig. 2A). This molecule is larger than the previously reported B cell markers in the same molecular weight range, such as Bp39 or Bp45 35 (Zipf, et al., 1983, 0. Immunol. 131: 3064-3072; Kitner, et al., 1981, Nature 294 , 458-460; Clark, et al., 1986, in Leukocyte Typing II eds. Reinherz, et al., Springer Verlag, Berlin, Chap. 12 Vol. 2, 155-167; SI ovin, et al., 1982, Proc Nat. Acad Sci. USA 79: 2649-2653; Thorley-16 DK 173940 B1

Lawson, et al., 1985, J. Immunol. 134: 3007-3012, and FIG. 2B). The exposure time of this gel was chosen so that the molecular weights of the other B-cell markers could be easily compared with Bp50. The Bp39 marker is expressed in contrast to Bp50 on granulocytes, and Bp45, in contrast to Bp50, is restricted to B cell blasts. Antibodies against Bp39 (41-H16) and Bp45 (MNM6, Blast-1, Blast-2) made available from an international working meeting (Clark, et al., 1986, 1 Leukocyte Typing II, ed. Reinherz, et al. al., Springer Verlag, Berlin, Chapter 12, Vol. 2, 155-167) did not block the binding of fluorescent anti-Bp50 antibodies 10 to B cells. Thus, based on tissue distribution, biochemical analysis, and blocking studies, the G28-5 monoclonal antibody recognizes a 50-Kd structure different from other known B-cell antigens.

2.2 EXPRESSION OF Bp50 IS LIMITED TO B CELLS Both hematopoietic tissue and cell line distribution studies as well as 15 detailed two-color flow cytometric analysis showed that Bp50 is expressed only on B lymphocytes. As shown in Figure 3, Bp50 is expressed on a small subset of blood lymphocytes and on a large population of tonsillar lymphocytes. Practically all Bp50 + cells in both blood and tonsils also expressed Bp35 and HLA-DR, but did not express CD2 (Fig. 1) or CD3, T-20 cell molecules or the IgG Fc receptors found on NK cells. ConA-activated CD3 + T-cell blasts IL-2 receptors but did not express Bp50.

Two-color flow cytometric analysis enables quantitative measurement of the density relationship between two surface antigens. It has been previously shown that the dense, resting B cells in the sheath zone of secondary follicles express IgM and low levels of Bp35, whereas liquid, 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 subgroups expressed Bp50 in the same amounts, indicating that Bp50 is expressed on both resting B cells and in vivo activated B cells.

3. INCREASE OF B CELL PROLIFERATION DOWN ANTI-Bp50 ANTIBODY As previously explained, B cells can be activated with low doses of anti-u chain specific antibodies. It has recently been found that the B-35 cell specific marker Bp35 (B1), a 35-kDa polypeptide, can also function in early B-cell activation, with 1F5 mAb against Bp35, as well as low doses of anti-u antibody. , activates B cells to grow 1 cell volume and RNA content and become responsive to BCGF (Clark, et al., 1985, 17 DK 173940 B1

Proc. Night. Acad. Sci. USA 82: 1766-1770; Gollay, et al., 1985, J. Immunol. 135: 3,795 to 3,801). It was therefore of interest to compare the effect of anti-Bp50 mAb in the proliferation of untreated B cells or B cells activated with either anti-Bp35 or anti-u antibodies 5 (Table I). Anti-Bp35 1 solution or anti-u antibodies attached to Sepharose beads, alone under appropriate conditions, could stimulate any B cell proliferation (Table I, line 1). In contrast, anti-Bp50 antibodies alone did not stimulate proliferation (Table I, line 2). However, anti-Bp50 mAb significantly proliferated when cultured with anti-u beads or with anti-Bp35. In this regard, anti-Bp50 was similar to BCGF (Table I, line 3). It was therefore important to determine whether anti-Bp50 and BCGF could together induce B-cell proliferation. As shown in Table I, line 4, anti-Bp50 and BCGF together did not induce any proliferation, but increased proliferation of either anti-15u or anti-Bp35 activated cells somewhat more than each stimulant alone.

BCGF, over a three-log interval, when used with anti-Bp50 without other signals, had no effect on the proliferation of dense B cells, even when anti-Bp50 was used at 1 doses ranging from 0.1 to 10 pg / ml.

TABLE I

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

Mean proliferation ± standard S.A. of B-cell cultures with: 25

Line Co-Stimulant Medium_Anti-u Beads Anti-bp35_ 1 None 1,212 + 547 10.2191462 5.5391308 2 anti-Bp50 7191718 38.79211,329 25.4651616 3 BCGF 4561217 14.2171445 9.4431343 30 4 anti-Bp50 _ + BCGF 1.456 + 126 54.47

Proliferation of dense Er-tonsillar B cells (95% surface IgM + cells) was measured on day 3, as described. Briefly, 2 X 10 6 cells / 200 μl well were cultured in quadroplicate for 48 hours with RPMI 1640 medium containing 35 15% fetal bovine serum plus additives without antibody or with either 2C3 u-chain monoclonal antibody coupled to Sepharose beads ("anti-u beads", 50g / ml) or free 1F5 anti-Bp35 antibody (5 / tg / ml). Cultures containing medium, "anti-beads" or anti-Bp35 were cultured alone or with BCGF (5% final concentrate, Cytokine Technology, Buffalo, New York; have no detectable IL-1 or IL-2 activity) ) with anti-Bp50 (1: 1000 dilution of ascites) as co-stimulants. After 40 hours, 1m-3 pulse cells were treated with H-thymidine and incorporated counts were measured after 18 hours.

3.1 ANTI-Bp-50 mAb INCREASES ONLY PROLIFERATION AFTER B-CELLS ARE ACTIVATED WITH ANTI-Bp35 OR ANTI-U ANTIBODIES

The results in Table I suggest that the anti-Bp50 mAb could not in itself induce proliferation. As shown in Figure 4, doses of 3 anti-Bp50, ranging from 0.05 µg to 2.0 µg / ml had no effect on H-thymldine uptake. However, in the presence of optimal amounts of anti-Bp35 mAb, as little as 0.1 to 0.5 µg / ml of anti-Bp50 antibodies significantly increased proliferation. As much as 50,000 to 70,000 cpm was detectable at the optimum time of proliferation when highly purified B cells were cultured alone with anti-Bp35 plus anti-Bp50. A consistent observation was that higher doses of anti-Bp50 (greater than 2 to 5 µg / ml) were less effective than doses in the range 100 to 200 ng.

These results suggest that anti-Bp50 can only function after B cells are activated by other signals. Results shown in Figure 5 suggest that this is actually the case. If B cells were first activated with anti-Bp35, anti-Bp50 could be added as late as 24 to 48 hours later and still increase proliferation on day 4. In contrast, anti-Bp35 was when cells were first treated with anti-Bp35. -Bp50, effective only, if added within a few hours of the commencement of cultures. Similar results were found when using anti-u instead of anti-Bp35.

3.2 ANTI-Bp50 mAb DOES NOT ACTIVATE B CELLS OUT OF GQ IT INDUCES

30 ACTIVATED B CELLS TO ENTER CELL CYCLE

It has been previously found that, like low doses of anti-u antibodies, anti-Bp35 induces resting tonsillar B cells 1 GQ to grow (Clark, et al., 1986, Leukocyte Typing II eds., Reinherz, et al. al., Springer Verlag, Berlin, Vol. 2, 455-462) and to enter the Gj phase 35 of cell cycle (Gollay, et al., 1985, J. Immunol. 135: 3795-3801). It was therefore of interest to compare the ability of anti-Bp50 mAb with anti-Bp35 mAb for their effects on B cell activation. As shown in Figure 6A, unstimulated dense tonsillar B cells, even after 3 to 4 days, had a uniform RNA profile characteristic of cells in GQ {Darzynkiewicz, 1980, Proc. Night. Acad. Sci. USA 77: 6697-6702). Ca. However, 15-30% of cells stimulated with anti-Bp35 or anti-u had increased RNA content indicating entry into Gp In contrast, neither anti-Bp50 (Fig. 6B) nor BCGF (Fig. 6C) alone induced signi -ficant number of B cells to enter Gp For example, 2 days after activation, anti-Bp35 and anti-Ig mAb induced 13.5% and 20.9% tonsillar cells to enter Gp, respectively, whereas cells treated alone with anti-Bp50, (2.7%) or BCGF (3.2%) remained at medium control levels (2.2%). However, when neither anti-Bp50 nor BCGF was added together with anti-Bp35 or anti-u antibodies, the amount of cells entering Gp increased dramatically. Similarly, anti-Bp50 and BCGF alone did not induce B cells to enter S-phase (Table II), but together with either anti-Bp35 or anti-u, the number of S-phase 15 cells grows two- or three-fold.

TABLE II

The effect of anti-Bp50 and BCGF on cell cycle progression in ton- $ in Army lymphocytes 20 __

Competence-throughput Gq% cells S / G ^ / M

signal signal Gj medium none 89.9 7.1 2.5 anti-Bp35 none 80.4 14.5 3.7 anti-Ig none 65.6 27.6 5.7 medium anti-Bp50 83.6 12, 0 3.3 anti1-Bp35 anti-Bp50 54.1 35.5 9.7 30 anti-Ig anti-Bp50 43.6 36.2 16.2 medium BCGF 85.4 11.7 2.2 anti-Bp35 BCGF 56.6 32.6 11.6 Anti-ία BCGF 48.4 36.1 14.1 35 Percent cells in GQ, Gj or S and G2 determined by the acridine-orange stain method (Darzynkiewicz, et al., 1980 Proc. Nat. Acad. Sci. USA 77: 6697-6702); 1 x 10 6 dense tonsillar lymphocytes with anti-Bp35 (5 µg / ml), anti-u on beads (50 µg / ml), anti-Bp50 (0.4 µg / ml), BCGF (5%) 20 DK 173940 B1 or combinations, as shown.

3.3 OPTIMUM CONDITIONS FOR INCREASING B-CELL PROLIFERATION DOWN ANTI-Bp50 ANTIBODIES

By itself, anti-Bp50 antibodies have little or no detectable effect on dense dormant B cells (Table III). However, in the presence of agents capable of activating B cells such as anti-Ig, anti-Bp35 and TPA, anti-Bp50 mAb significantly increased proliferation. Anti-Bp50 did not co-stimulate with several interleukins, including purified IL-1, recombinant IL-2, and BCGF (low). A comparison of the effect of ant1-Bp50 with the effect of BCGF (low) showed that the drugs co-stimulatory with anti-Bp50 were also co-stimulatory with BCGF (low) (Table III). Of particular interest was that BCGF and anti-Bp50 together were still not co-stimulatory for resting cells.

TABLE III

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

Mean proliferation ± standard S.A. of B cell 20 were cultured with:

Anti-Bp50 BCGF

few.·; stimulant_tedlum_ (ZOjL.nq / mi) _ (5¾_ none 96 ± 1 267 ± 15 285 ± 74 25 anti-Ig 5.833 ± 391 41.634 ± 2,103 25.094 ± 61 anti-Bp35 (5 µg / ml) 457 ± 45 8.143 ± 280 1.733 ± 32 TPA (2 ng / ml) 7.361 ± 871 21.163 ± 871 13.064 ± 1.030 IL-1 (10 U / ml) 264 ± 2 308 ± 23 221 ± 8 30 IL-2 (100 U / ml) 204 ± 34 350 ± 7 220 ± 11 BCGF 15%) 220 ± 7 851 ± 28 270 ± 18 Dense Ertonsillary B cells (greater than 95¾ sIgM + cells) were cultured for 48 hours at 2 X 10 cells / well followed by 24 hours of Pulse Therapy. 3 ling with H-thymidine before counting.

35 _

The kinetics of increased proliferation with anti-Bp50 are shown in Figure I

7. The proliferation peak was reached on day 4, after which it dropped J

whether or not cells were activated with anti-Bp35 or other activators, such as anti-Ig or TPA. The kinetics of increased proliferation with BCGF or Bp50 were the same.

As little as 0.05 µg of anti-Bp50 antibodies increased proliferation. An optimal dose of 0.3 μς / ηιΐ was used in subsequent studies.

A consistent observation was that higher doses of anti-Bp50 (greater than 2-5 µg / ml) were less effective than doses of 0.1-0.5 Mg / ml when whole antibody molecules were used.

Human B cells are highly sensitive to the human effects mediated by Fc receptors that bind to surface Ig 10 (Parker, 1980, Immunol. Rev. 52: 115; Bjjesterbosch, et al., 1985, J. Exp.

Hot. 162: 18-25). Thus, it was important to compare the efficacy of the entire anti-Bp50 mAb with the anti-Bp50 Fiab ™ fragments. Over a hundred-fold dosing interval, the F (ab ') 2 ~ fragments were clearly as effective as, if not more effective than, whole antibodies for enhancing B cell proliferation (Table 1111). Thus, the Fc domain of anti-Bp50 mAb is not required for anti-Bp50 to exert its effect and, if anything, may have inhibitory properties. In other words, anti-Bp50, like BCGF, may appear to act as a soluble intermediate without the aid of FC receptor mediated access cell function.

20 22 DK 173940 B1

TABLE IIII

The Fc domain of anti-Bp50 antibodies 5 is not required to enhance B cell proliferation

Medium proliferation of B cells grown with:

Dosage

Anti -Bd50_ (gg / ml) _Medium_Anti-Bp35

None - 295 ± 16 269 ± 27

Whole Ab 0.125 278 ± 32 5.140 ± 20 15 1.25 275 ± 24 4.686 ± 342 12.5 163 ± 15 3.852 ± 203 F (ab ') 2 0.125 594 ± 21 10.635 ± 449 20 1.25 531 ± 3 10.893 ± 575 12.5 279 ± 8 9.411 ± 870

Cell culture conditions were as described in Table III.

3.4 DIFFERENCES BETWEEN ANTI-Bp50 and BCGF (Low) Activity Anti-Bp50 and BCGF (low) had a similar effect on B cells and were costimulatory with the same substances (Table III). However, several orders indicate that anti-Bp50 and the BCGF 30 used in this study appear to act through different signals. First, in contrast to BCGF (low) receptors (Bijsterbosch, et al., 1985, J. Exp. Med. 162: 1825), Bp50 molecules are expressed on resting blood B cells (Fig. 3). Second, although both anti-Bp5Q and BCGF (low) function most effectively when added anti-Bp35 or anti-Ig, 35 anti-Bp50 was clearly most effective when added 12 hours after the onset of culture (Figs. 8A). In contrast, BCGF (low) could be added as late as 24 hours after the onset of culture and still be able to optimally increase proliferation (Fig. 8B). These kinetic DK 173940 B1 experiments designed according to the method described by Howard and Paul (1983, Ann. Rev. Immunol. 1: 307) suggest that a Bp50-dependent signal can generally exert its effect before BCGF.

Both anti-Bp50 and BCGF (low) increased proliferation of B cells activated with anti-Bp35 or anti-Ig (Table III). However, the effect of anti-Bp50 and BCGF (low) was additive in many experiments (Fig. 7).

FIG. Figure 9 shows a titration of BCGF (low) in an experiment using anti-Bp50 at its optimum concentration (0.2 Mg / ml). BCGF (low) could further increase proliferation of quiescent B cells in the presence of 10 anti-Bp50 after activation with either anti-Ig or with anti-Bp35. Optimal concentrations of BCGF (low) were 5-10% while 25% was inhibitory. Therefore, when anti-Bp50 and BCGF (low) were both used at their optimum concentrations, they continued to have additive effects on B-cell proliferation. Finally, both normal and malignant B cell subgroups differed in their responses to anti-Bp50 and BCGF (low). For example, some blood B cells responded to BCGF (low) but did not respond to anti-Bp50 (Table IV). An additional activation signal such as anti-Bp35 (Table IV) or TPA (Fig. 10) was constantly needed for blood B cells to respond to anti-Bp50. While dense tonsillar B cells generally did not respond to either BCGF or anti-Bp50, liquid B cells responded (Table IV). B-cell malignancies also differed in their responsiveness to anti-Bp50 relative to BCGF. For example, some B-cell lymphomas responded to TPA plus BCGF (low) but not to TPA plus G28-5 anti-Bp50 (Figs. 10B and D). In contrast, dense 25 tonsillar B cells and peripheral blood B cells responded to TPA plus either BCGF (low) or anti-Bp50 (Figs. 10A and C).

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The ligands of the present invention can be used in vivo or in vitro, in their unmodified or modified forms, to modulate immunoreactions. For example, the ligands themselves may be used as an "adjuvant" to enhance an immunoreaction of a vaccine or to enhance the immunoreaction of an immunosuppressive individual. Alternatively, these modified ligands, if cytotoxins or anti-proliferative agents are coupled to the ligands, can be used to decrease an immunoreaction, e.g. in the case of an autoimmune disease 10 or in transplant patients to avoid graft rejection. These modified ligands may also be used to treat malignancies which comprise cells or tumors expressing the Bp50 antigen, whether or not the malignancy is originally a B cell.

Both the ligands of the present invention and / or Bp50 themselves can be used in vitro. Such applications include in vitro studies such as immunoassays to detect cells expressing the Bp50 antigen and / or to detect possibly shed Bp50 antigen in body fluids. In this case, the ligand or Bp50 can be labeled with a radiolabelling, fluorine, enzyme, enzyme substrate, dye, etc. Also, the ligands can be used for separation and / or identification of cells expressing the Bp5G antigen, in which case the ligand can be linked immobile carrier or to a fluorine substance that can be used in a FACS (fluorescence-activated cell sorting apparatus).

The various uses of the ligands and Bp50 of the present invention are discussed in more detail below.

4.1 Bp50 RECEPTOR AND APPLICATIONS OF LIGANDS SUCH AS ANTI-Bp50 TO INCREASE B CELL PROLIFERATION

Previous studies have suggested that the factors involved in the induction of B cells from the Gq to Gj phase 1 cell cycle are 30 different from the factors or requirements that cause S-phase transmission. This model is primarily based on studies showing that agents such as low doses of ant1-Ig B-cell activation factors, or anti-Bp35 alone, have little or no effect on B-cell proliferation. Yet, the same agents can drive B cells to a point of cell activation where they are susceptible to growth factors. In contrast, growth factors such as BCGF or IL-2 alone have no effect on dormant B cells but enhance growth of activated B cells.

26 DK 173940 B1

Although the present invention should not be limited to any particular theory or explanation, the results presented herein provide further basis for a model for distinct regulation of B-cell activation and growth steps. Here, it has been demonstrated that activation and proliferation signals in human B cells can be transmitted through distinct cell surface structures. Although anti-Bp35 mAb activated B cells to enter the Gc phase of the cell cycle alone, it induced little or no proliferation. Anti-Bp50 mAb had the opposite effect in that it was unable to activate B cells, but by adding so late at 12-24 hours after 10 activation it could induce B cell growth.

The Bp50 molecule would probably normally function as either a receptor for a ligand such as a soluble growth factor or for a signal mediated through cell-cell contact (i.e., a ligand found on the surface of another cell). Previous studies have identified several 15 different T cell-derived BCGFs that, like anti-Bp50, enhance B cell proliferation. Both high and low molecular weight forms of B cell growth factors have been identified and various types have been shown to have additive effects (Kehrl, et al., 1984, Immunol. Rev. 18: 75-96; Kishlmoto, 1985, Ann. Rev. Immunol. 3: 133-157; Swain, et al. 1983, 20 J. Exp. Med. 158: 822-835; Howard et al., 1984, Immunol. Rev. 78: 185-210; Ambrus, et al. , J. Cl in Invest. 75: 732-739; Ambrus, 1985, J. Exp. Med. 162: 1319-1335). Bp50 could therefore be a receptor for one of these factors.

With the exception of IL-2 receptors and the C3d receptor, receptors 25 on B cells for growth signals have not yet been identified. mAb-AB-1 reacts with a B-cell marker that is expressed only on activated B cells and blocks BCGF-dependent proliferation, and could therefore possibly recognize the BCGF receptor or a related structure. Bp50 appears to be different from the AB-1 marker, since the AB-1 mAb does not block the binding of the G28-5 anti-Bp50 antibody and, in contrast to the G28-5 mAb, only reacts with activated B cells ( Jung, et al., 1984, J. Exp. Med. 160: 1919-1924). Bp50 is found on all B cells, which, based on absorption analysis and direct binding studies, does not appear to be the case for BCGF receptors. The available data indicates that Bp50 35 and the low molecular weight receptor BCGF are different structures. Using a rabbit heteroantiserum, Wang and co-workers (Wang, 1979, J. Exp. Med. 149: 1424-1433) previously described a 54-kDa glyco protein, gp54, which, like Bp50, is expressed on all B cells, but at 27 B 17 lower levels of blood B cells than tonsillar B cells. It is possible but unlikely that the rabbit heteroantiserum and anti-Bp50 recognize the same or related structures, but unlike the anti-Bp50 mAb, the rabbit antiserum against gp54 alone was sufficient to stimulate B-5 cell proliferation.

Anti-Bp35 alone, in contrast to anti-Bp50, can activate B cells from Gq to Gj and thus can be referred to as an inactivation signal. Whether Bp35 functions alone in early B cell activation has not yet been elucidated, since Bp35 antibodies can stimulate some B cells for 10 division (Clark et al., 1985, Proc. Nast. Acad. Sci. USA 82: 1766-1770). Similarly, Bp50 cannot properly function alone as a "growth" signal. , in that anti-Bp50 antibodies together with activation signals (anti-Bp35 or anti-u) not only increase proliferation, but also increase the total number of B cells entering Gj (Table II). anti-Bp50 as a stimulant to promote the progression of both activation (Gq to Gj) and growth (G to S) phases of the cell cycle. The BCGF used in these studies also had similar activity (Fig. 6C). and anti-Bp50 (or BCGF) therefore appears to be most analogous to "competence" and "progression" factors described in studies 20 regarding fibroblast growth regulation. How B cells respond to anti-Bp35 or anti-Bp50 may clearly depend on their differentiation or activation state.

It has been demonstrated here that two mAbs, anti-Bp35 (a "competence" signal) and anti-Bp50 (a "progression" signal) together can induce substantial proliferation of highly purified B cells in the absence of antigen or other known factors. The natural ligands for these structures are not yet known. However, since mAb against appropriate epitopes may resemble both soluble factors and signals mediated by cell-cell interactions, it may be possible to use appropriate combinations of mAb to control and regulate human B-cell proliferation or differentiation. This, in turn, will help to determine in vivo strategies to combat human diseases such as B-cell malignancies, immunodeficiency diseases and certain autoimmune diseases.

The novel monoclonal antibody, G-28-5, which reacts with a single chain polypeptide of ca. 50 Kd expressed on the surface of human B cells is only a particular embodiment of the ligands of the present invention which can enhance the proliferation of activated B cells. Since human B cell proliferation can be similarly increased by T cell derived BCGFs, including low and high molecular weight BCGF, the activity of anti-Bp50 G28-5 was compared with the activity of a BCGF preparation containing predominantly low molecular weight -BCGF. Anti-Bp50 G28-5 and BCGF (low) were very similar in that they were costimulatory with the same activating agents (anti-Ig, anti-Bp35 and TPA) but were not costimulatory with each other or with IL-1 or IL-1. 2nd Furthermore, the activity of anti-Bp50 G28-5 was not dependent on the Fc domain, with F (ab ') 2 fragments of G28-5 being functionally active. This suggests that soluble anti-Bp50, like soluble BCGF, does not require Fc receptor-bearing accessory cells to exert an effect. Furthermore, both anti-Bp50 and BCGF are effective alone in the presence of an activation stimulus. In other words, anti-Bp50 and BCGF are not "competence" factors, but rather they promote the "progression" of B cells through the cell cycle.

Although it is possible that Bp50 may act as a receptor for a ligand such as a B-cell growth factor, several results suggest that Bp50 is not the receptor at least for the BCGF (low) used in this study, as it is expressed on blood B cells, whereas BCGF (low) receptors do not. Possible structures of the BCGF (low) receptor, in contrast to Bp50, are also expressed only on activated B cells.

Furthermore, both normal and malignant B cell populations differ in their response to anti-Bp50 relative to BCGF (low) (Table IV and Fig. 10).

For example, B lymphomas propagate in response to BCGF (low) but not in response to anti-Bp50. Finally, in a series of experiments, optimal concentrations of anti-Bp50 and BCGF together induced more proliferation than each of them alone. Anti-Bp50 is similar to the activity of other BCGF such as BCGF (high) that is costimulatory with anti-IgM (Ambrus, et al., 1985, J. Exp. Med. 162: 1319; Ambrus, et al., 1985, J. Cl 1n Invest 75: 732). This suggests that Bp50 could act as the receptor for BCGF (high).

Alternatively, although Bp50 may be a receptor for a soluble ligand, Bp50 may act as a receptor for a cell-to-cell-mediated signal regulating BCGF receptor levels and / or autoerin production. The preference for differentiation antigens that serve as enhancers of an autocrine receptor pathway comes from studies with T cells. MAb against Lp220 general leukocyte antigen enhances proliferation by elevating IL-2 receptor expression on activated T cells (Ledbetter, et al., 1985, J. Immunol. 1935: 1819). An analogous mechanism may act with anti-Bp50 and expression of certain BCGF receptors. Bp50 and BCGF (low) are apparently under a coordinate control, as BCGF, like IL-1 and IL-2 receptors, increases expression of Bp50 on certain leukemia cells. The Bp50 molecule also bears similarities to the Tp44 molecule that acts by influencing IL-2 production. The present inventors and others have demonstrated that the 9.3 anti-5 Tp44 antibody enhances 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 enhances the proliferation of B cells activated with anti-Bp35 or TPA. The Tp44 signal acts by stimulating IL-2 production rather than by stimulating T cell growth. The Bp50 signal may probably act in an analogous manner by stimulating B-cell autocrine production (Gordon, et al., 1984, Nature, Lond. 310: 145).

4.2 MODIFIED LIGANDS USED FOR IMMUNOSUPPRESSION OR TREATMENT OF ORAL DISEASES

According to this embodiment, the ligand of the present invention can be modified by attaching an antiproliferative agent so that the resulting molecule can be used to kill cells expressing the Bp50 antigen. Such modified ligands can be used to treat autoimmune diseases to suppress the proliferation of 20 B cells and thereby suppress the autoimmune reaction. These modified ligands can also be used to immunosuppress a transplant patient to prevent rejection of a graft. Cytotoxic agents used to suppress immunoreactions can therefore be attached to the ligands of the invention. When ligands are used that enhance the proliferation of B cells, an enhanced effect should be obtained because the drug will target proliferating B cells.

In another embodiment, the ligands of the present invention, modified by attachment of an antiproliferative agent, can be used to treat malignant diseases in which tumors or cells express the Bp50 antigen. Attachment of these chemotherapeutic agents to the ligands of the invention should result in greater specificity of the drug against the malignant cells. Furthermore, a particular advantage should be obtained in treating a B-cell malignancy with a ligand coupled to a cytotoxin which is more effective in killing proliferating cells than non-proliferating cells, treatment with such a ligand should result in an enhancement of the action of the cytotoxin.

The chemotherapeutic agents or antiproliferative agents which can be coupled to the ligands of the present invention therefore comprise, but are not limited to, the agents listed in Table VI below, which are derived from Goodman and Gilman, The Pharmacological Basis of Therapeutics, 6th edition, MacMillan Publishing Co., Inc., New York, pp. 1249-1313, 1980, which is hereby incorporated into the present specification.

TABLE VI

Chemotherapeutic agents that can be coupled to anti-Bp50 ligands

Classes By means of 15 Alkylating agent Nitrogen mustard Mechlorethamine

Cyclophosphamide Melphalan Uracil Mustard Chlorambucil 20

Ethyleneimine- Thiotepa derivatives

Alkylsulfonater Busul of 25

Nitrosourea types Carmustin

Lomustln

semustine

Streptozocin 30

Triazener Dacarbazine

Antimetabolites Folic Acid Analog Methotrexate 35 Pyrimidine Analog Fluoruracil

cytarabine

Azaribin 31 DK 173940 B1

Purine analogue of Mercaptopurin

thioguanine

Natural products Vinca alkaloids Vinblastin 5 Vineristin

Antibiotics Dactinomycin

daunorubicin

Doxorubicin Bleomycin

mithramycin

mitomycin

Enzymes L-Asparaginase 15

Various agents Platinum-coordinated Cisplatin complexes

Substituted urea Hydroxyurea 20

Methyl hydrazine-Procarbazine derivative

Adrenocortical Mitotane 25 Suppressant

Hormones and Adrenocortico-Prednisone Antagonists Steroids 30 Progestins Hydroxyprogesterone Caproate

Medroprogesterone acetate

Megestrol acetate 35 Estrogens Diethylstilbestrol

Ethinyl Estradiol 32 DK 173940 B1

Anti estrogen Tamoxifen

Androgens Testosterone Propionate Fluoxymesterone

Radioactive Phosphorus Sodium Phosphate

isotopes 32P

10 solids Sodium solder 131 j

Any known method can be used to link the ligand to the chemotherapeutic or antiproliferative agent. Examples of such methods have been reviewed in advance (see above).

4.3 OTHER APPLICATIONS OF LIGANDS AND Bp50

In addition to the therapeutic applications, the ligands and Bp50 itself have other uses in both in vitro and in vivo diagnostic tests, 20 separation schemes, etc.

The Bp50 receptor can be used to prepare and / or form the ligands of the invention. Bp50 can also be used with the ligands of the invention in in vitro studies requiring a standard to quantify the amount of detectable Bp50 in a sample. Ultimately, 25 Bp50 itself can be useful as a soluble factor that mediates immunity, e.g. and lymphokine.

In addition to therapeutic treatment and diagnostic studies, the ligands of the present invention should be used to identify or separate cells expressing the Bp50 antigen. Furthermore, if an appropriate radiolabelling or radiopaque compound is bound to the ligand, it should be used for in vivo detection of tumors expressing the Bp50 antigen. Other uses should be apparent to those skilled in the art from the foregoing description.

DEPOSIT OF CELL LINES

35 The following hybridoma has been deposited with the American Type Cul ture Collection, Rockville, MD, and has been assigned the following accession number: 33 DK 173940 B1

Hybrid ATCC Accession Number G28-5 HB9110

The present invention should not be limited to the extent of the deposited hybridoma, since the deposited embodiment is intended as a single illustration of one aspect of the invention and any cell line which is functionally equivalent thereto falls within the scope of the invention. Of course, various modifications of the invention in addition to those shown and in the foregoing will be apparent to those skilled in the art from the present description and the appended drawings. Such modifications should be considered to fall within the scope of the invention.

Claims (16)

1. Essentially Pure Ligand, CHARACTERIZED IN Binding to Bp50, a 50 kilodalton B cell surface antigen defined by monoclonal antibody G28-5 produced by the hybridoma cell line HB9110 deposited with ATCC, or a mutant, a recombinant or a generic prepared derivative thereof, which ligand is selected from the group consisting of an antibody, or fragment thereof, and a lymphokine. A ligand according to claim 1, characterized in that, upon binding to a 10 activated B cell, it stimulates the activated B cell to undergo cell cycle, thus increasing proliferation of the B cell. A ligand according to claim 1 or 2, characterized in that it comprises an antibody molecule or a Fv, Fab, F (ab ') ^ or Fab' portion of the antibody molecule. The ligand according to claim 3, characterized in that the antibody molecule comprises a monoclonal antibody molecule or a Fv, Fab, Ftab 'or Fab' portion of the monoclonal antibody molecule. The ligand of claims 1 to 2, characterized in that it comprises a lymphokine. The ligand of claim 5, characterized in that the lymphokine comprises a B-cell growth factor. The ligand of claim 5, characterized in that the lymphokine is on the surface of a cell. Ligand according to claims 1 to 3 or 5, characterized in that it further comprises a compound linked to the ligand, which compound is preferably an antiproliterative agent, in particular an alkylating agent, an antimetabolite, an antibiotic, a vinca alkaloid, an enzyme. , a platinum-coordinated complex, a radioisotope, or a fluorescent compound. The ligand of claim 4, characterized in that the antibody molecule comprises G28-5 or any Fv, Fab, F (ab ') 2 or Fab' portion thereof. A ligand according to any one of the preceding claims for medical use. A ligand according to any one of the preceding claims for use in a method of increasing the proliferation of B cells. The ligand according to claims 1 to 10 for use in a method DK 173940 B1 for suppressing the proliferation of cells expressing Bp50. Use of a ligand according to claims 1 to 10 in the manufacture of a medicament for enhancing the proliferation of B cells. Use of a ligand according to claims 1 to 10 in the preparation 5 of a drug for suppressing the proliferation of cells expressing Bp50. Essentially pure 50 kilodalton B cell surface antigen (Bp50), CHARACTERIZED in that it is defined by monoclonal antibody G28-5 produced by hybridoma cell 1 or in HB9110 deposited with ATCC, 10 or a mutant, a recombinant or a generic derivative thereof. Essentially pure 50 kilodalton antigen according to claim 15, characterized in that it comprises a polypeptide. 15