NL195022C - Antibodies and active fragments thereof that serve to enhance B cell expansion. - Google Patents

Description

c 1 195022 'Antibodies and active fragments thereof that serve to enhance B cell expansion

The present invention relates to a substantially pure antibody or active fragment thereof that binds to a polypeptide that is specifically present on the surface of human B cells.

Such an antibody is known from a publication in Proc. Natl. Acad Sci USA vol 82, pp. 1766-1770 (1985) from E.A. Clark et al. This publication lists monoclonal antibodies (mABs) 2H7 and 1F5 against a 35-kDa polypeptide (Bp35), which is attached to the surface of human B cells. The mAB 1F5 can stimulate the expansion of human B cells. Fab fragments of this antibody do not have that effect.

In a publication by J.T. Golay et al (where one of the authors is EA Clark) in Journal of Immunology 135 3795-3801 (1985), it is stated that the monoclonal antibody 1F5 against Bp35 resting B cells increases and the RNA synthesis thereof rapidly increases, which shows that the human B cells are activated from the G0 to the G1 phase. Although the [3 H] -thymidine incorporation is increased 5- to 10-fold by this, no cell division of the B cells occurs, so that it is clear that they do not pass into the S phase.

1F5 prevents the formation of immunoglobulins by B cells.

In the relevant art, new means are desired that can be used to stimulate human B cell expansion.

It has been found that for such purpose a new antibody or active fragment thereof can be used that binds to another polypeptide that is specifically present on the surface of B cells.

The invention is characterized in that (a) the antibody binds to a 50 kDa human B cell specific surface polypeptide (Bp50), (such an antibody is the monoclonal antibody G28-5 produced by hybridoma cell line ATCC HB91120), and (b) after binding to Bp50, which is present on the surface of an B-cell activated with anti-Bp35 or anti-1g, the extension of the activated B-cell as measured in vitro by pH-thymidine incorporation, at a concentration of 0.1 to 0.5 pg / ml. Cell division of the B cells does occur here.

1. Background of the invention

The activation of resting B cells by transition from the G0 to the G1 phase of the cell cytosus and the subsequent induction of activated B cells to extend into the S phase are distinct steps that require different control mechanisms. According to A.L. De Franco et al. J. Immunol. 135 91) 87-94 (1985), B cells in the G0 phase are brought into the G1 phase by anti-IgM antibodies (anti-μ) in low concentration (1-5 35 pg / ml). To bring the B cells into the S phase, 5 to 50 times higher concentrations are needed.

E.M. Rabin et al. Proc. Natl. Aced. Sci USA 82 2935-2939 (1985) mentions a T cell-derived B cell stimulating factor 1 (BSF-1) that acts on mouse B cells. Due to the efficacy of BSF-1, resting B cells move from the G0 phase to the G1 phase. In the presence of BSF-1, low 40 concentrations of anti-IgM antibodies (5 pg / ml) are sufficient to bring the B cells into the S phase.

Y Noma et al Nature 319 640-646 (1986) describe cloning and sequencing a cDNA encoding a mouse-derived IgG1 induction factor presumably similar to BSF1.

G.D. Wetzel et al. J. Immunol. 133 (5) 23272332 (1984) mention LPS and DXS as activators for B cells. 45 Under the influence of LPS, the B cells go through the entire cycle, while DXS removes the cells from the G0 phase.

According to S.A. Mutaguchi et al. J. Immunol 132,176-180 (1984), human B cells are moved from the G0 to the G1 phase by anti-IgM antibodies (anti p), while a monoclonal B cell growth factor, BCGF, derived from human TT hybridoma brings these B cells from the G to the S phase.

J.H. Kehrl et al Immun Rev 1984 No. 78 pp. 75-96 discuss the stimulatory effect of various 50 factors on human B cells including anti p, an anti-lg reagent from a Staphylococcus aureus strain (SAC), the human B cell growth factor BCGF, human B cell differentiation factor BCDF, Interleukin 1 (IL1), IL2 and interferon. The direct effect of IL2 on B cells is being questioned.

By L.K.L. Jung et al: J. Exp. Med. 160 1597-1602 (1984), however, it is demonstrated with the aid of a monoclo-55 sewing antibody that resting B cells do not, but with the aid of anti-IgM activated B cells, contain a receptor for IL2 and that the IL2 enhances this activated B cells also, but not as strong as BCEF (BSF).

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Also by R.H. Zubler: J. Exp. Med. 160, 1170-1183 (1984), it is found that resting B cells and B cells that were activated only by lipofolysaccharide (LPS) did not respond to IL-2, but that IL-2 induced expansion in B cells that were lysed were stimulated together with anti-IgM.

The literature mentioned above already shows that there are a number of factors that promote the growth of B cells, both for mouse B cells and for human B cells. These include B cell growth factors (BCGF), which are derived from a variety of sources including T cell lines or hybridomas, B cell lines, or dendrite cells. Although both interleukin-1 (IL-1) and interleukin-2 (IL-2) have proven to enhance B cell growth, they are apparently different from certain BCGFs. For example, monoclonal antibodies (mAb) to a murine BCGF (J. Ohara, et al., 1985, Nature (Lond.) 315: 333-336) or human BCGF (high) give (JL Ambrus, et al., 1985 , J. Exp. Med. 162: 1319-1335) blocking the BCGF activity but not the IL-1 or IL-2 activity. The BCGFs themselves appear to be heterogeneous. For example, 60-kilodalton (kDa) high molecular weight human BCGF, BCGF (high) has been identified that is different from a 12-kDa low molecular weight form, BCGF (low) (JL Ambrus, et al., 1985, J. Clin. Invest. 75) : 732-739).

These activation and growth signals presumably regulate B cells through interaction with specific B cell surface structures. In addition to the antigen-specific signal via surface IgM, several other candidate B cell surface polypeptides have been identified that may play a role in the activation or growth of B cells in any way. Recently functional IL-2 receptors on B cells have been identified (R. H. Zuibler, et al., 1984, J. Exp. Med. 160: 1170-1183; L. K. L. Jung, et al., 1984, J. Exp.

20 Med. 160: 1597-1602; A. Muraguchi, et al., 1985, J. Exp. Med. 161: 181-197).

Several candidate B cell surface polypeptides have been identified that may play a role in B cell activation or growth. For example, B-Subbarao and Mosier (DE Subbarao, et al., 1983, Immunol. Rev. 69: 81-97) have found that monoclonal antibodies (mAb) to the mouse B-cell antigen can Lyb2 activation of B cells and evidence has recently emerged that Lyb2 may be the receptor for BSF-1 (H. Yakura, 1985, Fed. Proc. 44: 1532). Similarly, we have found that suitable mAb (1F5) against a 35 kDa polypeptide, Bp35, activates human B cells from G0 to G, (EA Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770; JT Golay, et al., J. Immunol. 135: 3795-3801). Aggregated C3d or antibody to the 140 kDa C3d receptor, Bp140, cause expansion of B cells that are T cell dependent (F. Melchers, et al., 1985, Nature 317: 264-30 267; GR Nemerow, et al. 1985, J. Immunol. 135: 3068-3073; R. Frade, et al., 1985, Eur. J. Immunol. 15: 73-76). Although BCGFs have been identified in both mouse and human, the receptors for these factors have not yet been isolated. A polyclonal antiserum was prepared by Wang and co-workers (Wang, et al., 1979, J. Exp. Med. 149: 1424-1433) that identified a 54 kDa polypeptide (gp54) on human B cells and was shown to be rabbits antiserum against gp54 tonsillary B cells induced to division. Recently, L.K.L. Jung and S.M. Fu (Jung, et al., 1984, J. Exp. Med. 160: 1919-1924) isolated an mAb (AB-1) against a 55-kDa antigen restricted to activated B cells that blocks BCGF-dependent extension. Whether or not anti-gp54 or AB-1 recognizes a BCGF receptor, is not yet known.

2. Summary of the invention

The present invention is directed to antibodies or fragments thereof that (a) bind to Bp50, a 50kDa B cell specific surface polypeptide described herein, and (b) enhance the expansion of activated B cells.

The present invention includes antibodies, monoclonal antibodies and the fragment F (ab% of 45 of these antibody molecules that contain the antigen combining site. They can be used to stimulate human B cell expansion.

The present invention is based on the finding that two human B cell differentiation antigens, Bp35 and the B cell antigen described herein, Bp50, apparently have distinct functions as signal receptors in B cell activation. Monoclonal antibodies (mAb) to Bp35 and Pb50 both give positive 50 signals to B cells that stimulate their cycle cycle. Monoclonal antibodies to Bp35, as well as anti-IgM antibodies, function in principle by activating resting B cells to enter the Gn phase of the cell cycle. In contrast, a monoclonal antibody described herein or its fragment against Bp50, a 50 kDa polypeptide that is expressed on all B cells, functions by stimulating activated B cells until cycle of cell 55 and enhances the expansion of activated B cells . Monoclonal antibodies to Bp35, as well as anti-IgM antibodies, activate tonsillary B cells and elicit low levels of B cell extension. In contrast, anti-Bp50 monoclonal antibody alone does not lead to B cell activation, nor to the generation of B cell expansion, but together with anti-Bp35 or anti-IgM antibodies, it enhances B cell expansion. In this respect, the effect of anti-Bp50 antibody appears to be on the activity of B cell growth factors (BCGF). Only a small amount of 0.05 pg / ml of anti-Bp50 is needed to enhance expansion, and just as BCGF is anti-Bp50. even effective when added with anti-IgM or anti-Bp35 12-24 hours after B cells 5 have been activated. Without additional exogenous signals, anti-Bp35 and anti-Bp50 antibodies together produce a strong expansion of purified resting B cells.

These results indicate that the Bp35 and Bp50 surface molecules play a role in the regulation of B cell activation and cell cycle progression. Because of the significance anti-Bp35 and similar molecules have on the effect and action of the ligands of the present invention, the publication of E.A. Clark et al., 1985, Proc. Natl. Acad. Sci. (USA) 82: 1766-1770 is hereby incorporated by reference.

Although the activity of anti-Bp50 is similar to that of BCGF (low) because both anti-Bp50 and BCGF (low) are constimulating with the same agents and both anti-Bp50 and BCGF (low) only influence activated B cells and in In a soluble form, the activity of anti-Bp50 can be distinguished from the activity of BCGF (low) in that the expansion of B cells with optimum amounts of anti-Bp50 and anti-Bp35 (or anti-IgM) ) is stimulated, can be further enhanced with BCGF (low) and because both B cells in the blood and certain B cell lymphomas respond differently to anti-Bp50 versus BCGF. For optimum activity, anti-Bp50 should be added within 12 hours after B cell activation, while BCGF (low) retains optimum activity even when added 24 hours after activation. In addition, Bp50 is expressed on all B cells, while receptors for BCGF (low) are limited to activated B cells. Therefore, anti-Bp50 and BCGF (low) can both regulate B cell growth in a coordinated manner, but apparently they do so through different signals.

In one embodiment of the present invention, the antibodies that bind to Bp50 and enhance the expansion of activated B cells can be used to enhance an immune response. For example, these antibodies that bind Bp50 can be used as an "adjuvant" to enhance an immune response to a vaccine. Alternatively, these antibodies can be used to enhance the immune response of an individual in a state of immunosuppression.

The antibodies or their fragments according to the invention can also be used in vitro to identify or separate cells expressing the Bp50 antigen and / or to examine body fluids for the presence of the Bp50 antigen that may or may not cell is bound. In addition, the antibodies of the invention can be used in vivo to obtain an image of cells or tumors expressing the Bp50 antigen.

35 2.1. Definitions

As used herein, the following abbreviations will have the indicated meanings: AO = acridine orange BCGF = B cell growth factor

BCGF (high) a 60 kDa human BCGF

40 BCGF (low) = a 12 kDa human BCGF

Bp35 = a 35 kDa B cell specific surface polypeptide (CD20) defined by mAb 1F5

Bp50 = a 50 kDa B cell specific surface polypeptide defined by mAb G28-5

Fv = the variable region or the antigen combining site of an antibody molecule. This can be any fragment containing the idiotype of the molecule, including 45 (without limitation) Fab, F (abO 2 · Fab ', and the like IF = immunofluorescence

Ig = immunoglobulins IL-1 = interleukin 1 IL-2 = interleukin 2 50 kDa = kilodalton mAb - monoclonal antibody SDS-PAGE = sodium dodecylsulfate-polyacrylamide gel electrophoresis TPA = 12-0-tetradecanoylforbol-13-acetate 55 3. Figure description

Figure 1. Bp50 expression is limited to Bp35 ± B cells. A two-color flow cytometric analysis of 50,000 cells was performed in the manner described by E.A. Clark, et al., 1985, Proc.

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Wet. Acad. Sci. USA 82: 1766-1770. The data are plotted as cell number versus the logarithm of green fluorescence and the logarithm of red fluorescence where 4-5 dots represent approximately a doubling of fluorescence. The data is provided to show autofluorescent negative cells. PE (red) -anti-Bp35 (1F5) versus FITC (green) -anti-Bp50 (G28-5) stain shows that all Bp50 + 5 cells are also Bp35 +.

Figure 2. Immunological comparison of Bp50 polypeptide with other B cell surface antigens. Immunoprecipitation of Bp50 from the surface of 125 I-labeled tonsillary cells was performed in the manner described. Isolated antigens were electrophoretically treated on 10% SDS polyacrylamide slab gels without reduction. The gels were visualized with autoradiography and enhancement screens.

Panel A: lane 1, anti-Bp50 (G28-5); lane 2, anti-Bp95 (G28-8); lane 3, sepharose-goat anti-mouse Ig alone.

Exposure time: 4 days.

Panel B: lane 1, anti-Bp50 (G28-5); lane 2, anti-Bp45 (BLAST-2); lane 3, anti-Bp39 (G28-1); lane 4, anti-Bp39 (41-H16); lane 5, sepharose-goat anti-mouse Ig alone.

An exposure time of 2 days was chosen so that the bands in lanes 2-4 were not overexposed and could be clearly distinguished with respect to Bp50.

Figure 3. Two-color immunofluorescence analysis of Bp50 expression. Peripheral blood or tonsillary mononuclear cells were isolated by centrifugation on Ficoll and stained with PE (red) -conjugated G28-5 (anti-Bp50) in combination with fluorescein (green) -conjugated reference-20 antibodies, including 2C3 (anti-IgM) ; 1F5 (anti-Bp35); HB10a (anti-DR); and 9.6 (anti-CD2), E receptor). Cells were analyzed with a FACS IV, equipped with four decade log amplifiers in both red and green dimensions. Forward and right-angle light scattering was used to exclude monocytes. Unstained cells are located at the rear of the grid; red fluorescence on the right and green fluorescence on the left.

Figure 4. Dose-response curves for enhancement of expansion of dense tonsillary Er-B cells by anti-Bp50 antibodies as indicated: Media only; anti-Bp50 mAb only; anti-Bp35 mAb (5 pg / ml) only; BCGF only; anti-Bp35 mAb plus BCGF; anti-Bp35 mAb plus tiered doses of anti-Bp50. Mean expansion ± standard error of quadruple samples was measured on day 3.

Figure 5. Anti-Bp50 mAb are very effective in enhancing expansion when added after a B cell activation signal. Dense tonsillary Er-B cells were incubated for 4 days with media alone, anti-Bp50 mAb (0.5 pg / ml) added at different times after incubation, anti-Bp35 mAb (5 pg / ml) added at different times after incubation; anti-Bp50 mAb kept constant to which anti-Bp35 mAb was later added at different times; anti-Bp35 mAb kept constant to which anti-Bp50 mAb was added to cultures at different time points. 3 H-thymidine was added during the last 10 hours and its uptake measured.

Figure 6. Comparison of the ability of anti-Bp35 and anti-Bp50 to induce resting tonsillary B cells to leave the G0 stage of the cell cycle. Day 3 after treatment media only (-), anti-Bp35 only (-); and anti-IgM only (.....), A, no additional additives; B, anti-Bp50 (0.5 pg / ml) added to each group; C, 5% BCGF added to each group. The data is plotted as a relative 40 cell number versus the logarithm of AO red fluorescence (RNA).

Figure 7. K cell expansion kinetics after stimulation with anti-Bp50 versus BCGF. Dense tonsillary E-B cells were stimulated with media alone; 10% BCGF only; anti-Bp35 only; anti-Bp50 only; anti-Bp35 + 10% BCGF; anti-Bp35 + anti-Bp50; and anti-Bp35 + anti-Bp50 + 10% BCGF. Expansion was measured on the indicated days 18 hours after a shot of 3 H-thymidine. Expansion was measured in quadruple 45 and standard errors are indicated. One of three experiments.

Figure 8. Times after stimulation with anti-Bp35 on which anti-Bp50 (A) or BCGF (B) optimally enhance the expansion, dense tonsillary E-B cells were stimulated in the manner shown and the extension was measured 18 hours after an impact of 3 H-thymidine on day 3. Media; anti-Bp35 only added at the indicated times; anti-Bp50 or BCGF only; anti-Bp35 added at the start of the culture 50 followed by addition of anti-Bp50 or BCGF at the indicated time points; anti-Bp50 or BCGF

added at the start of the culture followed by anti-Bp35. One of two experiments. The extension was measured in quadruple and standard errors are indicated. The doses used: anti-Bp35, 5 pg / ml; anti-Bp50, 0.2 pg / ml; GCGF (low) 5%. The concentrations used were as follows: anti-Bp35, 5 pg / ml; anti-Bp50, 0.2 pg / ml; BCGF, 5%.

55 Figure 9. Anti-Bp50 and BCGF have additive effects on B cell expansion. Dense tonsillary E-B cells were stimulated with tiered doses of BCGF (low) together with anti-Bp50 alone; anti-Bp35 only; anti-IgM beads only; anti-Bp35 + anti-Bp50; or anti-Bp50 + anti-IgM. The extension was measured at 5 195022 'on day 3 18 hours after stimulation with a 3 H-thymidine burst. The extension was measured in quadruple and standard errors are indicated. One of four experiments. The doses used for 10® cells: anti-Bp35, 5 pg / ml; anti-Bp50, 0.2 pg / ml; anti-IgM beads, 50 pg / ml.

Figure 10. Comparative effects of anti-Bp50 and BCGF on normal and malignant B cells. Peripheral blood E-B cells (A) or dense tonsillary E-B cells (C) were stimulated with or without TPA (75 ng / ml) in the presence of 10% BCGF or 1 pg / ml anti-Bp50. Two separate B cell lymphomas (panels B and D) were stimulated in the same way. The extension was measured on day 3 by incorporating 3 H-thymidine 12 hours after a burst of it was added. The extension was measured in quadruple and standard errors are indicated.

4. Detailed description of the invention

The present invention is directed to substantially pure antibodies that (a) bind to Bp50, a 50 kDA B cell specific surface polypeptide, and (b) enhance the expansion of activated B cells.

The present invention encompasses antibody molecules, monoclonal antibody molecules and fragments F (ab% of these antibody molecules containing the antigen combining site that bind to the Bp50 receptor.

When the antibody is a monoclonal antibody against Bp50 or the fragment F (ab02 thereof, it can be prepared by any technique that leads to the production of antibodies by a culture of continuous cell lines. For example, the hybridoma technique originally developed by Kohier 20 and Milstein (1975, Nature 256: 495-497) as well as other techniques that have become more recently available such as the human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72) and the EBV hybridoma technique for preparing human monoclonal antibodies (cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pages 77-96) and the like to the scope of the present invention.

An F (ab% fragment can be obtained by treating the antibody with pepsin. An F (ab ') 2 fragment can also be obtained by treating the antibody molecule with papain.

The subsections below describe the new, 50-kDa B cell surface marker, Bp50, which apparently plays a role in B cell expansion. Furthermore, according to the present invention, a monoclonal antibody defining Bp50 and its F (ab ') 2 fragments are described which, like BCGF, enhance B cell expansion. Unlike anti-Bp35 mAb, which can induce resting B cells in G0 to transition to G1 (anti-Bp50 mAb does not activate resting B cells. Anti-Bp35 and anti-Bp50 mAb together, without any additional exogenous signals, induce strong activation and expansion 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 low molecular weight BCGF because anti-Bp50 and low molecular weight BCGF are clearly additive and act differently on various B cell subsets or malignancies.

4.1. Methods used to characterize the Bp50 receptor Cell preparations Mononuclear cells were isolated from normal or leukemic, heparin-treated peripheral blood by Ficoll-Hypaque gradients (Pharmacia, Piscataway, NJ). Mononuclear cells were obtained from tonsillary tissues as described by E = (E. A. Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770). T cell depletion was achieved with AET-treated sheep erythrocyte rosette formation and Ficoll-Hypaque gradient separation. In some experiments, an enrichment of blood 45 B cells was achieved by isolating a nylon wool adherent cells. Monocytes were removed by incubation on plastic petri dishes, once or twice for 45 minutes at 37 ° C, unless otherwise indicated. Floating or dense tonsillary B cell fractions were isolated by Percoll step gradients as described by (E. A. Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770). dense tonsillary B cell preparations consistently contained more than 95% slg + Bp35 + cells. Preparations that were enriched 50 in blood B cells contained 60-85% slg + cells. B cell lymphoma cells were isolated by gently agitating lymphoma cells in the medium followed by Ficoll-Hypaque gradient centrifugation.

Monoclonal antibodies

The G28-5 anti-Bp50 antibody was obtained by immunizing BALB / c mice with human 55 E-tonillary lymphocytes and fusing immune spleen cells from these mice with the NS-1 myeloma (Kohier, et al., 1975, Nature 256: 495- 497; JA Ledbetter, et al., 1979, Immunol. Rev. 47: 63-82). Hybrid cell cultures secreting an antibody that reacted with tonsillary B cells and not with T cells were identified 195022 6 by indirect immunofluorescence (IF) and analysis with a FACS IV cell sorter; cultures with antibody giving similar histogram patterns as known mAb against pan B cell markers (e.g., Bp35) were cloned and selected for further investigation. The G28-5 clone produced an IgG, mAb that reacted only with normal or malignant B cells or B cell lines. Other mAb used in this study are described in detail (EA Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770; EA dark, et al., 1986, Human Immunol. 16: 100; JA Ledbetter, et al., 1986, Human Immunol. 15: 30-44; JA Ledbetter, et al., 1985, in Perspectives in Immunogenetics and Histocompatibility, ASHI, New York, 6, page 325 -340). These include 1F5 (IgG2a) anti-Bp35, HB10a (IgG2a). anti-HLA-DR, 2C3 (IgG,) anti-μ chain, G19-4 (IgG,) anti-CD3, FC-2 (IgG2a) anti-Fc receptor CD16, and 9.6 (IgG2a) anti-CD2 (E receptor 10 supplied by Dr. Paul J. Martin (Martin, et al., 1983, J. Immunol. 131: 180). The IgG, mAbs were purified by precipitation using 45% or 50% saturated ammonium sulfate and DEAE Sephacryl column chromatography, and the IgG2a mAbs were purified using protein A Sepharose columns. The F (ab ') 2 fragments of G28-5 were prepared by the method of Parham (Parham, et al., 1983, J. Immunol. 131: 2895), purified on a 2-meter-long Sephacryl S200 column, and tested for purity by SDS-PAGE (JA Ledbetter, et al., 1985, J. Immunol. 135: 1819). The 2C3 mAb against μ chains was conjugated to Sepharose 4B beads (Pharmacia Fine Chemicals, Uppsala, Sweden) using cyanogen-bromide coupling. Fluorescein and phycoeryryrin conjugations

Purified mAb was either directly bound to fluorescein by using fluorescein isothiocyanate (FITC; Molecular Probes) (green) according to the method of Goding (J.W.

Goding, et al., 1976, J. Immunol. Meth. 13: 215-226), or bound to R-phycoerythrin (PE) (red) using SPDP (Pharmacia) by a method described in detail by us in J.A. Ledbetter, et al., 1985, in Perspectives in Immunogenetics and Histocompatibility, ASHI, New York, 6, 119-129. 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 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 to split the beam and a 580 long-pass filter and 540 short-pass filter (Ditric Options, Hudson, MA) arranged for the red and green photomultiplier tubes respectively. In addition, a two-color compensator (T. Nozaki, Stanford University) was used to correct for minor overflow of green and red signals. For each two-color stain, data from 40,000 cells was collected and stored on floppy disks. Data are represented as cell number (vertical) versus the logarithm of green fluorescence versus the logarithm of red fluorescence on a 64 x 64 dot grid. About 4.5 dots represent a doubling of the fluorescence. Unstained cells are located at the rear corner of the lattice, red fluorescence is on the right and green fluorescence on the left. Our bi-color IF flow cytometry system with fluorescein and phycoerythrin has been described in more detail by J.A. Ledbetter, et al., In Perspectives in 40 Immunogenetics and Histocompatibility, ASHI, New York, 6.119-129 and 325-340.

Cell culture: B cells derived from bleeding or tonsillary lymphoid B cells were grown in quadruplicate from 5-10 x 105 cells per ml on 96 well microtiter plates containing 200 μΙ RPM1-1640 medium supplemented with 15% fetal bovine serum, antibiotics , flutamine and pyruvate (R15). After 1-7 days, all 3 were given a "ditch" of 3 H-thymidine of 0.5 μ Ci per well (New England Nuclear, 45.6, 7 Ci / mmol; 1 Ci = 37 x 10® disintegration / sec = 37 x 109 becquerel.

The cells were then separated after 18 hours on glass fiber filters and radioactivity, once before the extension, was measured with a scintillation counter. In some experiments, antibodies or other stimulating factors were added after the start of the culture. The cell expansion in these experiments was measured on day 3.

50 Cost-stimulating factors

Purified BCGF was purchased from Cytokine Technology (Buffalo, New York) and contained no detectable IL-1, IL-2 or interferon activity. This BCGF was prepared by the method according to A. Maizel and co-workers (A. Maizel, et al., 1982, Proc. Nat. Acad. Sci. USA 79: 5998-6002), who have demonstrated that the most important BCGF activity in this material is in a 12-kDA species that is further referred to as 55 "BCGF (low)" (SR Mehta, et al., 1985, J. Immunol. 135: 3298-3302). The purification steps include preparative scale DEAE affinity chromatography followed by Hydroxylapatite column chromatography IL-1 purified to homogeneity was a generous gift from Dr. Steven Dower (SK Dower, et al., 1985, J. Exp.

7 195022

"Med. 162: 501-515). The Cetus Corporation was kind enough to deliver recombinant IL-2. TPA

(12-O-tetradeconoylforbol 13-acetate) was purchased from Sigma.

Detection of cell activation

Cell volume changes evoked by mAb and / or other stimulating factors were measured with a cell sorter (cell sorter) and a forward angle light scattering (formed angle light scatter). Cell cycle changes in cellular RNA and DNA levels 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 of Z. Darzynkiewicz et al. (Z. Darzynkiewics , et al., 1980, Proc. Nat. Acad. Sci. USA 77: 6996-6699). Changes in relative levels of cell surface antigens were monitored by using mAb directly bound to fluorescein and then quantitatively determined by direct IF fluorescence levels with an Epics V cell sorter.

Immunological characterization of Bp50

Immunoprecipitation of Bp50 from 125 I-labeled tonsillary cells on the surface was performed as described (J. A. Ledbetter, et al., 1985, J. Immunol. 134: 4250-4254). Isolated antigens were electrophoresed on 10% SDS polyacrylamide slabs without reduction. The antigens in the gels were visualized using autoradiography at -70 ° C and Cronex exposure plus enhancement screens (Dupont).

4.2. Characterization of the Bp50 receptor The subsections below describe the results of the experiments performed with the methods described above.

4.2.1. 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 tonsillary lymphocytes and fusing immune spleen cells with the NS-1 myeloma. One clone, G28 * 5, produced a IgG1 mAb that did not contain the NS-1 light chain. After investigation with IF analysis, G28-5 was found to respond only with normal or malignant B cells or B cell lines. From an extensive study of normal tissues according to known methods (EA Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770; JA Ledbetter et al., 1986, Human Immunol. 15: 30- 44; JA Ledbetter, 1985, in Perspectives in Immunogenetics and Histocomality, ASHI, New York, 6, pp. 325-340), it was found that the G28-5 antibody reacts with E rosette-negative (Er-) cells from blood or tonsils but not with non-nylon wool adherent 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's lymphoma lines (Raji, Daudi, Namalwa), but not four T cell lines (CEM, HSB-2, JURKAT, and HPB-ALL). All tested chronic lymphocytic leukemias (3/3) and 90% (9/10) of tested B lymphomas responded positively to the Bp50 marker while only 28% (2/7) of non T, non B CALLA + acute lymphocytic leukemias Bp50 could be demonstrated.

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

40 Using an R-phycoerythrin (PE) -conjugated antibody (red) against the pan B cell antigen Bp35 (B1, CD20) and fluorescein-conjugated anti-Bp50 antibody (green), we found that Bp50 was only detected on Bp35 + B cells (Figure 1) in blood or tonsils. Blood B cells consistently demonstrated slightly lower levels of Bp50 than tonsillary B cells; this is consistent with HLA-DR expression, (JA Ledbetter, et al., 1986, Human Immunol. 15-30-40) and with gp54 expression 45 (Wang, et al., 1979, J. Exp. Med. 149: 1424-1433) which are also lower in blood B cells. Bp50 was detected at corresponding levels on tonsillary B cell subpopulations that were separated on Percoll gradients to supernatant and dense fractions. Using our PE-conjugated mAb against the T-cell marker, CD3 (T3), and NK cell-bound marker, CD16 (Fc receptor) (JA Ledbetter, et al., 1979, Immunol. Rev. 47: 63-82) , we found that Bp50 could not be detected on T cells or NK cells.

Using bi-color IF, we also found that CD3 + PHA blasts that were found to contain high levels of IL-2 receptors, Bp50 could not be detected.

The G28-5 antibody reacted with a single polypeptide on tonsillary lymphocytes, which according to SDS electrophoresis under non-reducing conditions had a molecular weight of about 50 Ka (Figure 2A). This molecule is larger than previously described B cell markers in the same molecular weight range such as Bp39 or Bp45 (Zipf, et al., 1983, J. Immunol. 131: 3064-3072; Ch. Kintner, et al., 1981, Nature 294, 458-460); E.A. Clark, et al., 1986, in Leukocyte Typing II, eds. Reinherz, et al., Springer Verlag,

Berlin, chapter 12 vol. 2, 155-167; Slovin, et al., 1982, Proc. Wet. Acad. Sci. USA 79: 2649-2653; D.A.

195022 8

Thorley-Lawson, et al., 1985, J. Immunol. 134: 3007-3012 and Figure 2B).

The exposure time for this gel was chosen so that the molecular weights of the other B cell markers could easily be compared to Bp50. The Bp39 marker, unlike Bp50, can be detected on granulocytes and Bp45, unlike Bp50, is limited to B cell blasts. Antibodies to Bp39 5 (41-H16) and BP45 (MNM6, Blast-1, Blast-2) that had become available through an international workshop (EA Clark, et al., 1986, in Leukocyte Typing II, eds. Reinherz, et al., Springer Verlag, Berlin, Chapter 12 Vol. 2,155-167) did not block the binding of fluoresceinated anti-Bp50 antibodies to B cells. Based on tissue distribution, biochemical analysis, and blocking studies, therefore, the G28-5 monoclonal antibody recognizes a 50 * Kd structure that differs from 10 other known B cell antigens.

4.2.2. The presence of Bp50 is limited to B cells

Both distribution studies on hematopoietic tissue and cell lines and detailed two-color flow cytometric analyzes showed that Bp50 can only be detected on B lymphocytes. As shown in Figure 3, the presence of Bp50 appeared on a small subset of blood lymphocytes and on a large population of tonsillary lymphocytes. Almost all Bp50 + cells in both blood and tonsils were also found to contain Bp35 and HLA-DR, but the CD2 (Figure 1) or CD3, T-cell molecules or the IgG Fc receptors found on NK cells could not be detected on this. . Furthermore, ConA-activated CD3 + T cell blasts showed the presence of IL-2 receptors, but no Bp50 could be detected on this.

Two-color flow cytometric analyzes allow a quantitative measurement of the density relationship between two antigens present on the surface. We have previously demonstrated that the dense, resting B cells in the mantle zone of secondary follicles were found to contain IgM and low levels of Bp35, while the superimposed, activated B cells in the germinal center are IgM negative and elevated levels of Bp35 showed (JA Ledbetter, et al., Human Immunol. 15:30).

Figure 3 shows that both IgM positive and IgM negative B cell subsets were found to contain Bp50 in equal amounts, indicating that Bp50 is expressed on both resting B cells and B cells activated in vivo.

4.3. Enhancement of B cell extension with anti-Bp50 antibody

As explained earlier, B cells can be activated with low doses of anti-μ chain-specific antibodies. We recently found that the B cell specific marker Bp35 (B1), a 35 kDa polypeptide, can also play a role in early B cell activation: the 1F5 mAb against Bp35 activates, as well as low doses of anti-μ antibody , B cells to an increase in cell volume and RNA content and to expansion using BCGF (EA Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770); J.T. Golay, et al., 1985, J. Immunol. 135: 3795-3801). It was therefore interesting to compare the effect of anti-Bp50 mAb in the extension of untreated B cells or B cells activated with either anti-Bp35 or anti-μ antibodies (Table A). Anti-Bp35 in solution or anti-μ antibodies bound to Sepharose beads could only stimulate some B cell extension under appropriate conditions (Table A, line 40 1); in contrast, anti-Bp50 antibodies alone did not stimulate expansion (Table A, Line 2). However, anti-Bp50 mAb significantly enhanced the extension when growing with anti-μ beads or with anti-Bp35. Anti-Bp50 or BCGF appeared in this respect (Table A, Line 3). It was therefore important to determine whether anti-Bp50 and BCGF together could elicit B cell expansion. As illustrated in Table A, line 4, anti-Bp50 and BCGF together did not elicit extension, but extension of either anti-μ or anti-BP35 45 activated cells was slightly more enhanced than by any stimulant alone. BCGF, when used with anti-Bp50 without activators, had no effect on the expansion of dense B cells, even when anti-Bp50 was used at doses ranging from 0.1 to 10 pg / ml.

TABLE A

50 Enhancement of anti-Ig or anti-Bp35 induced B cell extension with anti-BP50 antibodies

Average expansion ± S.E. of B cells (measured as cpm) grown with: control Co-stimulant media anti-p-correis anti-Bp35 55 ---- 1 none 1,212 ± 547 10,219 ± 462 5,539 ± 308 9 195022 TABLE A (continued)

Amplification of anti-Ig or anti-Bp35 induced B cell extension with anti-BP50 antibodies

Average expansion ± S.E. of B cells (measured as cpm) grown with: 5 ----- rule Co-stimulant media anti-p-beads anti-Bp35 2 anti-Bp50 719 ± 718 38,792 ± 1,329 25,465 ± 616 3 BCGF 456 ± 217 14,217 ± 445 9,443 ± 343 10 4 anti-Bp50 + BCGF 1,456 ± 126 54,393 ± 537 46,488 ± 3,387 Expansion of dense Ttonillary B cells (95% surface area of IgM * cells) was measured on Day 3 in the manner described. Briefly, 2x10 * cells / 200 µl well were grown in quadruplicate for 48 hours with RPM11640 medium containing 15% bovine bovine serum plus additives without antibody or with either 2C3 monoclonal antibody against μ chains linked to sepharose beads ("anti-μ 15 - grains (V50 pg / ml) or free 1F5 anti-BP35 antibody (5 pg / ml) Cultures containing media, "anti-μ grains", or anti-BP35 antibody were administered alone or with BCGF (5% final concentration, Cytokine Technology, Buffalo, New York, with no detectable IL-1 or IL-2 activity), with anti-BP50 (1: 1000 dilution of ascites) grown as co-stimulants, and after 40 hours a 3H thymidine, and counts per minute were measured after 18 hours.

4.3.1. Anti-BP50 mAb enhances expansion only after B cells are activated by anti-Bp35 or anti-μ antibodies

The results in Table 1 indicate that anti-Bp50 mAb is not in itself capable of inducing expansion. As shown in Figure 4, doses of anti-Bp50 mAb (G28-5) ranging from 0.05 pg to 2.0 pg / ml had no effect on 3 H-thymidine incorporation. However, in the presence of optimum levels of anti-Bp35 mAb, amounts of anti-Bp50 antibodies of only 0.1 to 0.5 pg / ml significantly enhanced the extension. Although not indicated in Figure 4, even 50,000 to 70,000 cpm were detectable at the optimum time of expansion when highly purified B cells were grown with anti-Bp35 antibodies plus anti-Bp50 antibodies. Furthermore, it was observed that doses of anti-Bp40 mAb greater than 2-5 pg / ml were less effective than doses in the range of 100-200 ng.

These results indicated that anti-Bp50 mAb can only function after B cells have been activated by other signals. The data shown in Figure 5 indicate that this is indeed the case. When B cells were first activated with anti-Bp35, the anti-Bp50 mAb could only be added after 24-48 hours later to enhance this expansion on day 4. In contrast, in the case that the cells were first treated with anti-Bp50, anti-Bp35 mAb was only effective if it was added within a few hours of the start of the culture. Similar results were found when anti-p instead of anti-BP35 mAb was used.

4.3.2. Anti-Bp50 mAb do not give activation of B cells from G0, but do induce activated B cells until the cell cycle continues, 40 We previously found that anti-Bp35 mAb, as well as small doses of anti-IgM, resting tonsillary B- to magnify cells in G0 (EA Clark, et al., 1986, Leukocyte Typing II, eds.,

Reinherz, et al., Springer Verlag, Berlin, Vol. 2, 455-462) and until transition of the G1 phase of the cell cycle (J. T. Golay, et al., 1985, J. Immunol. 135: 3795-3801). It was therefore interesting to compare the ability of anti-Bp50 mAb with that of anti-Bp35 mAb for their effects on 45 B cell activation. As shown in Figure 6A, unstimulated dense tonsillary B cells even after 3-4 days in culture had a uniform RNA profile characteristic of cells in G0 (Z. Darzynkiewicz, 1980, Proc. Nat. Acad. Sci. USA 77 : 6696-6699). However, about 15-30% of the anti-Bp35 mAb or anti-IgM stimulated cells had an increased RNA content indicating transition of Gv. In contrast, neither anti-Bp50 mAb (Figure 6B) nor BCGF (Figure 6C) induced only transition from significant 50 numbers of B cells to Gv For example, 2 days after activation, anti-Bp35 mAb and anti-IgM mAb induced 13.5% and 20.9% of tonsillary cells to transition to G ,, treated with anti-Bp50 mAb (2.7%) or BCGF (3.2%), remained at media control levels (2.2%). However, when either anti-Bp50 mAb or BCGF together with anti-Bp35 mAb or anti-IgM were added, the amount of cells that switched to G increased dramatically. Similarly, anti-Bp50 and 55 BCGF alone did not induce B cells to transition to the S phase (Table B), but together with either anti-B35 or anti-IgM they led to a two to three-fold increase in the number of cells in the S phase.

195022 10

TABLE B

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

Competence signal Progression G0% Cells G, S / Ga / M

5 signal media none 89.9 7.1 2.5 anti-BP35 none 80.4 14.5 3.7 anti-IgM none 65.6 27.6 5.7 10 media anti-BP50 83.6 12.0 3.3 anti-Bp35 anti-BP50 54.1 35.5 9.7 anti-IgM anti-BP50 43.6 36.2 16.2 media BCGF 85.4 11.7 2.2 anti-Bp35 BCGF 56, 6 32.6 11.6 15 anti-IgM BCGF 48.4 36.1 14.1

Percentage of cells in G0, Gv or S and G2 determined by the acridine orange staining procedure (Z. Darzynkiewicz, et al., 1980, Proc. Nat. Acad. Sci. USA 77: 6696-6699); 1 x 10® closed tonsillary lymphocytes with anti-Bp35 (r pg / ml), anti-IgM on beads (50 pg / ml), anti-BpSO (0.4 pg / ml), BCGF (5%) or combinations such as shown.

4.3.3. Optimal conditions for enhancement of B cell extension with anti-Bp50 antibodies Antibodies to Bp50 have little or no demonstrable effect on dense resting B cells (Table C). However, in the presence of agents that can activate B cells such as anti-IgM, anti-Bp35 and TPA, anti-Bp50 mAb clearly enhanced the extension.

However, Anti-Bp50 did not show marked enhancement of expansion in the presence of various interleukins, such as purified IL-1, recombinant IL-2 and BCGF (low). A comparison of the effects of anti-Bp50 with those of BCGF (low) showed that the same agents that gave anti-Bp50 mAb constimulation also provided costimulation with BCGF (low) (Table C). Of particular interest was the finding that BCGF and anti-Bp50 mAb together did not provide costimulation of resting cells.

30

TABLE C

Enhancement of B cell extension with anti-Bp50 antibodies or B cell growth factor

Average expansion ± S.E. of B cells (measured as cpm) characterized with: -

Co-stimulant media anti-Bp50 (200 ng / ml) BCGF (5%) none 96 ± 1 267 ± 15 285 ± 74 anti-IgM 5,833 ± 391 41,634 ± 2,103 25,094 ± 61 40 anti-Bp35 (5 pg / ml) 457 ± 45 8,143 ± 280 1,733 ± 32 TPA (2 ng / ml) 7,361 ± 537 21,163 ± 871 13,063 ± 1,030 IL-1 (10 U / ml) 264 ± 2 308 ± 23 221 ± 8 IL-2 (100 U / ml) 204 ± 34 350 ± 7 220 ± 11 BCGF (5%) 220 ± 7 851 ± 28 270 ± 18 45 -------

Dense Tetillaillary B cells (more than 95% slgM * cells) cultured for 48 hours at 2 x 10 5 cells / well, followed by a burst of 3 H-thymidine before counting after 24 hours.

The course of time of the expansion enhanced by anti-Bp50 mAb is shown in Figure 7. The largest expansion occurred on day 4 and then decreased regardless of whether the cells were activated with anti-Bp35 mAb or 50 other activators such as anti-IgM or TPA. The progress of the extension enhanced by BCGF or anti-Bp50 mAb was the same.

An amount of anti-Bp50 antibodies of only 0.05 pg enhanced the extension. An optimum dose of 0.3 pg / ml was used in subsequent studies. It was always observed that when whole antibody molecules were used, doses of anti-PB50 greater than 2 pg / ml 55 were less effective than doses in the preparation of 0.1-0.5 pg / ml.

Human B cells are extremely sensitive to inhibitory actions exerted via the Fc receptors of surface Ig binding antibodies (Parker, 1980, Immunol. Rev. 52: 115; M.K.

11, 195022, Bijsterbosch, et al., 1985, J. Exp. Med. 162: 1825-1836). It was therefore important to compare the efficacy of complete anti-Bp50 mAb with that of anti-Bp50 Fjab fragments. Over a 100-fold dose range, F (ab ') 2 fragments were clearly as effective as or more effective than full antibody in enhancing B cell expansion (Table D). Therefore, the Fc domain of anti-Bp50 mAb is not needed for anti-Bp50 to exert its effect and can, at most, have an inhibitory effect.

TABLE D

The Fc domain of anti-Bp50 antibodies is not required for enhancement of B cell expansion. Average expansion of B cells, measured as cpm grown with:

Anti-Bp50 Dose (pg / ml) media anti-Bp35 none - 295 ± 16 269 ± 27 15 complete Ab 0.125 278 ± 32 5.140 ± 20 1.25 275 ± 24 4,686 ± 342 12.5 163 ± 15 3,852 ± 203 F (ab ') 2) 0.125 594 ± 21 10.635 ± 449 1.25 531 ± 3 10.893 ± 575 20 12.5 279 ± 8 9.411 ± 870

The cell culture conditions were as described in Table C.

4.3.4. Differences between anti-Bp50 mAb and BCGF (low) activity

Anti-Bp50 mAb and BCGF (low) had a similar effect on B cells and were costimulatory with the same means (Table C). However, there are various indications that anti-Bp50 and the BCGF used in this study work via different signals. First, Bp50 molecules, other than BCGF (low) receptors, (MK Bijsterbosch, et al., 1985, J. Exp. Med. 162: 1825-1836) are already expressed on resting blood B cells (Figure 3). Secondly, although both anti-Bp50 and BCGF (low) function most effectively when added after anti-Bp35 or anti-IgM, 30 anti-Bp50 mAb was clearly most effective when it was 12 hours after the onset of culture was added (Figure 8A). In contrast, BCGF (low), even if it is added 24 hours after the start of the cultures, could still give an optimal enhancement of the extension (Figure 8B). These experiments, using the Howard and Paul approach (1983, Ann. Rev. Immunol. 1: 307), indicate that a Bp50 dependent signal can normally exert its effect on BCGF.

Both anti-Bp50 mAb and BCGF (low) enhanced extension of B cells that were activated with anti-Bp35 or anti-IgM (Table C). However, the effect of anti-Bp50 mAb and BCGF (low) was additive in many experiments (Figure 7). Figure 9 shows the influence of the concentration of BCGF (low) in an experiment in which anti-Bp50 mAb was used in its optimum concentration (0.2 pg / ml). BCGF (low) could further enhance the extension of resting B cells in the presence of anti-Bp50 after activation with anti-IgM 40 or with anti-Bp35. Optimal concentrations of BCGF (low) were 5-10%, while from 25% there was an inhibitory effect. Thus, when anti-Bp50 and BCGF (low) were both used at their optimum concentrations, they still showed additive effects on B cell expansion.

Finally, both normal and malignant B cell subsets differed in their responses to anti-Bp50 mAb and BCGF (low). For example, some blood B cells responded to BCGF (low) but not to anti-Bp50 45 mAb (Table E). An additional activation signal such as anti-Bp35 (Table E) or TPA (Figure 10) was consistently needed to allow blood B cells to respond to anti-Bp50 mAb. While dense tonsillary B cells generally did not respond to BCGF or anti-Bp50 mAb, supernatant B cells did respond (Table E). Malignant B cells also differed in their response to anti-Bp50 mAb versus BCGF. For example, some B cell lymphomas responded to TPA plus BCGF (low), but not to TPA plus G28-5 50 anti-Bp50 (Figure 10B and D). In contrast, dense tonsillary B cells and peripheral blood B cells responded to TPA plus either BCGF (low) or G28-5 anti-Pb50 (Figures 10A and C).

195022 12

TABLE E

B cell subsets differ in their response to anti-Bp50 or BCGF

Mean expansion (± S.E.M.) measured as cpm of lymphocytes from: 5 ------—-

Blood Tonsils

Incentive Exp. 1 Exp. 2 Exp. 3 floating above 10 none 527 ± 70 659 ± 133 906 ± 106 385 ± 43 387 ± 12 BCGF 13,918 ± 1,082 37,594 ± 2,023 3,347 ± 174 332 ± 33 1,451 ± 57 anti-BP50 (0.5 pg / ml) 519 ± 28 1,013 ± 81 1,151 ± 28 472 ± 8 1,543 ± 20 anti-Bp35 (5 pg / ml) 554 ± 90 645 ± 89 665 ± 115 2,415 ± 80 1,129 ± 68 anti-Bp50 + anti-Bp35 - 12,274 ± 546 15,667 ± 333 36,589 ± 1,335 4,843 ± 136 15 anti-Bp50 + BCGF - - 3,943 ± 115 1,342 ± 19 2,899 ± 91

The cell culture conditions were as described in Table A. Blood wool adhering lymphocytes (B cells plus monocytes) were depleted on monocytes by overnight incubation in plastic dishes before stimulation took place (Exp. 1 and Exp. 2). in Exp. 3, blood B lymphocytes were depleted on monocytes by incubation for 1 hour in plastic dishes prior to stimulation. Tonsillary B lymphocytes were fractionated using Percoll gradients into dense (pellet) or supernatant (fraction 1) subsets (32).

4.4. Applications of anti ~ Bp50 antibodies

The anti-Bp50 antibodies of the present invention can be used in vivo or in vitro as an "adjuvant" to enhance an immune response to a vaccine or to enhance the immune response of an individual in a state of immunosuppression.

The anti-Bp50 antibodies of the present invention can be used in vitro for the determination or secretion of cells containing the Bp50 antigen and / or for the detection of any released Bp50 in body fluids. In this case, the anti-Bp50 antibody could be labeled, for example, with a radiolabel, fluorescent compound, enzyme, enzyme substrate or a dye. For secreting cells containing the Bp50 antigen, the anti-Bp50 mAb can be coupled to a solid support.

4.4.1. Applications of anti-Bp50 antibody enhancement of B cell extension From previous studies it was found that the effects of B cell transition from G0 to G10 to G phase have little or no effect on B cell expansion. Yet these same agents can bring B cells to a point in cell activation where they are sensitive to growth factors. In contrast, growth factors such as BCGF or IL-2 alone do not have an effect on resting B cells, but they do enhance the growth of activated B cells.

Although anti-Bp35 mAb activated B cells until transitioning to the G ^ phase of the cell cycle, it only induced little or no expansion. Anti-Bp50 mAb had the opposite effect. It could not activate resting B cells, but even if it was not added until 12-24 hours after activation, it could induce B cell growth.

Previous studies have identified several t-cell-derived BCGFs that, as well as 45 anti-Bp50, enhance B cell expansion. Both high and low molecular weight forms of B cell growth factors have been identified and have been shown to have additive effects for various types (JH Kehrl, et al., 1984, Immunol. Rev. 78: 75-96; T. Kishimoto, 1985, Ann. Rev. Immunol 3: 133-157, SL Swain, et al., 1983, J. Exp, Med 158: 822-835, M. Howard, et al., 1984, Immunol Rev. 78: 185-210 JL Ambrus, et al., J. Clin Invest 75: 732-739; JL Ambrus, 1985, J. Exp. Med. 162: 1319-50 (1335). Bp50 could therefore be a receptor for one of these factors.

With the exception of IL-2 receptors and the C3d receptor, the receptors on B cells for growth signals have not yet been identified. The mAb AB-1 reacts with a B cell marker that blocks only expressed top activated B cells and BCGF-dependent extension, and thus could recognize the BCGF receptor or a related structure. Bp50 appears to differ from the AB-1 marker because the 55 AB-1 mAb does not block the binding of the G28-5 anti-Bp50 antibody, and unlike the G28-5 only reacts with activated B cells (LKL Jung, et. al., 1984, J. Exp. Med. 160: 1919-1924). Bp50 is present on all B cells, which appears to be not the case for BCGF receptors on the basis of absorption analysis and direct binding tests. Our current data indicate that Bp50 and the BCGF low molecular weight receptor are distinct structures. Using a rabbit heteroantiser, Wang and associates (Wang, 1979, J. Exp. Med. 149: 1424-1433) have previously described a 54-kDa glycoprotein, gp54, which, like Bp50, is expressed on all B cells but in lower levels on 5 blood B cells than on tonsillary B cells. It is possible, but unlikely, that the rabbit heteroantiser and anti-Bp50 recognize the same or related structures: unlike anti-Bp50 mAb, the rabbit antiserum against gp54 alone was sufficient to stimulate B cell expansion.

Anti-Bp35 alone can activate B cells from G0 to G other than anti-Bp50, and can therefore be referred to as an "activation" signal. Whether or not Bp35 functions in early B cell activation alone is not yet clear because anti-Bp35 antibodies can stimulate some B cells to divide (EA Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82 : 1766-1770). Similarly, Bp50 may not only function as a "growth" signal: anti-Bp50 antibodies together with activation signals (anti-Bp35 or "anti-μ) not only leads to enhancement of the extension but also to an increase in the total number of B cells passing into G1 (Table B). In other words, anti-Bp50 as a constimulant leads to promotion of the progression of both the activation (G0 to G.) and the growth (G to S) phases of the cell cycle. The BCGF used in these studies also had a similar activity (Figure 6C). Anti-Bp35 and anti-Pb50 (or BCGF) therefore appear to be very analogous to the "competence" and "progression" factors described in fibroblast growth regulation studies. How B cells respond to anti-Bp35 or anti-Bp50 can clearly depend on their state of differentiation or activation.

Here we have shown that two mAb, anti-Bp35 (a "competence" signal) and anti-Bp50 (a "progression" signal), together can significantly increase highly purified B cells in the absence of antigen or other known factors induce. The natural ligands for these structures are not yet known. However, since mAb against suitable epitopes can mimic both soluble factors and cell-mediated interactions, appropriate combinations of mAb may be used to control and regulate human B cell expansion or differentiation. This in turn will contribute to the design of in vivo strategies for the control of human diseases such as B cell malignancies, immunodeficiencies and certain autoimmune diseases.

The new monoclonal antibody G28-5, which reacts with a single chain polypeptide of approximately 50 Kd expressed on the surface of human B cells, is only one monoclonal antibody against Bp50. It can be expected that other anti-Bp50 antibodies can also enhance the expansion of activated B cells. Because human B cell expansion can also be enhanced by T cell derived BCGFs including low and high molecular weight BCGF, we compared the activity of anti-Bp50 G28-5 with that of a BCGF preparation that predominantly has low molecular weight BCGF contains. Anti-Bp50 G28-5 and BCGF (low) showed great similarity in that they gave constimulation with the same activating agents (anti-IgM, anti-Bp35 and TPA), but did not cause any constimulation with each other or with IL-1 or IL-2. Furthermore, the activity of anti-Bp50 G28-5 was not dependent on its Fc domain because Ftab fragments of G28-5 were functionally active. This indicates that soluble anti-Bp50 as well as soluble BCGF does not require Fc receptor-bearing accessory cells to exert an effect. Furthermore, both anti-Bp50 and BCGF are only effective in the presence of an activation stimulus. In other words, anti-Bp50 and BCGF are not "competence" factors, but rather promote the "progression" of B cells throughout the cell cycle.

Several results indicate that Bp50 is not the receptor for the BCGF (low) used in this study. Bp50 is expressed on unactivated B cells while BCGF (low) receptors are not expressed thereon. Candidate structures for the PCGF (low) receptor, unlike Bp50 alone, are expressed on activated B cells. Furthermore, both normal and malignant B cell populations differ in their response to anti-Bp50 versus BCGF (low) (Table E and Figure 10). For example, some B lymphomas show expansion in response to BCGF (low), but not in response to anti-Bp50. Finally, in a number of experiments, optimal concentrations of anti-Bp50 and BCGF together called for more expansion than either of them alone. Anti-Bp50 mimics the activity of other BCGF, such as BCGF (high) that give costimulation 50 with anti-IgM (JL Ambrus, et al., 1985, J. Exp. Med. 162: 1319; JL Ambrus, et al., 1985, J. Clin. Invest. 75: 732). This indicates that Bp50 could function as the receptor for BCGF (high).

While Bp50 may be a receptor for a soluble ligand, Bp50 may, on the other hand, function as a cell-cell-mediated signal receptor that regulates BCGF receptor levels and / or autocrine production. Indications for serving differentiation antigens such as enhancers of an autocrine-55 receptor pathway come from T cell studies. Monoclonal antibody to the Lp220 general leukocyte antigen enhances the extension by increasing the IL-2 receptor expression on activated T cells (J. A. Ledbetter, et al., 1985, J. Immunol. 135: 1819). An analogous mechanism could sag

Claims (2)

195022 14 with anti-Bp50 and expression of certain BCGF receptors. Bp50 and BCGF (low) are apparently under some coordinated control because BCGF, as well as IL-1 and IL-2 receptors, enhances the expression of Bp50 on certain leukemic cells. The Bp50 molecule also shows similarities with the Tp44 molecule that exerts an influence on IL-2 production. We and others have demonstrated that the anti-Tp44 antibody enhances the expansion of T cells activated by anti-CD3 or TPA (JA Ledbetter, et al., 1985, J. Immunol. 135: 2331; Hara, et. al., 1985, J. Exp. Med. 161: 1513). Similarly, anti-Bp50 enhances the expansion of B cells that are activated by anti-Bp35 or TPA. The Tp44 signal functions by stimulating IL-2 production rather than stimulating T cell growth. The Bp50 signal could presumably function in an analogous manner by stimulating B-cell autocrine production (Gordon, et al., 1984, Nature; Lond. 310: 145-147). 4.4.2. Other uses of anti-Bp50 antibodies In addition to therapeutic applications, anti-Bp50 antibodies themselves have other uses in both in vitro and in vivo diagnostic assays, separation schedules, etc. The Bp50 receptor can be used to prepare the antibodies of the invention. In addition to therapeutic treatment of diagnostic assays, the antibodies of the present invention could be used to identify or separate cells that express the Bp50 antigen. In addition, when a suitable radiolabel or radiopaque compound is bound to the ligand, the antibody could be used for in vivo imaging of tumors expressing the Bp50 antigen. Other applications will become apparent to those skilled in the art from the foregoing description. 5. Depots of cell lines The following hybridoma has been deposited with the American Type Culture Collection, Rockville, MD, and has been assigned the stated registration number: Hyhbridoma ATCC registration number G28-5 HB9110 The present invention is not limited in scope by the deposited hybridoma because the deposited embodiment is intended as a single illustration of one aspect of the invention and all cell lines that are functionally equivalent belong to the scope of the invention. 35 A substantially pure antibody or active fragment thereof that binds to a polypeptide that is specifically present on the surface of human B cells, characterized in that 40 (a) binds the antibody to a 50 kDa human B cell specific surface polypeptide (Bp50), (b) after binding to Bp50, which is present on the surface of an anti-Bp35 or anti-Ig activated B cell, the expansion of the activated B cell as measured in vitro by [3 H] - thymidine incorporation, at a concentration of 0.1 to 0.5 pg / ml. Antibody or active fragment thereof according to claim 1, characterized in that it is the G28-5 monoclonal antibody produced by hybrido-45 cell line ATCC HB 9110 or an active fragment thereof. Hereby 10 sheets of drawing