SE504675C2 - Ligands to Increase B-Cell Proliferation - Google Patents

Abstract

A new B-cell receptor, Bp50, a 50 kilodalton polypeptide, that functions in B-cell proliferation is described. Ligands such as lymphokines, antibody molecules or the Fv fragments of antibody molecules that bind to Bp50 and augment the proliferation of activated B-cells can be used to regulate B-cell proliferation or differentiation.

Description

504 675 2 10 15 20 25 30 35 they are obviously derived from certain B cell growth factors. For example, monoclonal antibodies (mAbs) block against a murine BCGF (O Hara et al., 1985, Nature (LondJ 315: 333) or against human BCGF (Ambrus et al., 1985, J. Exp. Med. 162: 139) BCGF activity but not IL-1 or IL-2 activity Although B cell growth factors differ from IL-1 or IL-2, they themselves appear to be heterogeneous based on biochemical data and differential activity in different B-cell subsets or For example, 60 kilodaltons (kDa) of high molecular weight human BCGF have been identified, BCGF (high), which differs from a 12-kDa low molecular weight form, BCGF (low) (Ambrus et al., 1985, J Clin. Invest. 75: 732) The cDNA encoding a 20-kDa murine BCGF, tentatively termed 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 activity but can also activate dormant B cells and induce differentiation of IgGI-producing cells; it differs from human BCGF (high) and BCGF (low) both in terms of molecular weight and its activity range.

These activation and growth signals probably regulate cells by interacting with specific B-cell surface structures. In addition to the antigen-specific signal through surface Ig, your other B-cell surface polypeptide candidates have been identified which may in some way exert activity upon activation or growth of B-cells. For example, cell surface receptors for IL-1 (Dower et al., 1985, J. Exp.

With. 160: 510) and IL-2 (Robb et al., 1984, J. Exp. Med. 160: 1126) and recently functional IL-2 receptors have been identified on B cells (Zubler et al., 1984, J Exp. Med. 16021170; Jung et al., 1984, J. Exp. Med. 160: 1597; Muraguchi et al., 1985, J. Exp. Med. 161: 181). Receptors for B cell growth and activation factors still need to be fully characterized. Several B cell surface polypeptide candidates have been identified which may in some way exert activity upon activation or growth of B cells. For example, Subbarao and Mosier (Subbarao et al., 1983, Immunol. Rev. 69: 81-97) found that monoclonal antibodies (mAbs) to the murine B cell antigen Lyb2 activate B cells and recently evidence has emerged that Lyb2 may be the receptor for BSF-pl (Yakura, 1985, Fed. Proc. 4411532). We have also found that an appropriate mAb (1F5) against a 35 kDa polypeptide, Bp35, activates human B cells from G0 to G1 (Clark et al., 1985, Proc. Nat. Acad. Sci. USA 8221766-1770 ; Gollay et al., 1985, J. Immunol. 135: 37 95-3801). Aggregated C3d or antibodies to the 140 kDa C3d receptor.

Bp140, causes proliferation of B cells that are T cell dependent (Melchers et al., 1985, Nature 317: 264-267; Nemerow et al., 1985, J. Immunol. 1353068-3073; 30 35 pp. 504 675 Frade et al., 1985, Eur. J. Immunol. 15: 73-76). Although B cell growth factors have been identified in both mice and humans, the receptors for these factors have not been isolated to date. Wang et al. (Wang et al., 1979, J. Exp. Med. 149: 1424-1433) prepared a polyclonal antiserum, which identified a 54-kDa polypeptide (gp54) on human B cells and showed rabbit antisera against gp54 in induced tonsil B cells for division. Recently, Jung and Fu (Jung et al., 1984, J. Exp. Med. 160: 1919-1924) isolated mAb (AB-1) against a 55-kDa antigen, which was restricted to activated B cells and blocks BCGF -dependent proliferation.

However, whether either anti-gp54 or AB-1 recognizes a BCGF receptor is not yet known. Summary of the Invention The present invention relates to a ligand which is characterized in that it (a) binds to Bp50, a 50 kilodalton B-cell surface antigen, and is defined by the monoclonal antibody G28-5; (b) upon binding to an activated B cell, stimulates it to undergo the cell cycle so as to increase the proliferation of the B cell; and (c) upon binding to dormant B cells, in the absence of other factors, does not stimulate proliferation, the ligand being a monoclonal antibody molecule or an Fv, Fab, F (ab ') 2 or Fab' fragment of the monoclonal antibody molecule containing the antigen combining site that binds to the Bp50 receptor, prepared by a hybridoma cell line, which has been deposited with the ATCC under the deposit number HB9110.

The ligands of the present invention may be chemically modified, for example, by linking or coupling a compound to the ligand. Such compounds include, but are not limited to, cytotoxic agents, therapeutic agents, chemotherapeutic agents, markers such as radiolabelers, dyes, enzymes, radiopaque compounds, and the like. The ligands of the present invention can be used in 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 the cell antigen Bp50, apparently plays crucial roles as signal receptors in B cell activation.

Monoclonal antibodies (mAbs) to Bp35 and Bp50 both deliver positive signals to B cells, which stimulate their passage through the cell cycle. MAb to bp35, like anti-Ig antibodies, essentially exerts an effect on activating dormant B cells to become competent to enter the G1 phase of the cell cycle. In contrast, a monoclonal antibody described herein or its F (ab ') 2 fragment against Bp50, a 50-kDa polypeptide expressed on all B cells, exerts an action to stimulate activated B cells. cells to undergo the cell cycle and increase the proliferation of activated B cells. Monoclonal antibodies to Bp35, like anti-Ig antibodies, activate tonsil B cells and induce low levels of B cell proliferation. In contrast, anti-Bp50 monoclonal antibody neither separately activates B cells nor induces B cells to proliferate but, together with anti-Bp35 or anti-Ig antibodies, increases B cell proliferation. In this respect, the action of the anti-Bp50 antibody is similar to the activity of B cell growth factors (BCGF).

As little as 0.05 pg / ml of anti-Bp5O is required to increase proliferation and, like BCGF, anti-Bp5O is effective even when added 12-24 hours after B cells have been activated with anti-Ig or anti-Bp5O. -Bp35. Without further exogenous signals, anti-Bp35 and anti-BpSO antibodies together induce strong proliferation of purified resting B cells. These results show that Bp35 and Bp50 surface molecules exert an effect on regulating B-cell activation and progression through the cell cycle. Due to the importance of anti-Bp35 and similar molecules in the effect and effect of the ligands of the present invention, Clark et al., 1985, Proc. Natl. Acad. Sci. (USA) 82: 1766-1770) by reference thereto.

Although the activity of anti-Bp5O is similar to the activity of BCGF (low) because both anti-Bp5O and BCGF (low) are co-stimulating with the same agent but not with each other and both anti-Bp5O and BCGF (low) only affect activated B cells and work in a soluble form, the activity of anti-Bp5O can be distinguished from the activity of BCGF (low) because the proliferation of B cells, which have been stimulated with optimal amounts of anti-Bp5O and anti-Bp35 (or anti-Ig), can be further increased with BCGF (low) and both blood B cells and some B cell lymphomas respond differently to anti-Bp5O versus BCGF. For optimal activity, anti-Bp5O should be added within 12 hours after B cell activation, while BCGF (low) maintains optimal activity even with addition 24 hours after activation. Furthermore, Bp5O is expressed on all B cells while receptors or BCGF (low) are limited to activated B cells. Thus, anti-Bp5O and BCGF (low) interact can regulate B cell growth but apparently do so via different signals.

According to one embodiment of the present invention, ligands that bind to Bp 50 and increase the proliferation of activated B cells can be used to enhance an immune response. For example, these ligands, which bind to Bp50, can be used as "adjuvants" to increase an immune response to a vaccine. Alternatively, these ligands can be used to increase the immune response of an individual with immunosuppression.

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 method could result in suppression of the immune response. For example, a cytotoxic drug coupled to a ligand of the present invention may be used in vivo to induce immunosuppression in order to cross histocompatibility barriers in transplant patients; alternatively, these modified ligands can be used to control autoimmune diseases.

According to another embodiment of the present invention, malignant cells, such as tumor cells, which express Bp50, can be treated using a ligand of the invention coupled to a chemotherapeutic agent useful for the treatment of such a neoplastic disease. These modified ligands can be used in vivo to direct the chemotherapeutic agent against any type of malignant cell that expresses the Bp50 antigen, including cells that are not B cells but that express Bp50. When using the ligands of the invention, which increase B cell proliferation, a particular advantage can be obtained in the treatment of malignant B cell conditions, where the chemotherapeutic agent coupled to the ligand includes one which is more effective in killing proliferative cells; in this case, it should be possible to achieve a potentiation of the drug effect.

Alternatively, the ligands of the invention may be used in vitro to identify or separate cells expressing the Bp 50 antigen, and / or to analyze body fluids for the presence of the Bp 50 antigen, which body fluid may have been drained. Furthermore, the ligands of the invention can be used. vivo for the purpose of imaging cells or tumors expressing the Bp50 antigen.

The purified Bp50 antigen can be used for the production of antibodies and for the production or construction of other ligands of the invention.

Furthermore, the Bp50 antigen can be used in assays such as diagnostic immune assays. In addition, Bp5O can itself be used as a mediator of cell immunity in viuo or in vitro. 3.1. Definitions The abbreviations used in the present context have the following meanings: 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 specific B cell surface polypeptide (CDZO) defined by mAb lF5 Bp50 = a 50 kDa specific B cell surface polypeptide defined by mAb G28-5 Fv = the variable region or antigen combining site of an antibody molecule. This can be any. preferably fragments containing the idiotype of the molecule including but not limited to Fab F (ab ') 2, Fab' and the like.

IF = immunofluorescence Ig = immunoglobulin IL-1 = interleukin 1 IL-2 = interleukin 2 kDa = kilodaltons mAb = monoclonal antibody SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis TPA = 12-O-tetradecanoylforbol-IB-Figure Fig. 1. Expression of Bp50 is limited to Bp35 in B cells. Cytometric two-color fate analysis of 50,000 cells was performed as described (Clark et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-17 70). Data intended as cell numbers against log of green fluorescence and log of red fl uorescence, where 4-5 points represent an approximately doubling of the fl uorescence. Data are presented to show autofluorescent negative cells. PE (red) -anti-Bp35 (1F5) -colouring versus FITC (green) -anti-Bp50 (G28-5) -colouring shows that all Bp50 + cells are also Bp35 +.

Fig. 2. Biochemical comparison of Bp50 polypeptide with other B cell surface antigens. Immunoprecipitation of Bp50 from 125 I-labeled tonsil surface cells was performed as described. Isolated antigens were electrophoresed on 10% SDS-polya crylarnid gel plates without reduction. The gels were visualized with autoradiography and intensifying screens. Panel A: lane 1, anti-Bp50 (G28-5); lane 2, anti- 10 15 20 25 30 35 7 504 675 Bp95 (G28-8); lane 3, sepharose-get-anti-mouse-Ig only. Exposure time: 4 days.

Panel B: lane 1, anti-Bp5O (G28-5); lane 2, anti-Bp45 (BLAST-2); lane 3, anti-Bp39 (G28-1); lane 4, anti-Bp39 (41-H16); lane 5, sepharose-get-anti-mouse-Ig only. An exposure time of two days was chosen so that the bands in lanes 2-4 were not overexposed and could be easily distinguished from Bp50. One of three attempts.

Fig. 3. Two-color immunofluorescence analysis of Bp50 expression. Peripheral blood cells or mononuclear tonic cells were isolated by centrifugation on Ficoll and stained with PE (red-conjugated G28-5_ (anti-Bp50) antibodies in combination with fluorescein (green) -conjugated reference antibodies comprising 2C3 (anti-IgM); Bp35); HBIOa (anti-DR); and 9.6 (anti-CD2, E-receptor) .Cells were analyzed with a FACS IV equipped with four-decad log amplifier in both red and green dimensions. Forward and right-angled scattered light was used for to stain monocytes.Colored cells are located on the back of the grid, red fluorescence is on the right and green fluorescence is on the left.

Fig. 4. Dose-response curves for increasing the proliferation of dense tonsil Er-B cells with anti-Bp50 antibodies as indicated; medium only; anti-Bp5O only; anti-Bp35 (5 reg / ml only; BCGF only; anti-Bp35 plus BCGF; anti-Bp35 plus graded doses of anti-Bp50. Mean proliferation z standard error in quadruplicate samples was measured on day 3.

Fig. 5. Anti-Bp50 mAbs are extremely effective in increasing proliferation upon addition after a B cell activation signal. Compact tonsil-Er-B cells were incubated for 4 days with medium only, anti-Bp5O (0.5 lig / ml) added at different times after the incubation, anti-Bp35 (5 leg / ml) added at different times points after the incubation; constant-level anti-Bp50 to which anti-Bp35 was later added at various times; anti-Bp35 at a constant level to which anti-Bp5O was added to cultures at different times. During the last 10 hours, 3H-thymidine was added and its incorporation was measured.

Fig. 6. Comparison of the ability of anti-Bp35 and anti-Bp5O to induce dormant tonsil B cells to leave the GO stage in the cell cycle. Day 3 after treatment only medium (), anti-Bp35 (-) only; and only Ig (.....), A, no added additives; B, anti-Bp 50 (0.5 leg / ml) added to each group; C, 5% BCGF added to each group. Data are plotted as relative cell numbers versus logs of AO red fluorescence (RNA).

Fig. 7. The kinetics of B-cell proliferation after stimulation with anti-Bp5O versus BCGF. Compact tonsil II-E-B cells were stimulated with medium alone; 10% BCGF alone; anti-Bp35 only; anti Bp5O only; anti-Bp35 plus 10% BCGF; 504 675 8 5 10 15 20 25 30 35 anti-Bp35 plus anti-BpöO; and anti-Bp35 plus anti-Bp5O plus 10% BCGF. The proliferation was measured on the indicated days by an 18-hour pulse of 3 H-thymidine. Proliferation was measured in quadruple samples and standard errors are shown.

One of three attempts.

Fig. 8. Timing after anti-Bp35 stimulation at optimal anti-Bp50 (A) or BCGF '(B) increase in proliferation. Compact tonsil E-B cells were stimulated as shown and proliferation was measured by an 18-hour pulse of 3 H-thymidine on day 3. Medium; anti-Bp35 added only at specified times; anti-Bp50 or BCGF alone; anti-Bp35 added at the beginning of the culture followed by an addition of anti-Bp5O or BCGF at specified times; anti-Bp5O or BCGF added at the beginning of the culture followed by anti-Bp35. One of two attempts. The proliferation was measured in quadruple samples and the standard errors are shown. Doses used: anti-Bp35, 5 / cg / ml; anti-Bp50, 0.2 μg / ml; BCGF (low) 5%. Concentrations used were as follows: anti-Bp35, 5 ug / ml; anti-BpöO, 0.2 pg / ml; BCGF, 5%.

Fig. 9. Anti-Bp5O and BCGF have additive effects on B-cell proliferation.

Compact tonsil E-B cells were stimulated with graduated doses of BCGF (low) along with anti-Bp50 alone; anti-Bp35 only; anti-Ig beads only; anti-Bp35 plus anti-Bp50; or anti-Bp50 plus anti-Ig. Proliferation was measured on day 3 after stimulation with an 18-hour pulse of αH-thymidine. Proliferation was measured in quadruple samples and standard errors are shown. One of four attempts. The doses utilized 106 cells: anti-Bp35, 5 μg / ml; anti-Bp50, 0.2 ug / ml; anti-Ig beads, 50 μg / ml.

Fig. 10. Comparative effects of anti-Bp5O and BCGF on normal and malignant B cells. Peripheral blood E-B cells (A) or compact tonsil 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 lš-cell lymphomas (panels B and D) were stimulated in the same way. Proliferation was measured on day three by incorporation of 3 H-thymidine during a 12-hour pulse. Proliferation was measured in quadruple samples and standard errors are shown. Detailed Description of the Invention The present invention relates to a ligand, characterized in that it (a) binds to Bp50, a 50 kilodalton B-cell surface antigen, and is defined by the monoclonal antibody G28-5; (b) upon binding to an activated B cell, stimulates it to undergo the cell cycle so as to increase the proliferation of the B cell; and (c) upon binding to dormant B cells, in the absence of other factors, does not stimulate proliferation, wherein the ligand is a monoclonal antibody molecule or an Fv-, Fab-, F (ab ' ) 2 or er Fab fragment of the monoclonal antibody molecule containing the antigen combining site that binds to the Bp50 receptor, prepared by a hybridoma cell line, which has been deposited with the ATCC under the deposit number HB9110.

The ligands of the present invention comprise antibody monoclonal molecules and fragments of these antibody molecules, which contain the antigen-binding site that binds to the Bp50 receptor, including chemically modified antibodies and fragments; such fragments include Fv, Fab, F (ab ') 2 and Fab1. The ligands of the invention can be used in their modified or unmodified form to modulate and regulate immune responses and in the therapy of malignant conditions, which express the Bp50 antigen. These uses are discussed in more detail in section 5.4 below. When the ligand is a monoclonal antibody or a fragment thereof, the monoclonal antibody can be prepared against Bp 50 using any technique that allows the production of antibody molecules by continuous cell lines in culture. For example, the present invention encompasses the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256: 495-497) as well as other techniques which have become more recently available, such as the human B cell hybridoma technique (Kozbor m. fl., 1983, Immunology 'Today 4:72) and the EBV hybridoma technique for producing human monoclonal antibodies (Cole m. fl., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., p. 77- 96) and the like.

Antibody fragments, which contain the idiotype of the molecule, can be generated by known techniques. For example, such fragments include: the F (ab ') 2 fragment, which can be generated by treating the antibody molecule with pepsin; The fabric fragment which can be generated by reducing the 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 molecule with papain and a reducing agent to reduce the disulfide bridges.

Depending on the intended use, the ligand or suitable fragments of the ligand may be chemically modified by coupling to the ligand of any of a variety of compounds using coupling techniques known in the art. Such techniques may include, but are not limited to, the use of carbodiimide, cyanogen bromide, bifunctional reagents such as glutaraldehyde, N-504 675 10 10 succinimidyl B- (Z-pyridyldithio) propionate (SPDP), shipbuilding reactions , coupling to sulfhydryl groups, the use of sodium isothiocyanate or enzymatic coupling, to name a few. When a radioisotope is to be attached to the ligand, this can also be achieved by enzymatic means, by oxidative substitution, complex formation, etc. For a summary of the chemical reagents that can be used for protein modification, see Lundblad and Noyes, Chemical Reagents for Protein Modification, Vol II, CRC Press, Inc., Boca Raton, Florida, chap. 5, pp. 123-139, 1984.

The chemical linkage or coupling of a compound to the ligand can be directed to a site on the ligand that does not participate in the binding to Bp50. This can be accomplished by protecting the binding site in the ligand before performing the coupling reaction. For example, the ligand may first bind to Bp 50 in order to protect the Bp 50 binding site and then the coupling reaction may be performed to link the desired compound to available reactive sites on the ligand-Bp 50 complex. When the coupling reaction is completed, the complex can decompose, thereby producing a modified ligand to which the desired compound is coupled, so that the BpSO binding site in the molecule is affected to a minimum. When the ligand comprises a monoclonal antibody, such as G28-5, in which the Fc domain of the molecule is not required for the ligand to exert its action (see section 5.3.3 below), it may be advantageous to direct the coupling of the desired compounds to the molecule. Fc domain.

The subsections below describe the new 50-kDa B cell surface marker, Bp50, which apparently exerts an effect on B-cell proliferation, as well as ligands that bind to the new 50 kDa receptor and their uses. Examples of ligands of the present invention also describe a monoclonal antibody, which defines Bp50, and its F (ab ') 2 fragment, which, like BCGF, increases B-cell proliferation. In contrast to anti-Bp35 mAb, which can induce dormant B cells in G0 to enter G1, anti-Bp50 mAb does not activate dormant B cells. Anti-Bp35 and anti-Bp5O mAb together, without any additional exogenous signals, induce vigorous activation and proliferation of purified B cells.

The experiments described below also show that the anti-Bp50 activity is similar to the BCGF activity but that anti-Bp5O is different from a BCGF, since anti-Bp5O and low molecule are BCGF are clearly additive and act differently on different B-cell subsets or malignant B-cell conditions. Bp5O can be a receptor for a distinct BCGF or for a transmembrane signal that modulates BCGF production or BCGF receptor expression. 10 15 20 25 30 35 11 _ 5 Û 4 6 7 5 5.1. Methods used to characterize the BQEO receptor Cell preparation. Mononuclear cells were isolated from normal or leukemically heparinized peripheral blood by the Ficoll-Hypaque gradient (Pharmacia, Piscataway, NJ). Mononuclear cells were obtained from tonsil tissue as described (Clark et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770). T cells were consumed by AET-treated sclerotic erythrocyte rosette and Ficoll-Hypaque gradient separation. In some experiments, blood B cells were enriched by "isolating nylon wool adhering cells. Monocytes were removed by incubation in plastic petri dishes once or twice at 37 ° for 45 minutes, unless otherwise indicated. Liquid or compact tonsil B cell fractions were isolated. by Percoll step gradients as described (Clark et al., 1985, Proc. Nat. Acad. Sci. USA 8221766-1770) Compact tonsil B cell preparations consistently had more than 95% sIg + Bp35 + cells. Blood B cell-enriched preparations had 60-857: sigi cells B-cell lymphoma cells were isolated by gently "irritating" lymphoma cells in medium followed by Ficoll-Hypaque gradient centrifugation.

Monoclonal antibodies. The G28-5 antibody to Bp5O was generated by immunizing BALB / c mice with E-human tonsil lymphocytes and fusing immune cells with NS-1 myeloma (Kohler et al., 1975, Nature 256495-497; Ledbetter et al. ,., 19.79, Immunol. Rev. 47: 63-82). Hybrid cell cultures that secreted antibody reactive with tonsil B cells and not with T cells were identified using indirect immunorescence (IF) and assayed with a FACS IV cell sorter; cultures with antibody, which gave histogram patterns similar to known mAb against pan-B cell markers (eg Bp35) were cloned and selected for further investigation. The G28-5 clone produced an IgG1 mAb, which 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. Sci. USA 821766-1770; Clark et al., 1986, Human Immunol, 16: 100; Ledbetter m 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 (IgGza) anti-Bp35, HB10a (IgGga), anti-HLA-DR, 2C3, (IgGI) anti-u chain, G19-4 (IgG1) anti-CD3, FC-2 (IgGza) anti- Fc receptor CD16 and 9.6 (IgGza) anti-CD2 (E receptor) provided by Dr.

Paul Martín (Martin et al., 1983, J. Immunol. 131: 180). IgGI mAb was purified by precipitation Linder using 45% or 50% saturated ammonium sulfate and DEAE-Sephacryl column chromatography and IgG2a mAb were purified using protein A-Sepharose columns. The F (ab ') 2 fragment of G28-5 504 675 H was prepared by a method specified by Parham (Parham et al., 1983, J.

Immunol. 131: 2895), which was purified on a 2 meter long Sephacryl S200 column and analyzed for purity by SDS-PAGE (Ledbetter et al., 1985, J. Immunol. 135: 1819). 2C3-mb mAb to u-chains was conjugated to Sepharose 4B beads (Pharmacia Fine Chemicals, Uppsala) using cyanogen bromide coupling.

Fluorescein and phycoerythrin conjugations. Purified mAb was either conjugated directly to fluorescein using fluorescein isothiocyanate (FlTC; Molecular Probes) (green) by the method described by Goding (Goding et al., 1976, J. Immunol. Meth. 131215-226) or conjugated to R-phycoerythrin (PE) (red) using SPDP (Pharmacia) by the method we have described in detail in Ledbetter et al., 1985, in Perspectives in Immunogenetics and Histocompatibility, ASHI, New York, 6, \ 119 -129. Lymphoid cells were incubated in round-bottomed microtiter plates for 30 minutes with appropriate dilution of green and / or red mAb, washed twice and then analyzed 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) using a 560 nm dichroic mirror splitting mirror and a 580 long-pass filter and a 540 short-pass filter (Ditric Optics , Hudson, MA) in front of the red and green photomultiplier tubes, respectively. Furthermore, a two-color compensator (T. Nozaki, Stanford University) was used to correct for small excesses of green and red signals. For each two-colored ck corner, data were collected from 40,000 cells and stored on diskettes. Data are given as cell numbers (vertical) versus log green. fluorescence versus log red fl uorescence on a 64 x 64 dot grid. About 4.5 points represent a doubling of the ores uorescence.

Unstained cells are located at the posterior corner of the grid; red fluorescence is on the right and green fluorescence is on the left. Our flow cytometry system for two-color IF with fl uorescein 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 tonsillymphoid cells were grown at 5-10 x 105 ml in quadruplicate samples in 96-well microtiter plates containing 200 μl of RPMI-1640 medium supplemented with 15% bovine fetal serum, antibiotics, glutamine and pyruvate (R15). After 1-7 days, the cells were pulsed with 0.5 μCi of sH-thymidine per 7550 compartment (New England Nuclear, 6.7 Ci / mmol; 1 ci = 37) for 18 hours. The cells were then harvested on a glass fiber filter with a cell harvester and the radioactivity was measured in a scintillation counter. In some experiments, antibodies or factors were added at different times after the start of culture; the proliferation in these trials was measured on day 3.

Co-stimulating factors. Purified BCGF was purchased from Cytokine Technology (Buffalo, New York) and did not contain any detectable IL-1, II-2 or interferon activity. This BCGF was prepared by the method set forth by Maizel et al. (Maizel et al., 1982, Proc. Nat. Acad. Sci. USA 7915998), which has shown that the main activity of BCGF in this material is attributable to an IZ -kDa fragment, hereinafter referred to as "BCGF (low)" (Metha et al., 1985, J. Immunol. 1353298). The purification steps included preparative scale DEAE affinity chromatography, followed by hydroxylapatide column chromatography. IL-1 purified to homogeneity constituted a generous gift from Dr. Steven Dower (Dower et al., 1985, J. Exp. Med. 162: 501). Recombinant IL-2 was kindly provided by Cetus Corporation. TPA (12-O-tetradecanoylphorbol-13-acetate) was purchased from Sigma.

Detection of cell activation. Changes in cell volume induced by mAb and / or other factors were measured using a cell sorter and front angle light diffuser. Cell cycle changes at the cellular RNA and DNA level were measured by staining activated cells with acridine orange and measuring the relative cellular RNA (red) and DNA (green) content with a cell sorter according to the method set forth by Darzynkiewicz et al., (Darzynkiewicz m. fi., 1980, Proc. Nat. Arari. sei. UsA vvreesv-svoz). Changes in relative levels of cell surface antigens were monitored using mAbs directly conjugated to fluorescein and then quantified by direct IF fluorescence levels with an Epícs V cell sorter.

Biochemical characterization of Bp50. Immunoprecipitation of Bp50 from 1251-labeled tonsil cells was performed as described (Ledbetter et al., 1985, J.

Immunol. 134: 4250-4254). Isolated antigens were electrophoresed on 10% SDS-polyacrylamide gel plates without reduction. The gels were visualized using autoradiography at -70 ° C and Cronex brightening and intensifying screens (Dupont). 504 675 14 10 15 20 25 30 35 5.2. Characterization of the BQEO receptor The subsections below describe the results of the experiments performed using the methods described above. 5.2.1. Identification of a specific 50 kDa-B cell surface marker, Bp5O A mAb to Bp5O was prepared by immunizing BALB / c mice with human tonsil lymphocytes and fusing immune spleen cells with the NS-α myeloma. One clone, G28-5, produced an IgGI mAb, which did not contain the NS-1 light chain. Upon close examination by IS analysis, G28-5 was found to react only with normal or malignant B cells or B cell lines. A comprehensive screening of normal tissues by accepted methods (Clark et al., 1985, Proc. Nat. Acad. Sci. USA 8211766-1770; Ledbetter et al., 1986, Human Immunol. 15: 30-44; Ledbetter, 1985, in Perspectives in Immunogenetics and Histocompatibility, ASHI, New York, 6, pp. 325-340) revealed that the G28-5 antibody reacts with E-rosette negative (Dr) cells from blood or tonsils but not with non- nylon wool adhesive T cells, PHA-induced T cell blasts or with blood granulocytes, monocytes, red cells or platelets. It reacted strongly with all seven B-lymphoblastoid cell lines tested and with three Burkitt lymphoma lines (Raji, Daudi, Namalwa) but not with four T-cell lines (CEM, HSB-2, JURKAT and HPB-ALL). All chronic lymphocytic leukemias tested (3/3) and 90% (9/10) of B lymphomas tested expressed the Bp50 marker while only 28% (2/7) of acute lymphocytic non-T, non-B-CALLA + - leukemias expressed Bp50.

The limited distribution of Bp5O in normal tissues has been further confirmed by quantitative two-color immuno-fluorescence (IF) assays.

Using an R-phycoerythrin (PE) -conjugated antibody (red) to the pan-B cell antigen Bp35 (B1, CD20) and fluorescein-conjugated anti-Bp50 antibody (green), we found that Bp5O was expressed only in Bp35 + B cells (Fig. 1) in blood or tonsils. Blood B cells consistently expressed slightly lower levels of Bp5O than tonsil B cells; this is similar to HLA-DR expression (Ledbetter et al., 1986, Human Immunol. 1530-44) and gp54 expression (Wang et al., 1979, J. Exp. Med. 1491424-1433), which is also lower in blood B cells. Bp50 was expressed at similar levels in tonsil1-B cell subpopulations separated by Percoll gradients into liquid and compact fractions. Using our PE-conjugated mAb to the T cell marker, CDS (T3), and the NK cell-associated marker, CD16 (Fc receptor) (Ledbetter et al., 1979, Immunol. Rev. 47: 63-82) , we have found that Bp5O is not expressed on T cells or N K cells. Using twoColor-IF, we have also found that CD3 + PHA blasts, which expressed high levels of IL-Z receptors, did not express Bp50.

The G28-5 antibody reacted with a single polypeptide on tonsil lymphocytes, which migrated at approximately 50 Kd under non-reducing conditions (Fig. 2A).

This molecule is larger than previously reported B cell markers within the same molecular weight range, such as Bp39 or Bp45 (Zipf et al., 1983, J. Immunol. 13113064-3072; Kitner et al., 1981, Nature 294, 458-460; Clark et al., 1986, in Leukocyte Typing II, ed. Reinherz et al., Springer Verlag, Berlin, chap. 12 vol. 2, 155-167; Slovin et al., 1982, Proc. Nat. Acad Sci. USA 79: 2649-2653; Thorley-Lawson et al., 1985, J. Immunol. 134: 3007-3012 and Fig. 2B). The exposure time 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 Bp5O on granulocytes and Bp45 in contrast to Bp5O is limited to B cell blasts. Antibodies to Bp39 (41-H16) and Bp45 (MNM6, Blast-1, Blast-2), which have been made available through an international working group (Clark et al., 1986 in Leukocyte Typing II, ed. Reinherz et al. et al., Springer Verlag, Berlin, chap. 12 vol. 2, 155-167), did not block the binding of fluoresceinized anti-Bp50 antibodies to B cells.

Thus, based on tissue distribution, biochemical analysis and blocking studies, the G28-5 monoclonal antibody recognizes a 50-Kd structure that differs from other known B-cell antigens. 5.2.2. Egression of ßgä fl is limited to B cells Both hematopoietic tissue and cell line distribution studies and detailed bivalent flow cytometric analyzes have revealed that Bp5O is expressed only on B lymphocytes. As illustrated in Fig. 3, Bp5O is expressed on a small subset of blood lymphocytes and on a large population of tonsill lymphocytes. Virtually all Bp50 + cells in both blood and tonsils also expressed Bp35 and HLA-DR but did not express CD2 (Fig. 1) or CD3, T cell molecules or the IgG FC receptors found on N K cells. Furthermore, ConA-activated CD3 + T cell blasts expressed IL-Z receptors but did not express Bp50.

Cytometric two-color flow analyzes allow quantitative determination of the density ratio between two surface ntigens. We have previously shown that the compact dormant B cells in the mantle zone of secondary follicles express IgM and low levels of Bp35 while the 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 subsets expressed Bp50 in equal amounts, suggesting that Bp50 is expressed on both dormant B cells and B cells activated in viuo. 5.3. Increasing B Cell Groliferation with Anti-Bgö fl Antibody As previously explained, B cells can be activated with low doses of specific anti-u chain antibodies. We have recently found that the B-cell specific marker Bp35 (B1), a 35-kDa polypeptide, can also exert action on early B-cell activation: 1F5-mAb against Bp35 activates similarly to low doses of anti-u antibody B cells to increase in cell volume and in RNA content and to respond 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 to compare the effects of anti-Bp50 mAb in the proliferation of untreated B cells or B cells activating those with either anti-Bp35 or ahti-u antibodies (Table 1). Anti-Bp35 in solution, or anti-u antibodies bound to Sepharose beads, could, under appropriate conditions, separately stimulate certain B-cell proliferation (Table 1, row 1); in contrast, anti-Bp50 antibodies did not stimulate separate proliferation (Table 1, row 2). However, anti-Bp50 mAb proliferation increased significantly when grown with anti-u beads or with anti-Bp35. In this respect, anti-Bp50-BCGF was similar (Table 1, row 3). Thus, it was essential to determine whether anti-Bp50 and BCGF together could induce B-cell proliferation. As illustrated in Table 1, row 4, anti-Bp50 and BCGF together did not induce any proliferation but increased the proliferation of either anti-u or anti-Bp35-activated cells slightly more than either the stimulant alone. Within a three-range range when used with anti-Bp50 without other signals, BCGF had no effect on the proliferation of compact B cells, even when anti-Bp50 was used at doses ranging from 0.1 to 10 layers / ml.

Table I Increase in anti-Ig or anti-BpBS-induced B cell proliferation with anti-Bp50 antibodies. 10 15 20 25 30 17 V 504 675 Mean proliferation in standard errors for B cells cultured with: Line Co-stimulus. Medium Anti-u beads Anti-Bp35 agent 1 none 1212 1547 10219: 462 55391308 2 anti-Bp50 7191718 3879211329 25465: 616 3 BCGF 456 * _- 217 14217 * _- 445 9443 r 343 4 anti-Bp50 + BCGF 1456: 126 543931-2537 46488: 3387 \ Proliferation of compact Er-tonsill B cells (95% IgM + surface cells) was measured on day 3 as described. Briefly, this meant that 2 x 105 cells / 200 irl compartments were grown in quadruplicate samples for 48 hours with RPMI 1640 medium containing 15% bovine fetal serum plus antibody without antibody or with either monoclonal 2C3 antibody to U-chains linked to Sepharose beads ("anti-u beads", 50 icg / ml) or free 1F5 anti-Bp35 antibody (5 pg / ml). Cultures containing medium, "anti-u-beads" or anti-Bp35 were grown separately or with BCGF (5% final concentration, Cytokine Technology, Buffalo, New York; has no detectable IL-1 or IL-2 activity) with anti -Bp50 (1: 1000 dilution of ascites) as a co-stimulant. After 40 hours, the cells were pulsed with βH-thymidine and incorporated pulse rates were measured after 18 hours. 5.3.1. Anti-Bg50 mAb increases groliferation only after B cells have been activated with anti-Bg35 or anti-Wang 'eyes. The results in Table 1 show that anti-Bp50 mAb could not induce proliferation itself. As shown in Fig. 4, doses of anti-Bp50 ranging from 0.05 .mu.g to 2.0 .mu.g / ml had no effect on αH-thymidine uptake. However, in the presence of optimal levels of anti-Bp35 mAb, as little as 0.1-0.5.5 pg / ml of anti-Bp50 antibodies significantly increased proliferation. As much as 50,000-70,000 pulses per minute could be detected at the optimal proliferation time when highly purified B cells were cultured with anti-Bp35 only plus anti-Bp50. A consistent observation was that higher doses of anti-Bp50 (above 2-5 ug / ml) were less effective than doses in the range of 100-200 ng. 504 675 w 10 15 20 These results showed that anti-Bp50 can exert action only after B cells have been activated with other signals. The data in Fig. 5 show that this is indeed the case. If the B cells have first been activated with anti-Bp35, anti-Bp50 could be added as late as 24-48 hours later and still increase proliferation on day 4. When cells were first treated with anti-Bp50 in which anti-Bp35 was only effective if it was added within a few hours after the start of cultivation.

Similar results were obtained when anti-u instead of anti-Bp35 was used. 5.3.2. Anti-Bg50 mAb does not activate B cells from Go but induces activated B cells to go through the cell cycle We have previously found that anti-Bp35, like low doses of anti-u antibodies, induces dormant tonsil B cells in G0 to enlarge (Clark et al., 1986, Leukocyte Typing II ed., Reinherzl 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. 13513795-3801). Thus, it was of interest to compare the ability of anti-Bp50 mAb and of anti-BpRS mAb to exert an effect on B cell activation. As shown in Fig. 6A, even after 3-4 days of culture, unstimulated compact tonsil B cells had uniform RNA-profile characteristics of cells in G0 (Darzynkiewicz, 1980, Proc.

Nat Acad. Sci. USA 7 7: 6697-6702). However, approximately 15-30% of the cells that had been stimulated with anti-Bp35 or anti-u had increased RNA levels, which indicated entry into G1. In contrast, neither anti-Bp50 (Fig. 6B) nor BCGF (Fig. 6G) separately induced a significant number of B cells to enter G1. For example, 2 days after activation, anti-Bp35 and anti-Ig mAb induced 13.5 and 20.97, respectively, of tonsil cells to enter G1, while cells were treated with only anti-Bp50 (2.7 ° 7r) or BCGF ( 3.2%) remained at medium control levels (2.2%). However, when either anti-Bp50 or BCGF was added along with anti-Bp35 or anti-u antibodies, the number of cells entering G1 drastically increased. Similarly, anti-BpSO and BCGF did not induce separate B cells to enter the S-phase (Table 2), but together with either anti-Bp35 or anti-u, the number of S-phases increased 2-fold to S-fold. 5 10 20 25 30 ,, 504 675 Table 2 Effect of anti-Bp5O and BCGF on cell cycle progression in tonsil lymphocytes Competence- Progression% cells S / Gz / M signal signal GO G1 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-Bp5O 83.6 12.0 3.3 anti-Bp35 anti-Bp5O - 54.1 35 5.7 anti-Ig anti-Bp5O 43.6 36.2 16.2 16.2 medium BCGF 85.4 11.7 2.2 anti-Bp35 BCGF 56.6 32.6 11.6 anti-Ig BCGF 48, 4 36.1 14.1 The percentage of cells in G0, G1 or S and G2 was determined using the acridine orange staining procedure (Darzynkiewicz et al., 1980, Proc.

Nat Acad. Sci. USA 7 7: 6697-6702); 1 x 106 compact tonsil lymphocytes with anti-Bp35 (5 / eg / ml), anti-u on beads (50 pg / ml), anti-Bp5O (0.4 gig / ml), BCGF (5%) or combinations as shown. 5.3.3. Optimal conditions for enhancing B cell cell proliferation with anti-Bp50 antibodies As such, antibodies to Bp5O have little or no detectable effect on compact dormant B cells (Table 3). However, in the presence of agents that can activate B cells, such as anti-Ig, anti-Bp35 and TPA, anti-Bp50 mAb clearly increases proliferation. Anti-Bp5O exerts non-stimulating action with several interleukins, including purified IL-1, recombinant IL-2 and BCGF (low). A comparison of the effect of anti-Bp5O with the effect of BCGF (low) showed that the same agents that exerted a stimulant effect with anti-Bp5O also exerted a stimulant effect with BCGF (low) (Table 3). Of particular interest was the discovery that BCGF and anti-Bp5O together still did not exert any co-stimulating effect on dormant cells.

Table 3 Increase in B-cell proliferation with anti-Bp50 antibodies or B-cell growth factor Mean proliferation 1 standard error for B-cells cultured with: Co-stimulating Medium Anti-Bp50_ BCGF agent (200 ng / ml) (5%) none 9611 267115 285174 anti-Ig 58331391. 4163412103 25094161 anti-Bp35 l (5 pg / ml) '457 1 45 8143 1280 1733 1 32' FPA (2 ng / ml) 73611537 211631871 1306411030 IL-1 (10 U / ml) 26412 308123 22118 IL-2 (100 U / ml) 204134 35017 2201 11 BCGF (5%) 22017 851128 270118 Compact Er-tonsill B cells (more than 95% sIgM + cells) cultured for 48 hours with 2 x 105 cells / compartment followed by 24-hour pulse with 3 H-thymidine before pulse counting.

The kinetics of the proliferation increased with anti-Bp50 are shown in Fig. 7.

The peak of proliferation occurred on day 4 and then decreased whether or not the cells were activated with anti-Bp35 or other activators such as anti-Ig or TPA.

The kinetics of the proliferation increased by BCGF or by anti-Bp50 were similar.

As little as 0.05 pg of anti-BpSO antibodies increased proliferation. An optimal dose of 0.3 / Ig / ml was used in subsequent studies. A consistent observation was that when using whole antibody molecules, higher doses of anti-Bp50 (greater than 2-5 pcg / ml) were less effective than doses in the range of 0.1-0.5 ug / ml.

Human B cells are extremely sensitive to inhibitory effects mediated by the Fc receptors of antibodies that bind to surface Ig (Parker, 1980, Immunol. Rev. 522115; Bijsterbosch et al., 1985, J. Exp. Med. 162 : 1825). Thus, it was essential to compare the efficacy of whole anti-Bp50 mAb with the efficacy of anti-Bp50-F (ab ') 2 fragments. Within a 100-fold dose range, the F (ab ') 2 fragments were clearly as effective as or more effective than whole antibody in increasing B cell proliferation (Table 4). Thus, the Fc domain of anti-Bp50 mAb is not required for anti-Bp50 to exert its action and may be potentially inhibitory. In other words, like BCGF, anti-Bp50 can obviously serve as a soluble mediator without the involvement of Fc receptor-mediated associated cell function.

Table 4 The Fc domain of anti-BpSO antibodies is not required to increase B cell proliferation Mean proliferation of B cells cultured with: \ Anti-Bp50 'Dose Medium Anti-Bp35 (ug / ml) None - 295 1 16 269 1 27 Hel Ab% 0.125 278132 5140120 1.24 275124 46861342 12.5 1631 15 38521203 F (ab ') 2 0.125 594 121 10635 1449 1.25 53113 108931575 12.5 27918 94111870 The cell culture conditions were those described in Table 3. 5.3 .4. Differences between anti-Bp50 and BCGFQ low activity Anti-Bp50 and BCGF (low) had similar effects on B cells and were co-stimulating with the same agent (Table 3). However, several clues suggest that the anti-Bp50 and the BCGF used in this study apparently work via different signals. First, Bp50 molecule is expressed in contrast to BCGF (low) receptors (Bijsterbosch et al., 1985, J. Exp. Med. 162: 1825) on dormant blood B cells (Fig. 3). Second, anti-Bp50 was clearly optimally effective when added 12 hours after the start of culture (Fig. 8A), although both anti-Bp50 and BCGF (low) exert their activity particularly effectively when added after anti-Bp35 or anti-Bp50. 15 20 25 Ig. In contrast, BCGF (low) could be added as late as 24 hours after the start of culture and still optimally increase proliferation (Fig. 8B). These kinetics experiments, which are based on a model according to Howard and Paul (1983, Ann. Rev.

Immunol. 1: 307), shows that a Bp50-dependent signal can normally exert its action before BCGF.

Both anti-Bp50 and BCGF (low) increased the proliferation of B cells activated with anti-Bp35 or anti-Ig (Table 3). However, the effect of anti-Bp50 and BCGF (low) was additive in many experiments (Fig. 7). Fig. 9 shows a titration of BCGF (low) in an experiment where anti-Bp50 was used at its optimal concentration (0.2 pg / ml). BCGF (low) could further increase the proliferation of dormant Bf cells in the presence of anti-Bp50 after activation with either anti-Ig or with anti-Bpßö.

Optimal concentrations of BCGF (low) were 5-10%, while 25% were inhibitory.

Thus, when anti-BpoF) and BCGF (low) were both used at their optimal concentrations, they still showed additive effects on B cell proliferation.

Finally, both normal and malignant B-cell subsets differed in their response to anti-Bp5O and to BCGF (low). For example, some blood B cells responded to BCGF (low) but did not respond to anti-Bp5O (Table 5).

An additional activation signal, such as anti-Bp35 (Table 5) or TPA (Fig. 10), was consistently necessary to make blood B cells respond to anti-Bp50. While compact tonsil B cells generally did not respond to either BCGF or anti-Bp50, liquid B cells did respond (Table 5). Malignant B-cell conditions also differed in their response to anti-Bp5O versus BCGF. For example, some B-cell lymphomas responded to TPA plus BCGF (low) but not to TPA plus G28-5 anti-Bp5O (Fig.

IOB and D). In contrast, compact tonsil B cells and peripheral blood B cells responded to TPA plus either BCGF (low) or anti-Bp50 (Fig. 10A and C). 23 504 675. ^ Nm. UMMC fi cuuwwmm al microns al cs __ co al uxmuuv møcmuæ f m uma f m Aum fifi MMV muxmmsox HHHH Hmucmwwmums flfi ooumm m Hcwcm>: m uwwc al mmwmuwcoHuxßuw umuæoouëæ fl | um | HHHm ob .m: HumHsEHum wunw mEEHu _ um fi mxmumm f m wa mcHumn x: H Eocmw uwuæoocøë: www umu> oøwE> H | m | @ 0Hn mm fl mwuwmn m xßmunm H .Am xmmuæm: oo _ xmmummv: wm: HuwHsEHum mumw cmuum: um> m um fi wxmumm fi m wa m: Huwnsx: H Eocmm umuæuocoë cwum mmvmHHwwA. :: oH> z ._ H fi mnmæ H muH> Huxmwn Eowwm ummHmmcHumnmm: HHwoHHwo + I _N mmæ N m_ H NHN _ m__ H MHN N 11 || Nøum + ommm-H »= ~ @N_ H MHN N mmm _ H mmm wm mmm H Nww m_ www H HNN N_ || mNmm | H »= ~. + ° mmm | H »= ~ Nm H NN_ _ oæ H m_« N m__ H www NN H m «w om H qmm .Ns \ @ = m. MNmm | H» = ~ ON H Nvm _ NH NNN _ NN H _m_ _ _m H N_ ° _ NN H N_m .He \ @ = m_Q_, _ .ommm | H »= m Nm H _m« _ NN H NNN «__ H NQNN NNO NW Hmm NN NNQ _ H @_N ~ _ .Nm_ Nwum N_ H Nwm _ NH H mwm @ ° _ H wow NN_ + mmm GN H »Nm. = @ m = H wu = m »> Nm mßxmmsox N xwmumm N xwmuwm __ xmmumm m = H ~ wH = eH» m ~ wH__m = oa. uoHm cmuw umuæuowäæ fi Hmm A fi wwwumwcmum HV: oHumumuHHoumHwum2 mUUm Hw flfl w ommm | Hucm wm um> m mcHm H um mHm um fifi wxw um @ cHcuuwmmmsHw fl c: | Hmm 10m 50m. Use of Anti-Bg01 Ligands and Bp5O The ligands of the present invention can be used in vivo or in vitro in their unmodified or modified forms to modulate immune responses.

For example, the ligands as such can be used as an "adjuvant" to increase an immune response to a vaccine or to increase the immune response of an immunosuppressive individual. Alternatively, if cytotoxins or anti-proliferative agents are coupled to the ligands, these modified ligands can be used to reduce an immune response, for example in autoimmune disease or in transplant patients to avoid rejection. These modified ligands could also be used to treat malignancies, which include cells or tumors that express the Bp50 antigen, whether or not the malignancy is in the original B cell.

Both the ligands of the present invention and / or Bp 50 as such can be used in vitro. Such applications include in vitro assays, such as immunoassays to detect cells that express the Bp50 antigen, and / or to detect any secreted Bp50 antigen in body fluids. In this case, the ligand or Bp 50 could be labeled with a radiolabel, or uor, enzyme, enzyme substrate, dye, etc. Furthermore, the ligands could be used to separate and / or identify cells expressing the Bp 50 antigen, in which case the ligand may connected to a non-mobile carrier or to fluorine, which can be used in a FACS (fluorescence-activated cell sorter).

The various applications and uses of the ligands and Bp 50 of the present invention are discussed in more detail below. 5.4.1. Bpäs receptor and: objections of ligands such as anti-B 250 to increase B cell groliferation Previous studies have shown that the factors involved in the induction of B cells from the G0 to G1 phase of the cell cycle differ from the factors required for entry into the S-phase. This model is mainly based on studies which show that such agents as low doses of anti-Ig-B cell activating factors or anti-Bp35 alone have little or no effect on B cell proliferation. However, the same agent can drive the B cells to a stage of cell activation where they are sensitive to growth factors. In contrast, growth factors such as BCGF or IL-2 not only have no effect on dormant B cells but increase the growth of activated B cells.

Although the present invention is not limited to any particular theory or explanation, the results presented herein provide further evidence for a model with specific control of B cell activation and growth steps. We have shown here that activation and proliferation signals in human B cells can be transmitted via specific cell surface structures. Although anti-Bp35 mAb activated B cells to enter the G1 phase of the cell cycle separately, it induced little or no proliferation. Anti-Bp-SO-mAb had the opposite effect; it could not activate B cells but when added even as late as 12-24 hours after activation it could induce B cell growth. The Bp50 molecule could probably normally act as either a receptor for a ligand, such as a soluble growth factor, or for a signal mediated by cell-cell contact (ie, a ligand found on the surface of another cell). Previous studies have identified several T-cell hair-bearing B-cell growth factors which, like anti-Bp50, increase B-cell proliferation. Both high molecular weight and low molecular weight forms of B cell growth factors have been identified and different types have been shown to have additive effects (Kehrl et al., 1984, Immunol. Rev. 18175-96; Kishimoto, 1985, Ann. Rev. Immunol 3133-157; 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. 16211319-1335). Thus, Bp50 may be a receptor for one of these factors.

With the exception of IL-Z receptors and C3d receptors, receptors on B cells for growth signals have not yet been identified. mAb-Ab-1 reacts with a B cell marker, which is expressed only on activated B cells and blocks BCGF-dependent proliferation and thus could recognize the BCGF receptor or a related structure. Bp50 appears to differ from the AB-1 marker because AB-1 mAb does not block the binding of the G28-5 anti-Bp50 antibody and, unlike G28-5 mAb, only reacts with activated B cells (Jung et al. ., 1984, J. Exp.

With. 1601919-1924). Bp50 is found on all B cells, which based on absorption assay and direct binding assay does not appear to be the case for BCGF receptors.

Our current data suggest that Bp50 and the receptor for low molecular weight BCGF are different structures. Using a rabbit heteroantiserum, previous Wang et al. (Wang, 1979, J. Exp. Med. 149: 1424-1433) have described a 54-kDa glycoprotein, gp54, which, like Bp50, is expressed on all B cells but at lower levels in blood B cells than in tonsil B cells. It is possible, but not likely, that the rabbit heteroantiserum and anti-Bp50 recognize the same or related structures: in contrast to anti-Bp50 mAb, the rabbit antiserum to gp54 alone was sufficient to stimulate B cell proliferation. 504 675 26 10 15 20 25 30 35 Anti-Bp35 can only, in contrast to anti-Bp50, activate B cells from G0 to G1 and can thus be referred to as an "activation" signal. Whether or not Bp35 acts only upon early B cell activation is not yet clear, as anti-Bp35 antibodies can stimulate certain B cells to divide (Clark et al., 1985; Proc. Nat. Acad. Sci. USA 8221766 -1770). Similarly, Bp50 cannot strictly exert action only as a "growth" signal: 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 G1 (Table 2). In other words, anti-Bp50 acts as a stimulant to promote the progression of both the activating IgO to G1 and growth (G to S) phases of the cell cycle. The BCGF used in these studies also had similar activity (Fig. -6C). Thus, anti-Bp35 and anti-Bp50 (or BCGF) appear to be highly analogous to the "competence" and "progression" factors described in fibroblast growth regulation studies. How B cells respond to anti-Bp35 or anti-Bp50 may obviously be due to their state of differentiation or activation. Here we have shown that two mAbs, anti-Bp35 (a "competence" signal) and anti-Bp50 (a " progression "signal) together can induce significant 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 mAbs for appropriate epitopes can mimic both soluble factors and signals mediated by cell-cell interactions, it may be possible to use appropriate combinations of mAbs to control and regulate human B-cell proliferation or differentiation. in turn will help to guide strategies in. uiuo for the control of human diseases such as malignant B-cell conditions, in immunodeficiency diseases and certain autoimmune diseases.

The novel monoclonal antibody, G28-5, which reacts with a single chain polypeptide of about 50 Kd expressed on the surface of human B cells, is only a particular embodiment of the ligands of the present invention, which can increase the proliferation of activated B cells. cells. Since human B cell proliferation can also be increased with T cell-derived B cell growth factors including low and high molecular weight BCGF, we have compared the activity of anti-Bp50-G28-5 with the activity of a BCGF preparation containing mainly low molecular weight BCGF.

Anti-Bp50-G28-5 and BCGF (low) were very similar in that they exerted stimulants with the same activating agent (anti-Ig, anti-Bp35 and TPA) but did not exert stimulants with each other or with IL-1 or Ilf2. Furthermore, the activity of anti-Bp50-G28-5 was not dependent on its Fc domain because Ffabiz fragments of G28-5 were functionally active. This suggests that soluble anti-Bp50, like soluble BCGF, does not require Fc receptor-bearing additional cells to exert action. Furthermore, both anti-Bp5O and BCGF are effective only in the presence of an activating stimulant. In other words, anti-Bp5O and BCGF are not "competence" factors but instead promote the "progression" of B cells through the cell cycle.

Although it is possible that Bp5O may act as a receptor for such a ligand as a B cell growth factor, several results suggest that Bp5O is not the receptor at least for the BCGF (low) used in this study: it is expressed on blood B cells while BCGF (low) receptors obviously do not.

Candidate structures for 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-Bp5O versus BCGF (low) (Table 5 and Fig. 10). For example, some B lymphomas multiply in response to BCGF (low) but not in response to anti-Bp50. Finally, in a number of experiments, optimal concentrations of anti-Bp5O and BCGF together induced more proliferation than either of these separately. Anti-Bp5O mimics the activity of another BCGF, such as BCGF (high), which exerts a stimulating effect on anti-IgM (Ambrus et al., 1985, J. Exp. Med. 162: 1319; Ambrus et al., 1985, J. Clin.

Invest. 75: 7 32). This suggests that Bp5O could act as a receptor for BCGF (high).

Although Bp5O may be a receptor for a soluble ligand, alternatively Bp5O may act as a receptor for a cell-cell-mediated signal, which regulates BCGF receptor levels and / or autocrine production. Preference for differentiation antigens, which serve as enhancers of an autocrine receptor pathway, comes from T cell studies. MAb against the common leukocyte antigen Lp22O increases proliferation by increasing IL-Z receptor expression on activated T cells (Ledbetter, et al., 1985, J. Immunol. 135: 1819). An analogous mechanical can take place with anti-Bp5O and expression of certain BCGF receptors. Bp5O and BCGF (low) are obviously under some coordinate control because BCGF, like IL-1 and IL-Z receptors, increases the expression of Bp5O on certain leukemic cells. The Bp50 molecule also shows similarities to the Tp44 molecule, which has an effect on IL-Z production. We and others have shown that the 9zß anti-Tp44 antibody increases the proliferation of T cells activated by anti-CD3 or TPA (Ledbetter, et al., 1985, J.

Immunl. 1352331; Hara, et al., 1985, J. Exp. With. 161: 1513). Likewise, the anti-504 675 28 10 15 20 25 30 30 Bp 50 proliferates the B cells activated by anti-Bp35 or TPA. The Tp44 signal exerts action by stimulating IL-Z production instead of stimulating T cell growth. The Bp50 signal could probably exert action in an analogous manner by stimulating autocrine B cell production (Gordon, et al., 1984, Nature, rank 3101145). 5.4.2. Modified ligands used for immunosugression or treatment of malignancies In this embodiment, the ligand of the present invention can be modified by coupling an antiproliferative agent so that the resulting molecule can be used to kill cells expressing the Bp50 antigen.

Such modified ligands can be used in the treatment of autoimmune diseases in order to suppress the proliferation of B cells and thereby suppress the autoimmune response. These modified ligands can also be used for immunosuppression of a transplant patient to prevent rejection of a transplant. Accordingly, cytotoxic agents used to suppress immune responses can be coupled to the ligands of the invention. When using ligands, which increase the proliferation of B cells, an increased effect should be obtained as the drug is directed against proliferating B cells.

According to another mode of administration, the ligands of the present invention, which are modified by coupling of an antiproliferative agent, can be used to treat malignant conditions in which tumors or cells express the Bp50 antigen. The coupling of these chemotherapeutic agents to the ligands of the invention should result in a greater specificity of the drug with respect to the malignant cells. Furthermore, a particular advantage should be obtained in treating a malignant B-cell condition with a ligand which is linked to a cytotoxin which is more effective in killing proliferating cells than non-proliferating cells; treatment with such a ligand should result in a potentiation of the cytotoxin's action.

Accordingly, the chemotherapeutic agents or antiproliferative agents that can be coupled to the ligands of the present invention include those listed in Table 6 below, derived from Goodnab and Gilman, The Pharmacological Basis of Therapeutics, 6th Edition, MacMillan Publishing Co., Inc., New York, p. 1249-1313, 1980, which is incorporated herein by reference. 10 15 20 25 30 Table 6 29 504 675 Chemotherapeutic agents that can be linked to anti-BQSO ligands Class Alkylating agents Antimetabolites Natural products Various agents TYPE Nitrogen monoxide Ethyleneimine derivatives Alkylsulfonates Nitrosocarbamides Triazenes Folic acid analogues "'Purimine alkaloids Antibiotics Enzyrner Platinum coordination complexes Substituted urea Methyl hydrazine derivatives average Mek] oretamin Cyclophosphamide Melphalan Uracil / mustard Chlorambucil Thiotepa Busulfan Carmustine Lomustine Semustine Streptozocin Dacarbazine Methotrexate Fluorouracil Cytarabine azaribine Mercaptopurine Thioguanine Vinblastine Vincristine Dactinomycin Daunorubicin Doxorubicin Bleomycin Mithramycin Mitomycin L-asparaginase cisplatin Hydroxyurea Procarbazine 10 15 20 25 30 5 0 4 6 7 5 30 i Adrenocortical Mitotane Suppression Agents Hormones and Adrenocortico- Prednisone antagonists steroids Progestins Hydroxyprogesterone caproate Medroprogesterone acetate Megestrol acetate Estrogens Diethylstilbestrol Ethinyl ester adiol Antiestrogen Tamoxifen Androgens Testosterone propionate Fluoxmesterone Radioactive isotopes Phosphorus \ Sodium phosphate 32P Iodine Sodium modidide 1311 Any method known in the art can be used to couple the ligand to the chemotherapeutic or antiproliferative agent. Examples of such methods have previously been listed (see section 5 above). 5.4.3. Other: objections of ligands and BQO In addition to the therapeutic applications, the ligands and Bp5O as such have other applications in diagnostic assays, separation scheme, etc. both in. uitro och in uivo.

The Bp50 receptor can be used to prepare and / or design the ligands of the invention. Bp5O can also be used with the ligands of the invention - in vitro assay methods, which require a standard to quantify the amount of Bp5O detected in a sample. Finally, Bp5O as such may be useful as a soluble factor, which mediates immunity, for example a lymphokine. In addition to therapeutic treatment and diagnostic assays, the ligands of the present invention could be used to identify or separate cells that express the Bp50 antigen. Furthermore, if a suitable radiolabel or radiopaque compound is attached to the ligand, the ligand could be used for in vivo imaging of tumors expressing the BpÖO antigen. Other uses will be apparent to those skilled in the art in light of the above description. 6. Deposition of Cell Lines The following hybridomas have been deposited with the American Type Culture Collection, Rockville, MD and have been granted the following deposit numbers: Hybridoma ATCC Deposit No. G28-5 HB91 The present invention is not limited to the scope of the deposited hybridoma because the deposited the embodiment is intended as a single illustration of an aspect of the invention and any cell lines that are functionally equivalent fall within the scope of this invention. In fact, numerous modifications of the invention, in addition to those described and shown above, will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims (3)

'so4 675 32 10 15 PATENT REQUIREMENTS Essentially pure ligand, characterized in that it (a) binds to Bp50, a 50 kilogram odalton B-cell surface antigen, and is defined by the monoclonal antibody G28-5; (b) upon binding to an activated B cell, stimulates it to undergo the cell cycle so as to increase the proliferation of the B cell; and (c) upon binding to dormant B cells, in the absence of other factors, does not stimulate proliferation, the ligand being a monoclonal antibody molecule or an Fv, Fab, F (ab ') 2 or Fab' fragment of the monoclonal antibody molecule containing the antigen combining site that binds to the Bp50 receptor, prepared by a hybridoma cell line, which has been deposited with the ATCC under the deposit number HB9110. The ligand of claim 1, characterized in that it further comprises a compound linked to the ligand. The ligand of claim 2, characterized in that the compound coupled to the ligand is an antiproliferative agent, an alkylating agent, an antimetabolite, an antibiotic, a vinca alkaloid, an enzyme, a platinum-coordinated complex, a radioisotope or a fluorescent compound.