IE60486B1 - Ligands and methods for augmenting b-cell proliferation - Google Patents

Abstract

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

Description

Λβ 1. INTRODUCTION The present invention is directed to ligands, such as antibody molecules or fragments of antibody molecules or other ligands such as lymphokines which bind to a SOkDa Bcell surface marker, herein referred to as Bp50, that functions in B-cell proliferation but not in early B-cell activation. The present invention is also directed to the Bp50 3-cell antigen itself. In a particular embodiment of the present invention, a monoclonal antibody, G28-5, is described that defines 3p50 and appears to play a role in the proliferation of activated B-cells but has no detectable effect on the proliferation of resting B-cells.

The ligands, such as antibodies, lymphokines and fragments thereof of the present invention can be used t© direct and regulate human 3-cell proliferation and/or differentiation. In addition, the ligands of the present invention may be modified by the attachment of other compounds which can be used in th® treatment and/or detection of malignant cells that express the SpSO antigen. 2. BACKGROUND OF THE INVENTION Th® activation of resting B-cells from to G^ phase ©f th® cell cycle and the subsequent induction of activated B-cells to proliferate are distinct steps requiring distinct regulatory mechanisms. Some agents, including murine B-cell stimulating factor-pl (BSF-pl) (Rabin, et al., 1985, Proc. Nat. Acad. Sci. USA 82, 2935-2939) or low doses of antiimmunoglobulin (anti-Ig) (DeFranco, et al., 1955, J.

Immunol. 135:37-94; Wettel, et si., 1984, J. Immunol. 133:2327-2332; DeFranco, et al., 1982, J. Exp. Med155:1523-1536; Muraguchi, et al., 1984, J. Immunol. 132:176-180), are activation* or ’competence* factors.

That is, they induce 3-cells to enlarge, synthesise more RNA, and enter G,, but alone they do not induce DNA synthesis in 3-cells. Other ''growth* factors, such as human B-celX growth factor (BCGF) and interleukin-2 (IL-2) cause activated B-cells to traverse the cell cycle and enter S phase but do not trigger resting B-cells (Xehrl, et al., 1984? Immunol. Hev. 13:75-96,: Muraguchi, et al. 1934, J.

Immunol. 132:176-180;; Zubler, et al. 1984, σ. Exp. Med. 160:1170-1183? Jung, et ah, 1984, J. Exp. Med. 160:15971604).

A number of factors that promote the growth of B-cells have now been described by investigators of both «urine and human systems. These include B-cell growth factors (BCGF) derived from several different sources including T-cell lines or hybridomas, B-cell lines, or dendritic cells.

Although both interleukin-1 (IL-1) and interleukin-2 (IL-2) have been shown to augment B-cell growth, they apparently are distinct from certain BCGFs. For instance, monoclonal antibodies (mAh, to a aurin® BCGF (O'Hara, et al., 1985, Nature (Lond.) 315:333) or human BCGF (Ambrus, et al., 1985, J. Exp. Med. 162:1319) block BCGF activity but sot IL-1 or IL-2 activity,.. Although distinct from IL.-1 or IL-2, the SCGFs themselves appear to be heterogeneous based on biochemical data and differential activity on different Bcell subsets or costimulation assays. For instance, 60kilodalton (kDa) high-molecular-weight human BCGF, BCGF (high), has been identified that is distinct from a 12-kDa low-molecular-veight fora, BCGF (low) (Ambrus, et ai., 1985, J. Clin. Invest. 75:732). The. cDNA encoding a 20-kDa murine BCGF, tentatively designated B-cell stimulating factor pi (BSF-pl,, has recently been cloned and sequenced (Soma et al., 1986, Nature 319:640). The recombinant lymphokine not only has BCGF activity but can also activate resting B-cells and induce the differentiation of IgG, producing cells; thus it differs from human BCGF (high) and BCGF (low) both in its molecular weight and in its range of activity.

These activation and growth signals presumably regulate S cells by interacting with specific 3-cell surface structures. In addition to the antigen-specific signal -'-through surface Ig, several other candidate 5-cell surface polypeptides have been identified that nay in some way function in the activation or growth of B-cells- For 10 instance, the cell surface receptors for IL-1 (Dower, et al., 1985, J- Exp. Med. 160:501) and IL-2 (Robb et al., 1984, J. Exp. Med. 160:1128) have been characterized, and recently functional XL-2 receptors have been identified on B-cells (Zubler, et al., 1934, J. Exp. Med. 160:1170; Jung, et al. „ 1984,, J. Exp. Med. 160:1597; Muraguchi, et al.,, 1985, J. Exp. Med. 161:181). However, receptors for B-cell growth and activation factors have yet to be fully characterised. Several candidate B-cell surface polypeptides have been identified that may is some way function in the activation or growth of B-cells. For example, Subbarao and Mosier (Subbarao, et al., 1983, Immunol. Rev. 69:81-97) found that monoclonal antibodies (mAb) to the murine B-cell antigen Lyb2 activate B-cells, and recently evidence has been presented suggesting Lyb2 may 2g be the receptor for BSF-pl (¥akura, 1985» Fed. Proc. 44:1532). Similarly, we have found that appropriate aAb (1F5) to a 35 kDa polypeptide, 3p35e, activates human B-cells from Gg into G, (Clark, et al.» 1985, Proc. Sat. Acad. Sci. USA 82:1766-1770; Gollay, et al., 1935, J. Immunol. 135:3795-3301). Aggregated C3d or antibodies to the 140 RD& C3d receptor, Bpl40, cause proliferation of B-cells that ar© T-cell dependent (Melchers, et al., 1985, Mature 317:264267; Memerow, et al., 19Θ5» J. Immunol. 135:3068-3073; Frade _et al., 1985, Eur. J. Immunol. 15:73-76). Although BCGFs have been identified in both mouse and man, the receptors for these factors have not yet been isolated. Wang and coworkers (Wang, et al., 1979, J. Exp. Med. 149:1424-1433) made a polyclonal antisera that identified a 54-kDa polypeptide (gpS4) on human B-cells and showed that the rabbit antisera to gpS4 induced tonsillar 3-cells to divide. Recently, Jung and Eu (Jung, et al., 1984, J. Exp. Med. 160:1919-1924) isolated a sAb (AB-1) to a 55-kDa antigen restricted to activated B-cells that blocks BCGF-dependent proliferation. However, whether or not either anti-gp54 or AB-1 recognise a BCG? receptor is not yet known. 3. SUMMARY OF THE INVENTION The present invention is directed to substantially pure ligands which (a) bind to BpSO, s 50kDa B-cell specific surface antigen described herein, and (b) augment the proliferation of activated B-cells. The invention is also directed to the BpSO antigen itself, which is defined by monoclonal antibody G28-5 and functions in proliferation of activated B-cells.

In addition the invention is directed to ligands which bind to BpSO, but do not demonstrate a biological effect or function such as augmentation of the proliferation of activated B-cells.

The ligands of the present invention include antibody. molecules, monoclonal antibody molecules and fragments of these antibody molecules which contain the antigen combining site or chemically modified antibodies and fragments; such fragments include but are not limited fo the Pv, Fab, P(ab'),( Fab·*1 and the like. Xn addition, the ligands of the present invention comprise lymphokines, which can include but are not limited to human B-cell growth factors as well as chemically modified lymphokines. The ligands of the present invention can be chemically modified, for example by linking or coupling a compound to the ligand.

Such compounds include but are not limited to cytotoxic agents# therapeutic agents# chemotherapeutic agents# labels such as radiolabels, dyes# enzymes# radioopaque compounds# and th® like. The ligands of the present invention can in their modified or unmodified form# be used to direct# regulate and modify human 3-cell proliferation and/or differentiation.

The present Invention is based upon the discovery that two human B-cell differentiation antigens# Bp35 and the ΒΙΟ cell antigen described herein, BpSO# apparently play distinctive roles as signal receptors in 3-cell activation. Monoclonal antibodies (mAh) to Bp35 and 3p50 both deliver positive signals to B-cells that stimulate their transition through the cell cycle. MAb to Bp35, like anti-Ig antibodies# functions principally to activate resting Bcells to become competent to enter the G, phase of the cell cycle. In contrast, a monoclonal antibody described herein or its Fiab’)^ fragment to BpSO, a 50-kDs polypeptide expressed on all B-cells, functions to stimulate activated 2q 3-cells to traverse th® cell cycle and augments the proliferation of activated 3-cells. Monoclonal antibodies to 3p35# like anti-lg antibodies# activate tonsillar B-cells and induce low levels of 3-cell proliferation. Xn contrast# anti-BpSO monoclonal antibody alone neither activates B25 cells nor induces 3-cells to proliferate, but together with anti-Bp3S or anti-Xg antibodies, augments 3-cell proliferation. Xn this respect the action off anti-BpS© antibody resembles the activity of B-cell growth factors (BCGF). As little as 0.05 ug/ml of anti-BpSO Is needed to 2Q augment proliferation and, like BCGF, anti-BpSO is effective even when added 12 to 24 hours after B-cells are activated with anti-Xg or anti-Bp35. Without additional exogenous signals, antl-3p35 and antI-3p50 antibodies together Induce strong proliferation of purified resting B-cells. These results suggest that the Bp3S and Bp50 surface molecules function in th® regulatory control of B-cell activation and progression through the cell cycle. The significance anti~Bp35 end like molecules have on the effect and action of the ligands of the present invention, is discussed in Clerk et _al., 1985, Proc. Natl. Acad. Sci. (USA) 82:1766-1770.

Although the activity of anti-Sp50 resembles that of BCGF (low) since both anti-BpSO and BCGF (low) are costinulatory with the same agents but not with each other and both anti-3p50 and BCGF (low) affect only activated 3cells end work in a soluble form, the activity of anti-BpSO can fce distinguished from the activity of BCGF (low), since the proliferation of B-cells stimulated with optimal amounts of anti-BpSO and anti-Bp35 (or anti-lg) can be augmented further with BCGF (low) and both blood B-cells and certain B-cell lymphomas respond differently to anti-BpSO versus BCGF. For optimal activity, anti-BpSO should be added within 12 hours of 3-cell activation, whereas BCGF (low) retains optimal activity even when added 34 hours after activation. Xn addition, 3p»Q is expressed on all B-cells while receptors or BCGF (low) are restricted to activated B-cells. Thus anti-BpSO and BCGF (low) aay coordinately regulate B-cell growth, but apparently do so through distinct signals.

Xn one embodiment of the present invention, the ligands which bind to BpSQ and augment the proliferation of activated B-cells can be used to increase an immune response» For example, these ligands which bind 3pS0 can b® used as an *adjuvant* to increase an immune response to a vaccine. Alternatively, these ligands can be used to increase the immune response of an immunosuppressed individual.

Xn another embodiment, the ligands of the invention can be chemically modified so that the eells to which the ligands bind are killed. Since all 3-cells express the Bp50 antigen, this approach would result in suppression of the immune response. For example, a cytotoxic drug linked to a ligand of the present invention can he used in vivo to cause Immunosuppression in order to cross histocompatibility barriers in transplant patients; alternatively, these modified ligands may be used to control autoimmune diseases.

In another embodiment of the present invention, malignancies such as tumor cells that express BpSO can be treated using a ligand of the invention linked to a chemotherapeutic agent useful In treating such neoplastic disease. These modified ligands can be used in vivo to direct the chemotherapeutic agent to any type of malignant cell which expresses the 3p50 antigen including cells which are not B-cells but which do express BpSO. When using the ligands of the invention which augment 0-cell proliferation, a particular advantage should be realized when treating Β» cell malignancies where the chemotherapeutic agent linked to the ligand comprises one that Is more effective in killing proliferating cell©; in this instance a potentiation of the drug action should be obtained.

Alternatively, the ligands of the invention can be used in vitro to identify or separate cells which express the BpSO antigen and/or to assay body fluids for the presence of the BpSO antigen which may or may not be shed. In addition, the ligands, of the invention can be used in vivo In order to image cells or tumors which express the BpSO antigen.

The purified BpSO antigen of the present invention can be used to make antibodies and to make or design other ligand© of the invention. In addition the BpSO antigen could be used in assays such as diagnostic immunoassays. Moreover, BpSO Itself aay be used as a mediator of cell Immunity in vivo or in vitro. 3.1. DEFINITIONS As used herein, the following abbreviations will have th® meanings indicated: AO - acridine orange 5 BCGF " 3-cell growth factor BCGF (high) « a 60 kDa human BCGF BCGF (low) » a 12 kDa human BCGF Bq35 « a 35 kDa B-celX specific surface polypeptide (CD20) -defined by mAh 1F5 BpSO * a 50 kDa 3-cell specific surface polypeptide defined by mAb G28-5 Fv « the variable region or antigen-combining site off an antibody molecule. This may be any fragment which contains the idiotype of jg the molecule including but not limited to the Fab, F(ab*),, Fab', and the like.

XF « immunofluorescence Ig « immunoglobulin IL-l « interleukin 1 .jq IL-2 " interleukin 2 kDa kilodalton mAb « monoclonal antibody SDS-PAGE « sodium dodecyl sulphate-polyacrylamide gel electrophoresis 2q TFA « l2-0-tetradecanoylphorbol-13 acetate 4. DESCRIPTION OF TBS FIGURES Fig. I- Expression of 3p50 is restricted to Bp35* Bcells. Two-color flow cytometric analysis of 50,000 cells was performed as described (Clark, et al., 1935, Proc. Nat. Acad. Sci. USA 82:1765-1770). The data are plotted as cell number versus log of green fluorescence and log of red fluorescence where 4-5 dots represent approximately a i ο doubling of fluorescence. The date are presented to show autofluorescent negative cells. PE (red) -anti-Bp35 (1F5) versus FITC (green) -anti-Bp50 (G28-5) staining shows that all Bp50+ cells are also 3p3S*.

S Fig. 2. Biochemical comparison of BpSO polypeptide with other B-cell surface antigens. Immunoprecipitation of 1^5 BpSO from surface **_I-labeled tonsillar cells was performed as described. Isolated antigens were electrophoresed on 10¾ SDS polyacrylamide slab gels without reduction. Gels were HO visualized with autoradiography and intensifying screens. Panel A: lane 1, anti~Bp50 (G28-5); lane 2, anti-Bp95 (G28-S); lane 3, sepharose-goat anti-mouse Ig only.

Exposure time: 4 days. Panel 3: lane 1, anti-3p50 (G28S); lane 2, anti-Bp4S (BLAST-2); lane 3, anti-Bp39 (G28-1); lane 4, anti-Bp39 (41-H16); lane 5, sepharose-goat antimouse Ig only. An exposure time of 2 days was selected so that the bands in lanes 2 to 4 were not overexposed and could be clearly distinguished relative to BpSO. one of three experiments.

Fig. 3. Two-color immunofluorescence analysis of 3p50 expression. Peripheral blood or tonsillar mononuclear cells were isolated by centrifugation on Ficoll and stained with PE (red)-conjugated G28-5 (anti-BpSO) in combination with fluorescein (green)-conjugated reference antibodies, including 2C3 (anti-IgM); 1F5 (anti-Bp35)? HBlOa (anti-DP); and 9.6 (anti-CD2, E receptor). Cells were analyzed with a FACS IV fitted with four decade log amplifiers in both red and green dimensions. Forward and right angle light scatter was used to gate out monocytes. Unstained cells are positioned at the back of the grid; red fluorescence Is to the right and green fluorescence is to the left. 1 Fig. 4. Dose response curves for augmentation of proliferation of dense tonsillar Er- B-cells by anti-BpSO antibodies as indicated: Media only, anti~Sp5Q only anti3p35 (5 ug/ml) only; BCGF only; anti-3p35 plus 3CGF; anti5 Bp35 plus graded doses of anti-Bp50. Mean proliferation + standard error of quadruplicate samples was measured on day 3.

Fig. 5. Anti-BpSO mAb are most effective at augmenting proliferation if added after a E-cell activation signal.

HO Dense tonsillar Sr- 3-cells were incubated for 4, days with media only, anti-BpSO (0.5 ug/ral) added at different tines after incubation, anti-Bp35 (5 ug/ral) added at different times after incubation; anti-BpSO kept constant to which anti-Bp35 was added later at different times; anti-Bp35 kept constant to which anti-BpSO was added to cultures at different tines. During the last 10 hr H-thyraidine was added and its incorporation was measured.

Fig. 6. Comparison of the ability of anti-Bp35 and anti-BpSO to induce resting tonsillar B-cells to leave the Gq stage of the cell cycle. Day 3 post treatment media only _), ®nti-Sp3S only (——} · and Ig only A, no additional additives; B, anti-BpSO (O.S ug/ral) added to each group; C, SI BCGF added to each group. Data is plotted as relative cell number versus log of AG red 2g fluorescence (PNA).

Fig. 7. Kinetics of 3-cell proliferation after stimulation with anti-3pSG versus BCGP. Dense tonsillar BB-cells were stimulated with media alone; 10% 3CGF only; anti-Bp35 only; anti-Sp50 only; anti-Bp35 + 10% BCGF; anti3Q Bp35 + anti-BpSO; and anti-3p35 + anti-3p50 + 10% BCGF, Proliferation was measured on the days indicated by an 183 hour pulse of H thymidine. Proliferation was measured in quadruplicate and standard errors are shown. One of three experiments.

Fig. S. Times after anti-Bp35 stimulation when anti5 Bp50 (A) or BCGF (B) optimally augment proliferation. Dense tonsillar E- B-cells were stimulated as shown and proliferation was measured by an 10 hour pulse of 3H thymidine on day 3» Media? anti-Bp35 only added at times indicated; anti-3p50 or BCGF only; anti=3p3S added at start ©f culture followed by addition of anti-3p50 or BCGF at times indicated; anti-BpSO or BCGF added at start of culture followed by anti-3p35. One of two experiments.

Proliferation was measured in quadruplicate and standard errors are shown. Doses used: anti-BpSS, 5 ug/ml; anti15 Bp50, 0.2 ug/nl; BCGF {low) 5%. concentrations used were as follows: anti-3p35, 5 ug/ml; anti-BpSO, 0.2 ug/ml; BCGF, 5¾. Fig. 9. Anti-BpSO and BCGF have additive effects on Bcell proliferation. Dens® tonsillar S- 3-cells were stimulated with graded doses of BCGF (low) together with 2q anti-BpSO only; anti-Bp35 only; anti-Ig-beads only ; antiBp35 + anti-BpSO; or anti-BpSO + anti-Xg. Proliferation was measured on day 3 after stimulation with an 18-hour pulse of * S3 thymidine. Proliferation was measured in quadruplicate and standard errors are shown. Ons of four experiments. 7g Doses used 10a cells: anti-Bp35, 5 ug/ml; anti-BpSO, 0.2 ug/ml; anti-Ig-beads, 50 ug/ml.

Fig. 10. Comparative effects off anti-Bp50 and BCGF on normal and malignant B-cells. Peripheral blood E- B-cells (A) or dense tonsillar E- B-cells (C) were stimulated with 30 or without TPA (75 ng/ml) in the presence of 10¾ BCGF or 1 ug/ml anti-BpSO. Two separate 3-cell lymphomas (panels 3 and D) were stimulated in the same way. Proliferation was 3 measured on day 3 by incorporation of H thymidine during a 12-hour pulse. Proliferation was measured in quadruplicate and standard errors are shown.

. DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to ligands which (a) bind to BpSO, a SOkDa B-cell specific surface polypeptide, and (b) augment the proliferation ©f activated B-cells.

The invention is also directed to the Bp50 antigen itself, which is defined by aAb G28-5 and functions in B-cell proliferation. In addition, the invention is directed to ligands which bind to Sp50 but do not demonstrate a biological effect or function such as augmentation of proliferation of activated B-cells.

The ligands of the present invention include antibody molecules, monoclonal antibody molecules and fragments, of thes® antibody molecules which contain the antigen combining site that binds to the Bp50 receptor including chemically modified antibodies and fragments; such fragments include but are not limited to the Fw, Fab, F(ab*>2» Fab* and the like. In addition, the ligands of the present invention comprise lymphokines, which bind to th© BpSO receptorThese may include but are not limited t© BCSFs as well as chemically modified lymphokines and the like. The ligands of the invention can be used in their modified or unmodified forms to modulate and regulate immune responses and in the therapy of malignancies which express the BpSO antigen.

These ’asses, ar© discussed in more detail in Section 5.4 below.

Where the ligand is a monoclonal antibody, or a fragment thereof, the monoclonal antibody can be prepared against Bp50 using any technique which provides for the. production ©f antibody molecules by continuous cell lines in culture. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Mature 256:495-497) 4 as well as other techniques which have more recently become available, such as tha human B-cell hybridoma technique (Kozbor et al. 1983, Immunology Today 4:72) and the EBVhybridoma technique to produce human monoclonal antibodies S (Cola et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Lies, Znc., pp. 77-96) and the like are within, the scope of the present invention.

Antibody fragments which contain the. id iotype of the molecule could be generated by known techniques. For W example, such fragments include but are not limited to: the F(ab')^ fragment which can be generated by treating the antibody molecule with pepsin; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab*>2 fragment; the F(,ai3*>o 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.

Where the ligand that binds 3p50 is a lymphokine, the 2Q lymphokine may be obtained from natural sources or iff its amino acid sequence is known or deduced the lymphokine can be synthesised via chemical synthetic methods.

Alternatively, iff the gene sequence off the lymphokine is known, recombinant DNA techniques may be utilized to clone 25 the gene in an expression vector which provides for transcription and translation of the gene sequence in an appropriate host cell.

Depending upon its .intended use, the ligand or appropriate fragments of the ligand may be chemically •4Q modified by the attachment of any of a variety off compounds to the ligand using coupling techniqu.es known in the art. Such techniques may include but are not limited to the use off carbodiimide, cyanogen bromide, biffunctional reagents such as glutaraldehyde, W-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), Schiff base reactions, attachment to sulfhydryl moieties, the use of sodium isothiocyanate, or enzymatic linkage, to name but a few. Where a radioisotope is to be attached to the ligand this may also be accomplished via enzymatic means, oxidative substitution, chelation etc. For a review of the chemical reagents which can be used for protein modification see, Lundblad and Moves, chemical Reagents for Protein Modification, Volume II, CRC Pres®, Inc., Boca Raton, Florida, Ch» S, pp.123-139, 1984.

The chemical linkage or coupling of a compound to the ligand could be directed to a site on the ligand that does not participate in binding to BpSO. This could be accomplished by protecting th® binding site of the ligand prior to performing the coupling reaction. For example, the ligand can first be bound to BpSO in order to protect th® BpSO binding site, then the coupling reaction can be accomplished to link the desired compound to available reactive sites on the ligand"3p50 complex. Once the coupling faction is complete, the complex can be disrupted thereby generating a modified ligand to which the desired compound is attached so that the BpSO binding site of the molecule is minimally affected. Where th© ligand comprises a monoclonal antibody such as £28-5, in which the Fc domain of the molecule is not required for the ligand to exert its effect (see Section 5.3.3. infra) it may be advantageous to direct the coupling of desired compounds to the Fc domain of the molecule.

The subsections below describe the naw, 50-kDa S-cell surface marker, BpSO, which apparently functions in B-eell proliferation as well as ligands which bind to the new SOkDa receptor, and their uses. As an example of the ligands of the present invention a monoclonal antibody which defin.es BpSO and its F(ab*)? fragments are also described which. 6 like BCGF, augments B-cell proliferation. Unlike anti-Bp35 mAb, which can induce resting 3-cells In GQ to enter G,, anfci-3p50 mAb does not activate resting B-cells. Anti-Bp35 and anti-Bp50 mAb together, without any additional exogenous signals, induce strong activation and proliferation of purified B-cells.

The experiments described below also demonstrate that anti-3p50 activity resembles BCGF activity but that antiBpSO is distinct from one BCGF since anti-3p50 and low molecular weight BCGF are clearly additive and act differently on various B-cell subsets or malignancies. BpSO Bay be a receptor for a distinct BCGF or for a transmembrane signal that modulates BCGF production or BCGF receptor expression. .1. METHODS USED TO CHARACTERIZE THE 3p50 RECEPTOR Cell preparations. Mononuclear cells were isolated from normal or leukemic heparanized peripheral blood by Ficoll-Hypague gradients (Pharmacia, Piscataway, NJ). Mononuclear cells vers obtained from tonsillar tissues as described (Clark, et al.„ 1985, Proc. Nat. Acad. Sci. USA 82:1766-1770). T cells were depleted with AST-treated sheep erythrocyte resetting and Ficoll-Hypague gradient separation. In some experiments blood B-cells were enriched by isolating nylon wool adherent cells. Monocytes were removed by incubation on plastic petri dishes one or two times at 37®C for 45 minutes unless otherwise stated.

Buoyant or dense tonsillar S-cell fractions were Isolated by Percoll step gradients as described (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82:1766-1770). Dense tonsillar B-cell preparations consistently bad greater than 95% elg* Sp35* cells. Blood B-cell-enriched preparations had -60-85% slg+ cells. B-cell lymphoma cells were isolated by gently teasing lymphoma cells into medium followed by FicollHypaque gradient centrifugation.

Monoclonal antibodies. The G28-5 antibody to Bp5C was generated by immunizing BALB/c mice with human £- tonsillar lymphocytes and fusing immune spleen cells with the NS-l myeloma (Kohler, et al., 1975e, Nature 256:495-497; Ledbetter, et al.* 1979, Immunol. Rev. 47:63-32). Hybrid cell cultures secreting antibody reactive with tonsillar ΒΙΟ cells and not with T cells were identified by the use of indirect immunofluorescence (IF) and analysis with a FACS IV cell sorter; cultures with antibody giving histogram patterns similar to known mAb to pan B-cell markers (e.g., Bp35) were cloned and selected for further study. The G28-5 clone produced an IgG, mAh that reacted only with normal or malignant B-cells or 9-cell lines. Other s&b used in this study hav® been described in detail (Clark, et al.» 1985, Proc. Nat. Acad. Sci. USA 32:1766-1770; Clark et al., 1986, Human Immunol. 16:100; Ledbetter, et al., 1986, Human Immunol.15;30-44? Ledbetter, et al., 1985, in Perspectives in Immunocenetics and H1 stocompa t ibij,ity, ASHX, New ‘fork, 6, pp. 325-340). These include 1F5 (IgG,*) anti-B©35, HBlOa (IgG,&), anti-hXA-DR, 2C3 (XgG·,) anti-u chain, 3X9—4 (XgG,) anti-CD3, FO2 (IgG, ) anti-Fc receptor CD16, and 9.6 ^-G2sJ anti-CD2 (B receptor) provided by Dr. Paul Martin (Martin, et al., 1983, J. Immunol. 131:180),. The IgG., mAbs ver® purified by precipitation using 45% or 50% saturated ammonium sulfate and DEAE SEPHACRYL (Trade Hark) eolurai chromatography, and the Ig&jg niAbs were purified by the use of 2q protein A SEPHAROSE (Trade Mark) colums. The F(ab’)5 fragments of G28-5 were prepared by the method of Parham (Parham, et al., 1983, 3. Ismwol. 131:2895) purified on a 2-meter long SEPHACRYL* S2 00 coluwi, and assayed for purity by SDS-PAGE (Ledbetter, et_ * (Registered Trade Mark) _al.e 1985, 3. Immunol. 135:1819). The 2C3 mAb to u-chains was conjugated to Sepharose 48 beads (Pharmacia Fine Chemicals, Uppsala, Sweden) using cyanogen bromide coupling.

Fluorescein and phycoerythrin conjugations. Purified 5 nAb were either directly conjugated with fluorescein using fluorescein-isothiocyanate (FITC; Molecular Probes) (green) by the method of Goding (Goding, et _al., 1976» 3. Immunol. Meth- 13:215-226), or conjugated to R-phycoerythrin (PE) (red) by using SPDP (Pharmacia) with a method we have HO detailed In Ledbetter, et al., 1985, in Perspectives in Immunogenetics and Histocompatibiity, ASHI, New york, 6, 119-129. Lymphoid cells were incubated in round-bottom microtiter plates for 30 minutes with an appropriate dilution of green and/or red mAh, washed twice, and then analyzed on a FACS IV cell sorter.

Two-color immunofluorescence. Two-color studies were .done with a fluorescence-activated cell sorter (FACS IV: Becton-Dickinson, Mountain View, CA) by using a 560-nm dichroic mirror to split the beam and a 580 long-pass filter and a 540 short-pass filter (Ditric optics, Hudson, MA) in front of the red and green photomultiplier tubes, respectively, in addition, a two-color compensator (T. Mozaki, Stanford University) was used to correct for minor spillover of green and red signals. For each two-color stain, data from 40,000 cells were collected and stored on floppy disks. Data are presented as cell number (vertical) versus log green fluorescence versus log red fluorescence on a 64 x 54 dot grid. Approximately 4.5 dots represents a doubling of fluorescence. Unstained cells are positioned at the back corner of the grid; red fluorescence is to the right and green fluorescence is to the left. Our flow cytometry system for two-color IF with fluorescein and phycoerythrin is described in more detail (Ledbetter, _et al., 1985, in Perspectives in Immunogenetics and Histocompatibiity, ASKI, New York, S, 119-129 and 325-340).

Cell culture. Blood or tonsillar lymphoid cells were 5 cultured at 5-10 x 10 al in quadruplicate in 96-vell saicrotiter plates containing 200 ul RPM1-1640 medium supplemented with 15¾ fetal bovine serua, antibiotics, glutamine, and pyruvate (HIS)» After 1 to 7 days, cells were pulsed with 0.5 uCi of H thymidine per well (New England Nuclear, 6.7 Ci/mmol; 1 Ci=37) for 18 hours. Cells were then harvested onto glass-fiber filters with a cell harvester, and radioactivity was measured in a scintillation counter. In some experiments, antibodies or factors were added at various times after the start of cultures; proliferation in these experiments was measured on day 3.

Costimulatory 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 of Maizel and coworkers (Haizel, et_a_l-, 1982, Proc. Nat. Acad« Sci. USA 79:5993), who have shown that the major SCGF activity in this material resides in a 12-kDa species, hereinafter referred to as *SCGF (law)* (Mehta, et al., 1935, J. Immunol. 135:3298). The purification steps included preparative scale DEAE affinity chromatography followed by hydroxylapatite column chromatography. IL-X purified to homogeneity was the generous gift of Dr. Steven Dower (Dower, et al., 1985, J. Exp. Med. 152:501). Recombinant IL-2 was kindly provided by Cetus Corporation. TPA (12-0-tetradeconoyl phorbol 13acetate) was purchased from Sigma.

Detection_of cell activation. Changes in cell volume induced by aAb and/or factors were measured using a cell sorter and forward angle light-scatter. Cell cycle changes Ο in cellular RNA and DNA levels were measured by staining activated cells with acridine orange and measuring relative cellular RNA (red) and DNA (green) content with a cell sorter by th© method of Darzynkiewicz et al. (Darzynkiewicz, .5 et al., 1980, Proc. Nat. Acad. Sci. USA 77:6697-6702).

Changes in relative levels of cell surface antigens were monitored by use of mAb directly conjugated with fluorescein and then quantitated by direct IF fluorescence levels with an Epics V cell sorter.

Biochemical characterization,of BpSO. Xmmunoprecipi'155 tation of BpSO from surface - I-labeled tonsillar cells was performed as described (Ledbetter, et al., 1985, J. Immunol. 134:4250-4254). Isolated antigens were electrophoresed on % SDS polyacrylamide slab gels without reduction. Gels were visualized using autoradiography at -70°C and cronex lightening plus intensifying screens, (Dupont) .

S.2. CHARACTERIZATION OF THE SpSO RECEPTOR The subsections below describe the results of the experiments conducted using the methods described above. .2.1. IDENTIFICATION OF A B-CELL SPECIFIC SO kPA CELL SURFACE MARKER, BpSO.

A mAh to SpSO was raised by immunizing BALB/c mice with human tonsillar lymphocytes and fusing immune spleen cells with the NS-1 myeloma. One clone, G28-5, produced an IgG, mAh that did not contain the NS-1 light chain. Upon scrutiny by IF analysis, G28-S was found to react only with normal or malignant 3-cells ©r 3-cell lines. A comprehensive screening off normal tissue® by established methods (Clark, et al.# 1985, Proc. Nat- Acad. Sci. USA 82:1756-1770; Ledbetter et·al., 1986, Human Immunol.15:SO44; Ledbetter# 1935, in Perspectives in Immunogenetics and Histocompatibility, ASHI, New York, 6, pp. 325-340) revealed that the G20-5 antibody reacts with E rosette negative (Er-) cell® from blood ar tonsils but not with nylon wool nonadherent T cells, PHA-induced T-cell blasts, or with blood granulocytes, monocytes, red cells, or platelets. It reacted strongly with all seven 3 lymphoblastoid cell lines tested and with three Burkitt's lymphoma lines (Raji, Daudi, Namalwa), but not with four T cell lines (CSM, HSB-2, JURKAT, and HPB-AXX). All chronic lymphocytic leukemias tested (3/3) and 901 (9/10) of B lymphomas tested expressed the BpSO marker while only 28* ¢2/7) of non T, non B CALLA* acute lymphocytic leukemias expressed Bp50.

The restricted distribution of Bp50 on normal tissues was further confirmed by quantitative two-color Immunofluorescense (two color IF) analyses. Using an Rphycoerythrin (PS)-conjugated antibody (red) to the pan Bcell antigen 3p35 (BI, CD20) and fluorescein-conjugated anti-Bp50 antibody (green), we found that BpSO was expressed only on Βρ35+· 3-cells (Fig. 1) in blood or tonsils. Blood B-cells consistently expressed somewhat lower levels of BpSO than tonsillar B-cells; this is similar to HLA-DR expression, (Ledbetter et al.,, 1986, Human Immunol„15:50-44) and to gp54 expression (Wang, etal., 1979, J. Sxp. Hed. 149:1424-1433) which are also lower on blood B-cells. BpSO was expressed at similar levels on tonsillar 3-cell subpopulations separated on Percoll gradients into buoyant and dense fractions. Using our PE-conjugated mAb to the T cell marker, CD3(T3), and NX cell-associated marker, C016(Fc receptor) (Ledbetter, etal·» 1979, Immunol. Rev. 47:63-32), we found that BpSO Is not expressed on T cells or NX cells. Using two-color IF, we also found that CDS·*· P55A blasts that expressed high levels of IL-2 receptors did not express BpSO.

The 628-5 antibody reacted with a single polypeptide on tonsillar lymphocytes that migrated at approximately 50 Kd under non-reducing conditions (Fig. 2A). This molecule is larger than previously reported B-cell markers in the sane S molecular weight range such as Bp39 or Bp45 (Zipf, et al., 1983, J- Immunol. 131:3064-3072? Xitner, et_al.» 1981, Mature 294, 458-460; Clark, et al.» 1986» in Leukocyte Typing II» eds. Sieinherz, et al,., Springer Verlag, Berlin, Chap. 32 Vol., 2» 155-167,· Slovin, et al., 1982, Proc. Mat.

W Acad. Sci. USA 79:2649-2653; Thorley-Lawson, et a 1.,, 1995, J. Immunol. 134:3007-3012, and Fig. 23). The exposure time for this gel was selected so that the molecular weights of the other 3-cell markers could be readily compared with BpSO. The 3p39 marker,, unlike Bp50, is expressed on granulocytes and 3p45, unlike BpSO, is restricted to B-cell blasts. Antibodies to 3p39 (41-H16) and Bp4S (MNM6, Blast1, Blast-2) made available through an international workshop (Clark, et al., 1986, in Leukocyte Typing ll, eds. Reinherz, et_al., Springer Verlag, Berlin, Chap. 12 Vol. 2, 155-167) 2o did not block the binding of fluoresceinated anti-3p50 antibodies to B-cells. Thus, based on tissue distribution, biochemical analysis, and blocking studies»' the G28-5 monoclonal antibody recognizes a 50-Xd structure distinct from other known 3-cell antigens. .2.2. EXPRESSION OF BpSO IS RESTRICTED TO 3-CELLS Both hematopoietic tissue and cell-line distribution studies and detailed two-color flow cytometric analyses revealed that BpSO is expressed only on S lymphocytes. As illustrated in Fig. 3, BpSO is expressed on a small subset of blood lymphocytes and on a large population of tonsillar lymphocytes. Virtually all Bp50+ cells in both blood and tonsils also expressed Bp35 and HLA-DR, but did not express the CD2 (Fig. 1) or CDS, T-cell molecules or the IgG Fc receptors that are found on MK cells. Furthermore, ConA2 3 activated CD3* T-cell blasts expressed IL-2 receptors but did not express Sp5O.

Two-color flow cytometric analyses allow the quantitative measurement of the density relationship between two surface antigens. We previously showed that the dense, resting B-cells in the mantle sone of secondary follicles express IgM and low levels of Bp35, whereas the buoyant, activated B-cells in the germinal center are XgM-negative and, express elevated levels of Bp35 (Ledbetter, et al., Human Immunol. 15:30). Figure 3 shows that both IgMpositiv© and IgK-negative B-cell subsets expressed BpSO in equal amounts, indicating that BpSO is expressed on both resting 3-cells and 3-cells activated in vivo. .3,, AUGMENTATION OF B-CELL PROLIFERATION WITH ΑΝΤΙ-BpSO antibody____ As previously explained, B-cells can be activated with low doses of anti-u chain specific antibodies. We recently found that the 3-cell-specific marker Bp3S (31), a 35-kDa polypeptide, say also function in early 3-cell activation: the 1F5 mAh to 3p35£! like low doses of anti-u antibody, activates B-cells to increase in cell volume and RNA content and to become responsive to BCGF (Clark, et al.„ 1985, Proc. Nat. Acad. Sci. USA 82:1766-1770? Gollay, et al., 19SS, J. Immunol. 135:3755-3801). Therefore, it was of interest to compare the effect of anti-BpSO mAb in the proliferation of untreated B-cells or B-cells activated with either anti-Bp35 or anti-u antibodies (Table 1). Anti-3p35 in solution or anti-u antibodies attached to SBH4AROSE (Trade Mark) beads, under appropriate conditions alone, could stimulate sons B-cell proliferation (Table l, line 1) ; in contrast, anti-SpSO antibodies alone did not stimulate proliferation (Table l, line 2). However, anti-3p50 mAb augmented proliferation considerably when cultured with anti-u beads or with antiBp3S„ In this respect anti-BpSO resembled BCGF (Table 1, line 3) . Thus, it was important to determine whether 4 anti-Bp50 and BCGF together could induce. 3-cell proliferation. As illustrated in Table 1, line 4, anti-BpSO and BCGF together induced no proliferation, but did augment proliferation of either anti-u or anti~Bp35 activated cells somewhat more than either stimulant alone. BCGF over a three-log range, when used with anti-Bp50 without other signals, had no effect on proliferation of dense 3-cell® even when anti-3p50 was used at doses ranging from 0.1 to io ug/ml.

Table 1 Augmentation of Anti-Ig or Anti-3p35 Induced B Cell Proliferation with Anti-Bp50 Antibodies Mean Proliferation + S.B. of B Cells Cultured With: Line Co-stimulant Media Anti-u-beads Anti-3p35 1 None 1,212+547 10,219+452 5,539+308 2 Anti~Bp50 7X9+718 38,792+1,329 25,465+616 3 BCGF 456+217 14,217+445 9,443+343 4 Anti-BpSO + BCGF X „,456+128 54,393+2,537 46,488+3,387 Proliferation of dense Er- tonsillar B-cells ( 95¾ surface IgMv cells) was measured on day 3 as described. Briefly, x 10s cells/200 ul well were cultured in quadruplicate for 48 hrs with RPMI 1640 medium containing 15¾ fetal bovine serum plus additives without antibody or with either 2C3 monoclonal antibody to u chains coupled to sepharose beads <*anti-u beads,* 50 ug/ml) or free IPS anti-Bp35 antibody (5 ug/ml). Cultures containing media, anfci-u beads,* or anti-Bp35 were cultured alone or with BCGF (3¾ final concentration, cytokine Technology, Buffalo, New York; has 3Q no detectable IL-X or IL-2 activity), with anti-BpSG (1:1000 dilution -of ascites) as co-stimulants. After 40 hrs cells were pulsed with JH-thymidine, and counts incorporated were measured after 18 fers. 2S .3.1. ANTI-3p50 mAb AUGMENTS PROLIFERATION ONLY AFTER B-CELLS ARE ACTUATED 3Y ANTI-BO35 OR ANTI-U-ANTIBODIES The results in Table 1 suggest that anti-BpSO mAb could not induce proliferation by itself. As shown in Fig. 4, doses of anti-BpSO ranging from 0.05 ug to 2.0 ug/ml had no effect on "«-thymidine uptake. However, In the presence of optimal levels ©f anti=Bp33 mAb, as little as O„1 to Q.S ug/ml of anti-3p50 antibodies augmented proliferation substantially. As much as 50,000 to 70,000 cpra were detectable at the optimal time of proliferation when highly purified B-cells were cultured only with antiBp35 plus anti-BpSO- A consistent observation was that higher doses of anti-BpSO (greater than 2-5 ug/ml) were less effective than doses in the 100-200 ng range.

These results suggested that anti-8p50 may function only after B-cells are activated by other signals. Data shown in Fig. 5 suggest that this Is Indeed the case. If 3cells were first activated with anti-Bp35, anti-BpSO could be added as late as 24-48 hours later and still augment proliferation at day <4. In contrast, when cells were first treated with anti-Sp50, anti-Bp35 was effective only if added within a few hours after the start of cultures.

Similar results were found when anti-u rather than anti-Bp35 was used. .3.2. ANTX-Bo50 mAh DO NOT ACTIVATE S-CELLS OUT OF G_ BUT* DO INDUCE ACTIVATED B-CELLS TO PROGRESS THROUGH THE CELL CYCLE__,__ Previously, we have found that anti-Bp35, like low doses of anti-u antibodies, induce resting tonsillar B-cells in Gq to enlarge (Clark, _et al., 1935, Leukocyte Typing II, eds., Reinherz, et al., Springer Verlag, Berlin, Vol. 2, 455-462) and to enter the G, phase of the cell cycle (Gollay, et al., 1985, J. Immunol. 135:3795=3801). Thus, it wa© of interest to compare the ability of anti-BpSO mAb to anti-Bp35 mAb for their effects on B-cell activation. As 6 shown in Fig. 6A, unstimuleted dense tonsillar B-cells even after 3 to 4 days in culture had a uniform RNA profile characteristic of cells in GQ (Darzynkiewicz, 1980, Proc. Nat. Acad. Sci. USA 77:6697-6702). However, about 15-30¾ of cells stimulated with anti-Bp35 or anti-u had increased RNA content indicative of entry into G1. In contrast, neither anti-Bp50 (Fig. SB) nor BCGF (Fig. 6C) ©lone induced significant numbers of B-cells to enter G1. For instance, 2 days after activation, anti~3p35 and anti-Ig mAh induced respectively 13.5% and 20.9% of tonsillar cells to enter Gn, whereas cells treated with only anti-Bp50 (2.7%) or BCGF (3.2%) remained at media control levels (2.21). However, when either anti-Bp50 or BCGF were added together with anti-Bp35 or anti-u antibodies, the proportion of cells entering ΟΊ increased dramatically. Similarly, anti~Bp50 and BCGF alone did not induce B-celXs to enter S phase (Table 2), but together with either anti-Bp35 or anti-u did increase the number of S phase cells two- to threefold.

Table 2 Effect of Anti-3p5G and BCGF on Cell Cycle Progression in Tonsillar Lymphocytes competence Signal Progression Signal S % Cells S1 s/g2/h media none 89.9 7.1 2.5 anti-Sp35 80.4 14.5 3.7 anti-Ig 65. β 27.6 5.7 media anti-BpSO §3.6 12.0 3.3 anti-Bp35 & 54. a 35.S 9.7 anti-Ig 4 3.6 36.2 16.2 media BCGF 83.4 11.7 2.2 anti"Bp35 ί» 56.6 32.6 11.6 anti-Ig & 48.4 36.1 14.1 7 Percentage of cells in G , G1„ or S and Gt> determined with the use of the acridine orange-staining procedure (Darzynkiewicz, et al,., 1980 Proc. Nat. Acad. Sci. USA 77; 6697-6702) » X x 10° dense tonsillar lymphocytes with anti-Bp35 (5 ug/ml), anti-u on beads (50 ug/mlj» anti-ap50 (0.4 ug/ml), BCGF (5%) or combinations as shown. .3.3. OPTIMAL CONDITIONS FOR AUGMENTING B-CELL PROLIFERATION WITH ΑΝΤΙ-BpSO ANTIBODIES Antibodies to Bp50 by themselves havie little or so detectable effect on dense resting B-cells (Table 3). However, in th® presence of agents that can activate Bcells, such as anti-lg, anti-9p35 and TPA, anti-BpSO mAb clearly augmented proliferation. Anti~3p50 did not costimulate with several interleukins, Including purified IL-1, recombinant IL~2 and BCGF (low) . a comparison of th® effects of anti-BpSO with those of BCGF (low) showed that th® same agents that were costimulatory with anti-BpSO were also costimulatory with BCGF (low) (Table 3). Of particular interest was the finding that together BCGF and anti-BpSO still were not costimulatory for resting cells.

Table 3’ Augmentation of B-Cell Proliferation with Anti-BpSO Antibodies or 3-Cell Growth Factor Mean Proliferation + S.2. of B-Cells Cultured with: Anti-BpSO ' BCGF Co-stimulant_Media(200 ng/ml)(5¾) none @6 ± 1 267 + 15 285 * 74 anti-lg 5,333 ± 391 41,634 + 2,103 25,094 + 61 anti~Bp35 (5 ug/ml) 457 ± 45 8,143 2 280 1,733 i 32 TFA (2 ng/ml) 7,361 X 537 21,163 * 871 13,064 4· 1,030 IL-1 (10 U/ml) 254 * 2 308 ± 23 221 •r s^> s IL-2 (100 U/ml) 204 49 34 3S0 ± Ί 220 * 11 BCGF iS%) 220 + 7 851 * 28 270 IS Dense Er- tonsillar 3-cells /greater than 951 slgM’ cells) cultured for 48 hr at 2 x 10" cells/well followed by 24 hr pulse with Η-thymidine before counting.

The kinetics of proliferation augmented by anti-BpSO is shown in Fig. 7. The peak of proliferation occurred at day 4 and then waned whether or not cells were activated with anti~Bp35 or other activators such as anti~Xg or TPA. The kinetics of proliferation augmented by BCGF or by anti-Bp50 were similar.

As little as 0.05 ug of anti-Bp50 antibodies augmented proliferation. An optimal dose of 0.3 ug/ml was used in subsequent studies. A consistent observation was that when using whole antibody molecules, higher doses of &nti~Bp50 (greater than 2-5 ug/ml) were less effective than doses in the 0.1-0-5 ug/ml rangeHuman B-cells are exquisitely sensitive to inhibitory effects mediated by the Fc receptors of antibodies binding to surface Ig (Parker, 1980, Immunol. Rev. 52:115,- Bijsterbosch, et a 1.,, 1985, J. Exp. Med. 162:1825). Thus, it was important to compare the efficacy of whole anti~Bp50 mAb with that of anti-BpSO F(ab?)^ fragments. Over a 100-fold dose range F(ab*)2 fragments were clearly as effective as, or more effective than, whole antibody at augmenting B-cell proliferation (Table 4). Thus, the Fc domain of anti-BpSO mAb is not required for anti-BpSO to exert its effect and, if anything, may be inhibitory. In other words, anti-BpSO, like BCGF, apparently can act as a soluble mediator without the aid of Fc receptor-mediated accessory cell function. 9 Table 4 The Fc Domain of Anti-3p50 Antibodies is Not Required for Augmenting 3-Cell Proliferation Mean Proliferation of B Cells Cultured with: Anti-Bp50 Dose iHSZHll Media Anti9p35 none —— 295 + 16 269 ± 27 whole Ab 0.125 278 + 32 5,140 + 20 1.25 275 + 24 4,686 + 342 12.5 1S3 + 15 3,852 203 F(abi?) 2 0.125 594 i 21 10,635 4· 449 1.25 S31 4· 3 10,893 + 575 12.S 279 ± 8 9,411 T 870 Cell culture conditions were as described in Table 3. .3.4 DIFFERENCES BETWEEN ANTI-Bp50 AND 3CGF (LOW) ACTIVITY Anti~3p50 and BCGF (low) had a similar effect on Bcells and were costimulatory with the same agents (Table 3|. However, several lines of evidence indicate that anti~Bp50 and the BCGF used in this study apparently operate through different signals. First, SpSO molecules, unlike BCGF (low) receptors (Bijsterbosch, et al.1985, J- Exp· Med. 162: 1825), are expressed sn resting blood B-cells (Fig. 3J. Second, although both anti-BpSO and BCGF (low) function most effectively when added after anti-Bp35 or anti-Ig, anti-5p50 clearly was optimally effective when added 12 hours after cultures began (Fig. 8A). In contrast, BCGF (low) .could be added as long as 24 hours after start of cultures and still optimally augment proliferation (Fig. SB). These kinetic experiments, which are modeled after the approach of Howard and Paul (1983, Ann. Rev. Immunol. 1:307), suggest that a Bp50-dependent signal may normally exert its effect before BCGF.

Both anti-SpSG and BCGF (low) augmented proliferation of B-cells activated with anti-Bp35 or anti-Ig (Table 3). However, the effect of anti-BpSO and BCGF (low) were additive in many experiments (Figure 7). Figure 9 shows a titration of BCGF (low) in an experiment where anti-Bp50 was used at its optimal concentration (0.2 ug/ml). BCGF (low) could further augment proliferation of resting B-cells in the presence of anti-Bp50 after activation by either anti-Ig or by anti-Bp35. Optimal concentrations of BCGF (low) were 5-10%, while 25% was inhibitory. Thus, when anti-BpSO and BCGF (low) were both used at their optimal concentrations, they still showed additive effects on 3-cell proliferation.

Finally,, both normal and malignant B-cell subsets differed In their responses to anti-BpSO and to BCGF (low). For example, some blood B-cells responded to BCGF (low) but did not respond to anti-BpSO (Table 5). An additional activation signal such as anti-Bp35 (Table S) or TFA (Fig. ) was consistently necessary to allow blood B-cells to respond to anti-BpSO. While dense tonsillar B-cells generally did not respond to either BCGF or anti-BpSO, buoyant 3-cells did respond (Table 5). B-cell malignancies also differed in their responsiveness to anti-3p50 versus BCGF. For example, some B-cell lymphomas responded to TFA plus BCGF (low) but not to TPA plus G2S-5 anti-BpSO (Fig. 103 and D). In contrast, dense tonsillar B-cells and peripheral blood B-cells responded to TFA plus either BCGF (low) or anti-BpSO (Fig« XOA and C>, Table 5® Subocta Differ in Thoir Responsiveness to Anti-BpSO or BOGF Mean Proliferation (χ S.E.M.) of Lymphocytes Prom Seed Tozuih Stimulation Esp i Exp 2 Exp 1 Dense Buoyant none 527 ♦ 70 * 659 X 133 906 1. 106 315» « 317χ. 12 BCGF (3%) 13,911 χ 1,0« 37,399 χ 2,0?3 3,3 » 173 332» 33 ί,«1χ 37 anti«Sp30 (0-J· ug/ml) J19' * 21 1,013 χ Si 1,131 χ 2.1 *72χ 1 Ι»5«χ 20 a«li-Bp35 (J ug/ml) ss* * SO 6« t 89 US ♦ 1)3 ϊ,*13χ 10 1,1Ϊ9χ 61 anli»'Bp30 « anti.-Bp33 . s es· .12,.271 χ «6 .13,667 χ 333 36,319» 1,33.3 «Ί«χ 136 anti-BpSO » BCGF <& s i3 3,9« ♦ 113 ί,3«χ if 2,«99» 91 Cell culture conditions as described In Table L Blood Nylon wood adherent lymphocytes (B cells plus monocytes) were depleted ol Ϊ1 monocytes by Incubation on plastic dishes overnight prior to siimuIatien'iExps I and Exp® 2). In Exp, 3, blood E-lymplwcytes were depleted of monocytes by incubation on plastic dishes ior .1 hr® prior, to stimulation® Tonsillar Er lymphocyte» were iraeiionated using Percoll gradients into dense (pellet) or buoyant (traction I) subsets (32)a .4. USES OF ANTI-BpSO LIGANDS AND Bp50 The ligands of the present invention may be used in vivo or in vitro, in their unmodified or modified forms to modulate immune responses. For example, the ligands themselves may be used as an 'adjuvant* to increase an immune response to a vaccine or to increase the immune response of an immunosuppressed individual. Alternatively, if cytotoxins or anti-proliferative agents are coupled to the ligands, these modified ligands may be used to decrease an immune response, for example, in autoimmune disease or in transplant patients to obviate graft rejection. These modified ligands could also be used to treat malignancies that comprise cells or tumors which express th© Bp50 antigen whether or not the malignancy is 3-cell in origin.

Both the ligands of the present invention and/or 3p50 itself can be used in vitro. Such applications include in vitro assays, such as immunoassays for th® detection of cells which express the BpSO antigen and/or for the detection of shed Bp50 antigen, if any, in body fluids. In 2q this instance the ligand or BpSO could be labeled with a radiolabel, fluor, ensyme, enzyme substrate, dye, etc. In addition, the ligands may be used to separate and/or identify cells which express the BpSO antigen, in which case the ligand may be coupled to an immobile support, or to a fluor which can be used in a FACS (fluorescence activated cell sorter).

The various applications and uses of the ligands and BpSO of the present invention are discussed in more detail below. .4.1. BpSO RECEPTOR AND USES OF LIGANDS SUCH AS ANTI-3p50 TO AUGMENT B-CELL PROLIFERATION Previous studies have suggested that the factors involved In the induction of B-cells from GQ into the G, phase of the cell cycle are distinct from the factors or requirements for transit into the s phase. This model is based principally on studies showing that agents such as low doses of anti-Ig B-cell activation factors , or anti-Bp35 alone have little or no effect on 3-cell proliferation. Yet, these same agents can drive 3-cells to a point in cell activation where they are susceptible to growth factors. In contrast, growth factors such as BCGF or IL-2 alone have no effect on resting B-cells but do augment growth of activated B-cells.

While the present invention is not to be limited to any theory or explanation, th® results presented herein provide additional support for a modal of distinct regulation of 3cell activation and growth steps. Here we have shown that activation and proliferation signals in human B-cells may be transmitted through distinct cell surface structures.

Although anti-Bp35 mAb activated B-cells to enter the G, phase of th® cell cycle, alone, it induced little or no proliferation. Anui-3p5Q mAh had the opposite effect: it could not activate B-cells, but when added even as late as 12-24 hours after activation could induce B-cell growth.

The Sp50 molecule presumably could normally function as either a receptor for a ligand such as a soluble growth factor or for a signal mediated through cell-cell contact (i.e., a ligand found on the surface of another cell). Previous studies have identified several T cell-derived SCGFs that, like anti«3p50, augment B-cell proliferation. Both high and low molecular weight forms of B-cell growth factors have been identified and different types have been shown to have additive effects (Kehrl, eta)., 1984,,, Immunol. Rev. 10:75-96? Kishimoto, 1985, Ann. Rev. Immunol. 3:133-157; Swain, et al. 1983, J. Exp. Med. 158:822-835? Howard et al., 1984, Immunol. Rev. 78:185-210? Ambrus, et al,, Clin. 4 Invest. 75:732-739: Ambrus, 1985, J. Exp. Med. 162:13191335). Thus, Bp50 might be a receptor for one of these factors.

With the exception of IL-2 receptors and the C3d receptor, the receptors on 3-cells for growth signals have not yet been identified. The mAb A3-1 reacts with a 3~cell marker expressed only on activated 3-cells and blocks 3CGFdependent proliferation, and thus might recognize the BCGF receptor or a related structure. 3p50 appears to be distinct from the AB-1 marker since th© AB-1 mAb does not block the binding of the G28-5 anti-BpSO antibody, and unlike the G28-5 mAb, reacts only with activated 3-cells (Jung, et aj.„, 1984, J. Exp. Med. 160:1919-1924). 3p50 is on all B-cells, which based, on absorption analysis and direct binding assays appears not to be the case for BCGF receptors. Our current data indicate that BpSO and the receptor for low molecular weight BCGF are distinct structures. Using a rabbit heteroantiserum, Wang and coworkers (Wang, 1979, J. Exp. Med. 149:1424-1433) previously described a 54-kDa glycoprotein, gp54, that like BpSO is expressed on all 3-cells but at lower levels on blood B-cells than tonsillar B-cells. it is possible, but unlikely, that the rabbit heteroantiserum and anti-Sp50 recognize the saae or related structures: unlike anti-Bp50 »Ab, the rabbit antiserum to gp54 alone was sufficient to stimulate B-cell proliferation.

Anti-Bp3S alone, unlike anti-BpSO, can activate B-cells from Gq to G, and thus can be referred to as an 'activation* signal. Whether or not Bp35 functions only in early B-cell activation is not yet clear since anti~Bp35 antibodies can stimulate some B-cells to divide (Clark et al,.., 1985, Proc. Nat. Acad. Sci. 'USA 82:1766-1770). Similarly, BpSO may not strictly function only as a 'growth* signal: anti-3p50 antibodies together with activation signals (anti-3p35 or anti-u) not only augment proliferation but also augment the total number of 3-cells entering G, (Table 2). In other words, anti-BpSO as costimulant acts to promote the progression of both the activation (GQ to G,) and growth (G to S) phases of the cell cycle. The BCGF used in these studies also had similar activity (Fig. 6C). Thus, anti-3p35 and anti-3p50 (or 3CGF) appear to be most analogous to the competence* and progression* factors described in studies of fibroblast growth regulation. How B-cells respond to HO anti-3p35 or anti-BpSO clearly may depend on their state of differentiation or activation.

Here we have shown that two mAb, anti-Bp35 (a competence* signal) and anti-BpSO (a progression signal), together can induce substantial proliferation of highly IS purified B-cells in the absence of antigen or other known factors. The natural ligands for these structures are not yet known. However, since mAh to appropriate epitopes can mimic both soluble factors and signals mediated by cell-cell interactions, it nay be possible to use appropriate combinations of mAb to direct and regulate human B-cell proliferation or differentiation. This, in turn, will help in devising strategies in vivo for the control of human diseases such as B-cell malignancies, immunodeficiencies and certain autoimmune diseases.

The new monoclonal antibody, G2S-5, that reacts with a single-chain polypeptide of approximately 50 Kd expressed on the surface ©f human 3-cells is but a particular embodiment of the ligands of the present invention which can augment the proliferation of activated B-cells. Since human B-cell proliferation can be augmented similarly by T-c®11-derived BCGFs Including low- and high-molecular-weight BCGF we compared the activity of anti-Bp50 G28-5 with that of a BCGF preparation containing predominantly low-molecular-weight BCGF» Anti~Bp50 G23-5 and BCGF (low) were very similar in that they were costimulatory with the same activation agents (anti-Ig, anti-Bp35 and TFA) hut were not costimulatory with each other or with IL-1 or IL-2. Furthermore, the activity of anti-Bp50 G23-5 was not dependent on its Fc domain since F(ab*)2 fragments of G28-5 were functionally active. This suggests that soluble anti-Sp50, like soluble BCGF, does not require Fc-receptor-bearing accessory cells to exert an effect- Furthermore, both anti-Bp50 and BCGF are effective W only in the presence of an activation stimulus. In other words, anti-Bp50 and BCGF are not competence* factors, but rather promote th® progression'* of B-cells through the cell cycle» While it is possible that Bp50 may function as receptor for a ligand such as a 3-cell growth factor, several results suggest that Bp50 is not the receptor at least for the BCGF (low) used in this study: it is expressed on blood 3-cells while BCGF (low) receptors apparently are not» Candidate structures for the BCGF (low) receptor, unlike 3ρ50, are also 2q expressed only on activated B-cells. Furthermore, both normal and malignant B-cell populations differ in their responsiveness to anti-Bp50 versus BCGF (low) (Table 5 and Figure 10). For instance, some 3 lymphomas proliferate in response to BCGF (low), but not in response to anti-Bp50. «,g Finally, in a number of experiments, optimal concentrations of anti-3p50 and BCGF together induced more proliferation than either one alone. Anti-Bp50 mimics the activity of other BCGF, such as BCGF (high) that are co-stimulatory with anti-IgM (Ambrus, et al», 1985, J. Exp. Hed. 162:1319? Ambrus, et al., 1985, J. Clin. Invest. 75:732). This suggests that BpSO could function as the receptor for BCGF (high)» Although BpSO may be a receptor for a soluble ligand, alternatively, Bp50 may function as a receptor for a cellcell mediated signal that regulates BCGF receptor levels and/or autocrine production. Precedence for differentiation antigens serving as amplifiers of an autocrine-receptor pathway comes from studies with T cells. MAb to the Lp220 common leukocyte antigen augments proliferation by elevating SL-2 receptor expression on activated T cells (Ledbetter, et aj,, 1995, Immunol. 135:1819). An analogous mechanism may be operating with anti-BpSO and expression of certain BCGF receptors. 3p50 and BCGF (low) apparently are under some coordinate control since, like IL-1 and IL-2 receptors, BCGF augments expression of SpSO on certain leukemic cells. The BpSO molecule also shares similarities with the Tp44 molecule that functions to influence IL-2 production. Me and others have shown that the 9.3 anti-Tp44 antibody augments proliferation of T cells activated by anti-CD3 or TPA (Ledbetter, et al., 2,985, J. Immunol. 135:2331? Hara, et al., 1985, J. Exp. Med- 161:1513). Similarly, anti~3p50 augments the proliferation of B-cells activated by anti-3p35 or TPA. The Tp44 signal functions by stimulating IL-2 production rather than by stimulating T cell growth. The BpSO signal presumably could function in an analogous manner by stimulating B-cell autocrine production (Gordon, et al,, , 2984, Nature, Lend. 310i145). .4.2. MODIFIED LIGANDS OBSD FOR IMMUNOSUPPRESSION OR TREATMENT 0F MA LI GN AN A CI ES......

According to this embodiment, the ligand of the present invention can be modified by the attachment of an antiproliferative agent so that the resulting molecule can be used to kill cells which express the Bp50 antigen. Such modified ligands may b® used in the treatment of autoimmune disease in order to supress the proliferation of B-cells and • 38 thereby suppress th® autoimmune response. These modified ligands can also be used to immunosuppress a transplant patient to prevent rejection of a graft. Accordingly, cytotoxic agents which are used for the suppression of immune responses can be attached to the ligands of the invention. When using ligands which augment the proliferation of B-cells, an increased effect should result because the drug will be directed to proliferating B-cells, In another embodiment, the ligands of the present invention which are modified by the attachment of an antiproliferative agent can b® used to treat malignancies in which tumors or cells express th® Bp50 antigen. Attachment off these chemotherapeutic agents to the ligands of the invention should result in a greater specificity of the drug for the malignant cells. Moreover, a particular advantage should be obtained when treating a B-cell malignancy with a ligand coupled to a cytotoxin which is more effective in killing proliferating cells than non-proliferating cells,· treatment with such a ligand should result in a potentiation of the action of the cytotoxin, Accordingly, the chemotherapeutic agents or antiproliferative agents which can be coupled to the ligands of the present invention include but are not limited to the agents listed in Table 6 below which is derived from Goodman and Gilman, The Pharmacological Basis of Therapeutics, Sixth Edition, MacMillan Publishing Co,, Inc, New York,-pa. 12491313, IPSO, Table 6 Chemotherapeutic Agents Which to Anti-BpSO Liga Can be Coupled Class Type Alkylating Agent Nitrogen Mustard Ethylenimine Derivatives Alkyl Sulfonates Nitrosoureas Agent Meehlorethem ine Cyclophosphamide Melphalan Uracil Mustard Chlorambucil Thiotepa Antimstabolites Triazenres Folic Acid Analogs Pyrimidine Analogs Busulfan Carmustine Lomustine Semustine Streptozocin Dacarbazine Methotrexate Purine Anlogs Natural Products Vinca Alkaloids Antibiotics Fluorouracil Cytarabine Azaribine Me rcaptopurise Thioguanine vinblastine Vincristine Dactinomycin Daunorubicin Doxorubicin Bleomycin Mithraraycin Mitomycin L-Asparaginase Enzymes ο Miscellaneous Agents Platinum Coordinated Complexes Cisplatin Substituted Urea Hydroxyurea Methyl Hydratine Derivative Procarbazine Adrenocortical Suppressant Mitotane Hormones and Antagonists Ad renocort i ~ costeroids Prednisone Progestins Hydroxyprogesterom caproate Medroprogesterone acetate Megestrol acetate Estrogens Diethylstilbestrol Ethinyl estradiol Antiestrogen Tamoxifen Androgens Testosterone propionate Fluoxymesterone Radioactive Isotopes Phosphorous Sodium phosphate Iodine Iodide Any method known in th© art can be used to couple the ligand to the c hemotherapeutic or antiproliferative agent. Examples of such methods have been enumerated previously (see Section S, supra). 1 .4.3. OTHER USES OF LIGANDS AND 3p50 In addition to the therapeutic applications the ligands end BpSO itself have other applications in both in vitro and In viyo diagnostic assays, separation schemes, etc.

S The 3p50 receptor can be used to manufacture and/or design the ligands of the invention. BpSO can also be used with the ligands of the invention in assays in vitro which require a standard to quantify the amount of BpSO detected in a sample. Ultimately, Bp50, itself may be useful as a soluble factor which mediates immunity, e.g. a lymphokine.

In addition to therapeutic treatment and diagnostic assays, the ligands of th® present invention could be used for identifying or separating cells which express the BpSO antigen. In addition, if an appropriate radiolabel er Tig radio-opaque compound is linked to th© ligand, the ligand could bs used for in vivo imaging of tumors which express the BpSO antigen. Other uses should become apparent to those skilled in the art from the foregoing description. 6e DEPOSIT OF CELL .LINES, The following hybridoma has been deposited with tbs American Type Culture Collection, Rockville, MD, and has been assigned the listed accession number: Hybridoma ATCC Accession Number 25 G28-5 HB9120 The present invention Is not to be limited in scope by the hybridoma deposited since the deposited embodiment is Intended as a single illustration ©f one aspect of the invention and any cell lines which are functionally 2P equivalent are within the scope of this Invention. Indeed various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings» Such modifications are intended ts fall within th© scope of the appended claims.

Claims (57)

1. CLAIMS?

1. A substantially pure ligand that binds to BpSO, a 50 kiloDalton 3-cell surface antigen defined by monoclonal antibody G28-5.

2. A substantially pure ligand according to claim 1 which upon binding to an activated B-cell stimulates the activated B-cell to traverse the cell cycle so that proliferation of the B-cell is augmented.

3. A ligand according to either of claims 1 and 2 which comprises an antibody molecule or an Fv, Fab, F(ab') 9 or Fab' portion of the antibody molecule.

4. A ligand according to claim 3 in which the antibody molecule comprises a monoclonal antibody molecule or an Fv, Fab, Fiab'Jj ot Fab' portion of the monoclonal antibody molecule.

5. A ligand according to claim 4 in which the monoclonal antibody molecule comprises G28-5, or any Fv, Fab, Fiab')^ or Fab' portion thereof.

6. A ligand according to claim 4 in which the monoclonal antibody molecule has been produced by a hybridoma cell line as deposited with the ATCC having accession no. HB9110, or a mutant recombinant or genetically engineered derivative thereof.

7. A ligand according to either of claims land 2 which comprises a lymphokine. 3. A ligand according to claim 7 in which th® lymphokine comprises a 3-cell growth factor. 4 3

8. 9, A ligand of either of Claims 7 and 8 in which the lymphokine is on the surface of a cell.

9. 10. A ligand according to any one of Claims 1 to 9 which further comprises a compound coupled to the ligand.

10. 11» A ligand according to any one of Claims 1,3,4,5,6,7 and 8 in which the conpound coupled to the ligand comprises an antiproliferative agent.

11. 12» A ligand according to Claim 11 in which the compound coupled to the ligand comprises an alkylating agent.

12. 13» A ligand according to Claim 11 in which the compound coupled to the ligand comprises an antimetabolite.

13. 14» A ligand according to any one of Claims I to 9 inclusive in which the ccxpound coupled to the ligaxd ccnprises an antibiotic.

14. 15. A ligand according to any one of Claims 1-9 inclusive in which the compound coupled to the ligand comprises a vinca alkaloid»

15. 16» A ligand according to any one of Claims 1-9 inclusive in which the compound coupled to the ligand comprises an enzyme.

16. 17. A ligand according to any one of Claims 1-9 inclusive in which the compound coupled to the ligand emprises a platinum coordinated complex.

17. 18» A ligand according to Claim 11 in which the compound coupled to the ligand comprises a radioisotope.

18. 19. A ligand according to any one of Claims 1-9 inclusive in which the compound coupled to the ligand comprises a fluorescent ccnpound.

19. 20. A substantially pure 50 kiloDalton B-cell surface antigen which is defined by monoclonal antibody G28-5, 4 4

20. 21. A substantially pure 50 kiloDalton antigen according to claim 20 which comprises a polypeptide.

21. 22. A method for augmenting proliferation of B-cells comprising treating activated B-cells with an effective 5 dose of a ligand that binds to Bp50, a 50 kiloDalton B-cell surface antigen defined by monoclonal antibody G28-5, so that the activated B-cells traverse the cell cycle and proliferation is augmented.

22. 23. A method according to claim. 22 in which the B-cells 10 were activated by treatment with an effective dose of a second ligand that binds to Bp35, a 35 kiloDalton B-cell surface antigen, so that the B-cell progresses from the G q to G 1 stage of the cell cycle.

23. 24. Use of an effective dose according to either of Claims 22 15 and 23 in a composition for treatment of th© human or animal body.

24. 25. A method according to either of claims 22 or 23 is performed in vitro.

25. 26. A method according to any one of claims 22 to 25 in which the ligand comprises an antibody molecule that 20 binds to BpSO or an Fv, Fab, F(ab'), or Fab'* portion of the antibody molecule that binds fo BpSO.

26. 27. A method according to any one of claims 22 to 25 in which the ligand comprises a monoclonal antibody molecule that binds fco SpSO or an Fv, Fab, Fiab* 1 ’ ) 9 „ or 25 Fab*” portion of a monoclonal antibody molecule that binds to BpSO.

27. 28. A method according to claim 27 in which the monoclonal antibody molecule comprises G28-5, or any Fv, Fab, Flab),, or Fab portion thereof. 2S. A method according to claim 27 in which the monoclonal antibody is produced by a hybridoma cell line as deposited with the ATCC having accession number K89110, or a mutant, recombinant or genetically engineered derivative thereof.

28. 30. A method according to any one of claims 22 to 25 in which the ligand comprises a lymphokine.

29. 31. A method according to claim 30 in which the lymphokine comprises a B-cell growth factor.

30. 32. A method according to claim 30 in which the lymphokine is on the surface of a cell.

31. 33,, A method according to any one of claims 22 to 25 in which the second ligand comprises an antibody molecule that binds to Bp35.

32. 34. A method according to claim 33 in which the antibody that binds to Bp35 further comprises a monoclonal antibody.

33. 35. A method according to claim 23 in which fhe activated B-cells are treated with the ligand that binds to 3p50 within about 12 hours after activation by the second ligand that binds to Bp35.

34. 36. A method for suppressing proliferation of cells which express BpSO, a 50 kiloDalton B-cell surface antigen defined by monoclonal antibody G28-5, comprising treating the cells which express BpSO with an effective dose of a ligand that binds to BpSO, which ligand is coupled to an antiproliferative agent.

35. 37. A method according to claim 36 in which the cells which express BpSO comprise B-cells. 4 6

36. 38- A method according to claim 36 in which the cells which express Bp50 comprise malignant cells.

37. 39. Use of an effective dose according to any one of Claims 36 to 38 in a composition for treatment of the human or animal body.

38. 40. A method according to any one of claims 36 to 38 which is performed in vitro.

39. 41. A method according to claim 36 in which the ligand comprises an antibody molecule or an Fv, Fab, F(ab 0 ) 9 or Fab^ portion of the antibody molecule.

40. 42. A method according to claim 36 in which the antibody molecule comprises a monoclonal antibody molecule or an Fv, Fab, F(ab z )2 or Fab'' portion of the monoclonal antibody molecule.

41. 43. A method according to claim 42 in which the monoclonal antibody comprises G28-5„ or any Fv, Fab, FCab'’)^, or Fab'' portion thereof.

42. 44. A method according to claim 42 in which the monoclonal antibody is produced by a hybridoma cell line as deposited with the ATCC having accession number HB9110, or any mutant, recombinant or genetically engineered derivative thereof.

43. 45. A method according to claim 36 in which the ligand comprises a lymphokine.

44. 46. A method according to claim 45 in which the lymphokine comprises a 3-cell growth factor.

45. 47. A method according to any one of claims 36 to 46 in which the antiproliferative agent comprises an alkylating agent. 4 7

46. 48. A method according to any one of claims 36 to 46 in which the antiproliferative agent comprises an antimetabolite.

47. 49. A method according to any on© of claims 36 to 46 in which the antiproliferative agent comprises an antibiotic.

48. 50. A method according to any one of claims 36 to 46 in which the antiproliferative agent comprises a vinca alkaloid.

49. 51. A method according to any one of claims 36 to 46 in which the antiproliferative agent comprises an enzyme.

50. 52. A method according to any on® of claims 36 to 46 in which th® antiproliferative agent comprises a platinum coordinated complex.

51. 53. A method according to any one of claims 36 to 46 in which the antiproliferative agent comprises a radioisotope. 34. A substantially pure ligand as described in claim 1 substantially as hereinbefore specifically described.

52. 55. A substantially pure surface antigen according to claim 2G substantially as hereinbefore specifically described. ,1 V, 4 8

53. 56, a method according to claim 22 proliferation of 3-cells substantially specifically described. for augmenting as hereinbefore

54. 57. A method proliferation of described. according to claim 36 B-cells substantially for suppressing as hereinbefore

55. 58. use of a ligand that binds to BpSO, a 50 kiloDalton B-cell surface antigen defined by monoclonal antibody G28-5, for augmenting in vivo proliferation of 10 B-cells, by treating activated B-cells so that the activated B-cells traverse the cell cycle and prolifera tion is augmented.

56. 59. use of a ligand that binds to BpSO, a 50 kiloDalton B-cell surface antigen defined by monoclonal 15 antibody G28-5. which ligand is coupled to an anti-proliferative agent, for suppressing in vivo proliferation of cells which express BpSO.

57. 60. Use according to claim 58 or 59, substantially as hereinbefore described.