IE871563L - Process for producing natural heparan sulphate andTION their¹pharmaceutical use - Google Patents

Abstract

A new B-cell receptor, Bp50, a 50 kilodalton polypeptide, that functions in B-cell proliferation is described. Ligands such as lymphokines, antibody molecules or the Fv fragments of antibody molecules that bind to Bp50 and augment the proliferation of activated B-cells can be used to regulate B-cell proliferation or differentiation. [CA1338781C]

Description

6 0 4 8 6 i 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 B~ 5 cell surface suarkerf? herein referred to as Bp50, that functions in S~cell proliferation but not in early B-cell activation. The present invention is also directed to the 3p50 B-cell antigen itself, in a particular embodiment of the present invention,, a monoclonal antibody,, G2S-5, is 10 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 antibodiesf lymphokines and fragments thereof of the present invention can be used to 15 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 he used in the treatment and/or detection of malignant cells that express the BpSO antigen. 2* BACKGROUND OF THE INVENTION The activation of resting B-cells fro® G^ to G., phase of the sell cycle and the subsequent induction of activated B-cells to proliferate are distinct steps requiring distinct regulatory mechanisms* Some agents, including saurine B-cell 25 stimulating factor-pl CBSF~pl) (Rabin, et aj.., 1985,, Proc. Mat, Ji=cad„ Sei. USA 82, 2935-2939) or low doses of antiimmunoglobulin (anti-Tig) (DeFranco, et al-, 1985, J.

Immunol. 135:37-94 ; Wetzel, et al -, 3.984, J. Immunol. 133:2327-2332; DeFranco, et al_., 1982, J. Exp. Hed. 30 155:1523-1536; Muraguchi, et _al., 1934, J. Immunol. 1321176-180) , are "activation" or 'competence* factors.

That is, they induce 3-cells to enlarge, synthesize jnore RNA, and enter G,, but alone they do not induce DMA synthesis in B-cells. Other "growth" factors, such a© human B-cell 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 (Kehrl, et al., 1984, Immunol. Rev. 18s75-9Sj Muraguchi, et al., 1984, J,., Immunol. 132:176-180;; Zubler, et al. 1984, J. Exp. Bed. 160:1170-1183; Jung, et 8l_., 1984, J. Exp. Med. 160:1597-1604).

A number of factors that promote the growth of B-cells have now been described by investigators of both aurine 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 (XL-1) and interleukin-2 (IL-2) have been shown to augment B-cell growth, they apparently are distinct from certain BCGFs. For instance, sionoclon&l antibodies (mAb) to a murine BCGF (O'Hara, et al., 1985, Nature (Lond.) 315:333) or human BCGF (Ambrus, et al., 1935,, J. Exp. Med. 162:1319) block BCGF activity but sot 2L-1 or IL-2 activity,., Although distinct frosa 2L-1 or IL-2, the BCGFs themselves appear to be heterogeneous based on biochemical data and differential activity on different B~ cell subsets or costisaulation assays. For instance, 60-kilodslton (kDa) high-molecular-weight human SCGF, BCGF (high), has been identified that is distinct froa a 12-kDa low-molecular-weight fora, 9CGF (low) (Aabrus, et al., 1985, J. Clin. Invest. 75:732). The cDNA encoding a 20-kDa aurine BCGF, tentatively designated B~celi stimulating factor pi (BSf-pl), has recently been cloned and sequenced (Nona et al., 1986, Nature 319:640). The recombinant lymphokine not only has BCGF activity but can also activate resting B-cells 4 and induce the differentiation of XgG1 producing cells; thus it differs front 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 B-cell surface structures. Ira addition to the antigen-specific signal '-through surface Xg, several other candidate B-cell surface polypeptides have been identified that way 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 s J. Exp. Med. 160:1126) have been characterised,, and recently functional IL-2 receptors have been identified on B-cells (Zubler, et al.r 1934, J. EKp. Med, 160:1170; Jung, 15 et al., 198«», 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 say in some way 2o function in the activation or growth o£ B-cells. For example, Subbarao and Hosier (Subbarao, et ajU „ 1983,* Immunol. Hev„ S9sil-97| found that monoclonal antibodies (mAb) to the murine B-cell antigen Lyb2 activate B-cells, and recently evidence has beer, presented suggesting Lyb2 may 25 be the receptor for BSF-pl (Yakura, 19BS, Fed. Proc. 44:1532). Similarly, we have found that appropriate aAb (1F5) to a 35 kDa polypeptides Bp3Se, activates human B-cells from GQ into G, (Clark, et al.» 19BS, Proc. Sat. Acad, Sci. US& 32:1766-1770; Gollay, et slif 1985^ J. Immunol. 135:3795-3301}. Aggregated C3d or antibodies to the 240 kDa C3d receptor, 5pl4 0, cause proliferation of B-cells that are T-cell dependent (Melchers, _et al.f 1935, Nature 317:264-267; Nemerow, et al., 1985„ J. Immunol. 135:3063-3073? 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 bean isolated. Wang and coworkers (Wang, et al., 1979, J. Exp. Med. 149:1424-1433) 5 made a polyclonal antisera that identified a 54-kDa polypeptide C®PS4) human B-csllss and shoved that, the rabbit antisera to gpS4 induced tonsillar 3-cells to divide. Recently, Jung and Pu (Jung, et al., 1984, J. Exp. Med. 160:1919-1924) isolated a aAb (AB-1) to a 55-kDa antigen 10 restricted to activated B-cells "that blocks SCGF-dependent proliferation. However, whether or not either anti-gp54 or AB-1 recognise a BCGF receptor is not yet known. 3. SUMMARY OF THE INVENTION The present invention is directed to substantially pure ligands 15 which (a) bind to Bp50 t, s 50kDa B-cell specific surface antigen described herein, and (to) augment the proliferation of activated B-cells. The invention is also directed to the BpSO antigen itself, which is defined by monoclonal antibody G3S8-5 and functions in proliferation of activated B-cells. 20 In addition the invention is directed to ligands which bind to BpSO, but do not demonstrate a biologies! effect or function such as augmentation of the proliferation of activated B-cells.

The ligands of the present invention include antibody. 25 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 mot limited to the Fv, Fab*. F(ab#),, Fab' and the like. In addition, the ligands of 30 the present invention comprise: lymphokines, which can include but are not limited to human IB-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. 0 Such compounds include but are not limited to cytotoxic agents, therapeutic agents,, chemotherapeutic agents, labels such as radiolabels, dyes, enzymes, radioopaque compounds,, and the lik e. Tib© ligands of the present invent ion can in S their Modified or unmodified form, b® used to direct, regulate and modify human B-cell proliferation and/or dif ferentiation.

The present invention is based upon the discovery that two human B-cell differentiation antigens, Bp35 and the 8-10 cell antigen described herein, Bp50£, apparently play distinctive roles as signal receptors in B-cell activation. Monoclonal antibodies (mAb) to 8p35 and 3p50 both deliver positive signals to 3-cells that stimulate their transition through the cell cycle. HAb to Bp35, like anti-Ig 15 antibodies, functions principally to activate resting B- cells to become competent to enter the G, phase of the cell cycle. In contrast, a monoclonal antibody described herein or its F(ab#)0 fragment to 8pS0f, a 50-kDa polypeptide expressed on all 3-cells, functions to stimulate activated 2Q B-cells to traverse the cell cycle and augments ttie proliferation of activated B-cells. Monoclonal antibodies to 3p35, like anti-lg antibodies, activate tonsillar B-cells and induce low levels of 3-cell proliferation. In contrast, anti-BpSO monoclonal antibody alone neither activates B-2g cells nor induces B-cells to proliferate, but together with anti-Bp35 or anti-Ig antibodies, augments 3-cell proliferation, In this respect the action off anti-BpSO antibody resembles the activity of B-cell growth factors (BCGF). As little as 0.05 ug/ml off anti-BpSO is seeded to 2Q augment proliferation and, like BCGF, anti~3p5Q is effective even when added 12 to 24 hours after B-cells are activated with anti-Ig or anti-Bp35- Without additional exogenous signals,, anti-Bp35 and anti-BpSO antibodies together induce strong proliferation of purified resting B-cells. These ,r results suggest that the Bp35 and 3p50 surface ssolecules 7 function in the regulatory control of B-cell activation and progression through the call cycla. The significance anti~Bp3 5 end like molecules have on the effect and action of the ligands of the present invention, is discussed 5 in Clark et al., 1985, Proc. Natl. Acad. Sci„ (USA) 82;1766-1770.

Although the activity of anti-BpSO resembles that of BCGF (low) since both anti~Bp50 and BCGF (low) are costinulatory with the sa^e agents but not with each other and both In one embodiment of the present invention, the ligands 25 which bind to BpSO and augment the proliferation of activated B-cells can be us In another embodiment, the ligands of the invention can be chemically modified so that the sells to which the ligands bind are killed. Since all B-cells express the BpSO 8 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 be used in vivo to cause immunosuppresion in order to cross histocompatibility 5 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 HO chemothesrapeutic agent useful in treating such neoplastic disease. These modified ligands can be used in vivo to direct the cherootherapeutic agent to any type of, malignant cell which expresses the BpSO antigen including cells which are not B-cells but which do express Bp50. when using the 15 ligands of the invention which augment B-cell proliferation,, a particular advantage should be realised whan treating B~ •cell Malignancies where the chemotherapeutic agent linked to the ligand comprises one that is more effective in killing proliferating cells; 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 ligands 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. 0 3-1. DEFINITIONS As used herein,, the following abbreviations will have the Meanings indicated: AO - acridine orange 5 BCGF ™ 3-cell growth factor BCGF {high) * a W kDa human BCGF BCGF (low) " ® 3L2 human BCGF Bp35 86 a 35 kDa 3~cell specific surface polypeptide (CD20) defined by mAb IFS 10 BpSO ■ a 50 kDa B-cell specific swrface polypeptide defined by snAb G28-S Fv « the variable region or antigen-combining site off an antibody molecule. This may be any fragment which contains the idiotype of ig the molecule including but not limited to the FabP F(ab'),» Fab'# and the like. IF « immunofluorescence Ig « immunoglobulin IL-1 » interleukin 1 0q IL-2 « interleukin 2 kDa ■ kilodalton jnAb « monoclonal antibody SDS-PAGE » sodiua dodecyl sulp'nate-polyacrylamide gel electrophoresis «r TPA » l2-0-tetradecanoylphorbol~13 acetate 4. DESCRIPTION OF THE FIGURES Fig. 1. Expression of BpSO is restricted to Bp35+ B-cells. Two-color flow cytometric analysis of 50,000 cells 20 «as performed as described (Clark, Acad. Sci„ USA 82:17S6-17?0|. The data are plotted as cell number versus log of green fluorescence and log of red fluorescence where 4-5 dots represent approximately a 1 0 doubling of fluorescence™ The data 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 3p35+.

S Fig. 2. Biochemical comparison of BpSO polypeptide with other B-cell surface antigens.. Immunoprecipitation of 105 BpSO from surface I-labeled tonsillar sells was performed as described. Isolated antigens were electrophoresed on 1Q£ SDS polyacrylamide slab gels without reduction. Gels were 10 visualised with autoradiography and intensifying screens. Panel A: lane 1, anti-BpSO (G28-5); lane 2, anti~Bp95 (G28-8); lane 3, sepharose-goat anti-mouse Ig only.

Exposure time: 4 days. Panel B: lane 1„ anti-3p50 (G28-5); lane 2, anti-Bp4 5 (BLAST-2)? lane 3, anti-Bp39 (G28-1); 15 lane 4, anti-Bp39 (41-H16)s lane 5, sepharose-goat anti-mouse 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 BpSO expression. Peripheral blood or tonsillar aononuclear cells were isolated by centrifugation on Ficoll and stained with PE (red)-conjugated G23-5 (anti-BpSO) in combination with fluorescein (green)-conjugated reference antibodies, 25 including 2C3 (anti-IgH); 1F5 (anti-Bp35); HBlOa (anti-DR)1 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 ansd right angle light scatter was used to gate out aonocytes. Unstained cells are 20 positioned at the back off the grid? red fluorescence is to the right and green fluorescence is to the left.

Fig. 4. Dose response curves for augmentation of proliferation of dense tonsillar Er~ B-cells by anti-BpSO antibodies as indicated: Media only; anti~Bp50 only anti-Bp35 (5 ug/ml) only; 3C*GF only; anti-Bp35 plus BCGF; anti-S Bp35 plus graded doses of anti-BpSO. Mean proliferation ♦ standard error of guadruplicate samples was measured on day 3.

Fig. 5. Anti-BpSO aAb are aost effective at augmenting proliferation if added after a B-cell activation signal.

Dense tonsillar Er- 3-cells were incubated for 4 days with media only, anti-BpSO <0.S ug/al) added at different times after incubation, anti-Bp35 (5 ug/al) added at different times after incubation; anti-BpSO kept constant to which anti-Bp35 vas added later at different times; &nti-Sp3S 15 kept constant to which anti-BpSO was added to cultures at different times. During the last 10 hr H-thyaidine was added and its incorporation was measured.

Fig. S. Comparison of the ability of anti-Bp3S and anti~Bp50 to induce resting tonsillar B-cells to leave the 20 G0 stage of the cell cycle. Day 3 post treatment media only (_ ), anti~3p35 only f—; and Ig only A, no additional additives; B, anti~Bp50 (0.5 ug/ml) added to each group; c, 5% BCGF addled to each group. Data is plotted as relative cell number versus log of AO red 25 fluorescence (RNA).

Fig. 7. Kinetics of 3-cell proliferation after stimulation with aati-3pS0 versus BCGF. Dense tonsillar E~ B-cells %?ere stimulated with media alone; 10% BCGF only; anti~Bp33 only? anti-Bp50 only; anti~Bp35 + 3.0% BCGF; anti-30 Bp35 + anti-BpSO; and anti-Bp35 + anti-Bp50 + 10% BCGF.

Proliferation was measured on the days indicated by an 18- 3 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 anti-3p50 (A) or BCGF (B) optimally augment proliferation. Dense tonsillar E- B-cell© were stimulated as shown and proliferation was measured by an IB hour pulse of 3H thymidine on day 3. Media? anti-Bp35 only added at times indicated? anti-BpSO or BCGF only; anti-Bp35 added at start of culture followed by addition of anti-BpSO or BCGF at times indicated? anti-BpSO or BCGF added at start of culture followed by anti-Bp35. On® of two experiments. Proliferation was measured in quadruplicate and standard errors are shown. Doses used: anti-Bp35, 5 ug/ml; anti-Bp50, 0.2 ug/nl; BCGF (low) 54. Concentrations used were as follows: anti-3p35, 5 ug/ml; anti-Bp50, 0.2 ug/ml; bcgf, 54.

Fig. 9. Anti-BpSO and BCGF have additive effects on B-cell proliferation. Dense tonsillar E- B-cells were stimulated with graded doses of BCGF (low) together with anti-BpSO only; anti-Bp35 only; anti-Ig~beads only ; anti-Bp35 + anti-BpSO; or anti-BpSO ♦ anti-lg. Proliferation was measured on day 3 after stimulation with an 18-hour pulse of ~H thymidine. Proliferation was measured in quadruplicate and standard errors are shown. One of four experiments. Doses ?ased 10b cells: &nti~Sp3St, S ug/ml; anti-Bp50, 0.2 u@/ml; anti-Ig-beadsf 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 or without TPA (75 ng/sal) in the presence of 10% BCGF or 1 nag/ml a«ti~Bp50.. Two separate 3-cell lymphomas (panels B 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 BpSOs a SOkDa B-cell specific surface polypeptide, and (*>) augment the proliferation of activated B-cells. The invention is also directed to the BpSO antigen itself, which is defined by saAb G2S-5 and functions in B-cell proliferation. In addition, the invention is .directed to ligands which ibind to Bp50 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 wolecules, monoclonal antibody molecules and fragments of these antibody molecules which contain the antigen combining site that binds to the BpSO receptor including chemically saodifi€:d antibodies and fragments? such fragments include but are not limited to the pv, Fab,, F(ab'52# Fab* and the like. In addition, the ligands of the present invention comprise lymphokines, which bind to the BpSO receptor.

These isay include but are raoft limited to BCGFs as well as chemically modified lymphokines and the like. The ligands of the invention can be used in their modified or unmodified forms to modulate and regulate immune responses and in the therapy of malignancies which express the B®SO antigen.

These 'asses are discussed in more detail im Section 5-4 below.

Where the ligand is a monoclonal antibody, or a fragment thereof, the monoclonal antibody can be prepared against BpSO using any technique which provides for the production of antibody molecules by continuous cell lises in culture. For example,, the hybridoma technique originally developed by Kohler and Milstein (1975, Mature 256:495-497) as well as other techniques which have more recently become available, such as the human B-cell hybridoma technique (Kozbor _et zil., 1983*, Immunology Today 4:72) and the EBV-hybridoma technique to produce human monoclone1 antibodies (Cole et a_l. , 1985s Monoclonal Antibodies and Cancer Therapy, Alan H. Liss, 2nc.f pp. 77-96) and the like are within the scop® of present, invention.

Antibody fragments which contain the idiotype of the aolecule could be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')., fragment which car* 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(ab'), 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 Bp5Q is a lymphokine, the lymphokine may be obtained from natural sources or if its amino acid sequence is known or deduced the lymphokine can be synthesized via chemical synthetic methods.

Alternatively, if the gene sequence of the lymphokine is known, recombinant DMA techniques may be utilised to clone 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 iigarid or appropriate fragments of the ligand siay be chemically modified by the attachment of any of a variety of compounds to the ligand using coupling techniques known in the art. Such techniques ®ay include but are not limited to the use of carbodiimide, cyanogen bromide, bifunctional reagents such as glutaraldehyde, N-succinimidyl 3-(2-pyridvldithio) propionate fSPDP), 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,, 5 chelation etc. For a review of the chemical reagents which can be used for protein modification see, Lundblad and Moves, Chemical Reagent® for Protein Modification,, Volume IX, CRC Press,, 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 the binding site of the ligand prior to performing the coupling reaction. For example, the 15 ligand can first be bound to Bp50 in order to protect the 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 r&ction is complete,, the complex can be disrupted 20 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 the ligand comprises a monoclonal antibody such as G28-5, in which the Fc domain of the molecule is not required far the ligand to exert its 25 effect (see Section 5.3.3. infra) it may be advantageous to direct the coupling of desired compounds ta the Fc domain of the molecule.

The subsections bel©"^ describe the new, 50-fcDa B-eell surface marker, BpSO^, which apparently functions in B-eell 2q 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 defines BpSO and its Ffab^J^ fragments are also described which* 1 6 like BCGF, augments B-cell proliferation. Unlike anfci-Bp35 ®Ab, which can induce resting B~cells in GQ to enter G1, anti-Bp50 mAb does not activate resting B-cells. Anti~Bp3 5 and anti-BpSO mAb together, without any additional exogenous 5 signals, induce strong activation and proliferation of purified B-cells, The experiments described below also demonstrate that anti-BpSO activity resembles BCGF activity but that anti-Bp50 is distinct from one BCGF since anti-3p50 and low 10 molecular weight BCGF are clearly additive and act differently on various B-cell subsets or malignancies. 3p5Q may be a receptor for a distinct BCGF or for a transmembrane signal that modulates BCGF production or BCGF receptor expression. 5.1. METHODS USED TO CHARACTERIEE THE BpSO 3ECEPT0R Cell preparations. Mononuclear cells were isolated from normal or leukemic heparanized peripheral blood by Ficoll-Hypague gradients (Pharmacia,, Piscataway, NJ) .

Mononuclear cells were obtained from tonsillar tissues as described (Clark, etjsl., 1985, Proc. Nat. Acad* Sei» USA 82i1766-1770). T cells were depleted with AET-treated sheep erythrocyte resetting and Ficoll-Kypague 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 4,5 minutes unless otherwise stated- Buoyant or dense tonsillar B-cell fractions were isolated by Percoll step gradients as described (Clark, et _al., 1935, Proc. Mat., lead. Sci. USA 82:1766-17705. Senise tonsillar B-cell preparations consistently had greater than 95% «Xg* Bp35+ cells. Blood 3-cell-enriched preparations had 60-35% 1 7 slg-t- cells. B~cel1 lymphoma cells were isolated by gently teasing lymphoma cells into medium followed by Ficoll-Hypaque gradient centrifugation.

Monoclonal antibodies. The G28-5 antibody to Bp§0 was S generated by immunizing B&LB/c nice ujth bunian S- tonsillar lymphocytes and fusing immune spleen calls with the NS-1 myeloma (Kohler, et al., 1975, Mature 256:495-497; Ledbetter, cat al., 1979, Immunol. Heve 47:S3~82}„ Hybrid cell cultures secreting antibody reactive with tonsillar 8-10 cells and not with T calls were identified by the use of indirect immunofluorescence (IF) and analysis with a FACS IV call sorter; cultures with antibody giving histogram patterns similar to known mAb to pan S~cell markers (e.g., Bp35) were cloned and selected for further study. The G28-5 ^5 clone produced an IgG1 mAh that reacted only with normal or malignant B-cells sr B-cell lines. Other mAb ?j,sed in this study have been described in detail (Clark,, et_a_l.» 1985, Proc. Nat. Acad. Sci» USA 82:1766-1770.- Clark et al. . 1986, Human Immunol, las 100; Ledbetter, 1986, Human 2q Immunol.15:30-44; Ledbetter, et al., 1985, in Perspectives in Immunocenetics and Histocompatibility, ASHX, New York, 6, pp. 325-340). Thasa inclu.de XF5 anti-Bp35, HBlOa (IgG,a) , anti-HLA-DR, 2C3 (ISGi) anti-u chain, G19-4 (IgG,,) ar.ti-CD3, FC-2 (IgG,^) anti-Fc receptor GDI6, and 9.6 os (IcG_ ) anti-CD2 (S receptor) provided by Dr. Paul Martin " 4, 01 ('Martin, _et al_., 1983, J. Immunol. 131:180). The IgG, mAfas were purified by precipitation using 45% or SG% saturated asrroonium sulfate and DEAE SEPH&CHYL (Trade Mark) column chromatography, and the IgCrjg mftbs were purified by the use of 20 protein A SEPHMGSE (Trade Mark) columns. The F(ab8 )0 fragments of G28-5 were prepared by the method of Farham (Par ham, et al., 1983, «3. Immunol. 131s2895) purified on a 2-meter long SEPHAGRYL* S200 column, and assayed for purity by SDS-PAGE (Ledbetter, et_ * (Registered Trade Mark) 11 8 jil., 198 5, 3, Immunol. 13 5:1819). The 2C3 mAb to u-chains was conjugated to Sepharose 4B beads (Pharmacia Fine Chemicals, Uppsala, Swedan) using cyanogen brosnide coupling. Fluorescein and phycoerythrin conjugations. Purified S aAb were either directly conjugated with fluorescein using fluorescein-isothiocyanate (FITC; Molecular Probes) (green) by the aiethod of Goding CGo<3ing, et _al., 1976, J. Immunol* Heth... 13s215-226)# or conjugated to R-phycoerythrin (PE) (red) by using sPOP (Pharmacia) with a aiethod we have HO detailed in Ledbetter, et _al., 1935, in Perspectives in Immunoqenetics and Histocompatibi ity, ASHIf 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 mAb, washed twice, and then 15 analysed 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 dichroie mirror to split the beam and a 580 long-pass filter 2Q and a 54 0 short-pass filter (Ditric Optics, Hudson, MA) in front of the red and green photomultiplier tubes, respectively. In addition, a two-color compensator (T. Nozaki, Stanford University) was used to correct for minor spillover of green and red signals. For each two-color 25 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 64 dot grid. Approximately 4.5 dots represents a doubling of fluorescence. Unstained cells are positioned at the back corner of the grids red fluorescence is to the i^U right and green fluorescence is to the left- Our flow cytometry system for two-color IF with fluorescein and 1 9 phycoerythrin is described in snore detail (Ledbetter, _et al., 1985, in Perspectives in Immunoqenetics and H istocompa tibiity „ ASH I, Mew York,. 6, 119-129 and 325-340) .

Cell culture. Blood or tonsillar lymphoid cells vara 5 cultured at 5-10 x 10 al in quadruplicate in 96-well aicrotiter plates containing 200 ml RPM1-1640 medium supplemented with 15% fetal bovine serum, antibiotics, glutamine, and pyruvate (R15). After 1 to 7 days, cells were pulsed with 0.5 uCi of 3H thymidine per well (New 10 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; 15 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, 20 et_*l-# 1982, Proc. Mat. &ca4* Sci. USA 79:5993), who have shown that the Major BCGF activity in this material resides in a 12-kDa species, hereinafter referred to as "BCGF flow)* (Mehta, et al_., 1935, J. Immunol. 135:3293). The purification steps included preparative scale DEAE affinity chromatography followed by hydroxylapatite column chromatography.. IL-1 purified to homogeneity was the generous gift of Dr. Steven Dower (Dower, et jsi., 2.985,, J. Exp. Med. 162:501). Recombinant IL-2 was kindly provided by Cetus Corporation. TPA (12-0-tetradeconoyl phorbol 13-acetate) 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 2 0 in cellular RNA and DNA levels were measured by staining activated calls with acridine orange and measuring relative cellular RNA (red) and DNA (green) content with a cell sorter by the method of Darzynkiewicz et al. (Darzynkiewicz, ■ 5 et al.» 1980, Proc. Hat. Acad. Sei„ USA 77:6697-6702) .

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

Biochemical characterisation of BpSO. Immunoprecipi- '125 tation of BpSO from surface " I-labeied tonsillar cells was performed as described (Ledbetter, et al., 1985,, J. Immunol. 134:4250-4254) . Isolated antigens were electrophoresed on 10% SDS poly aery lcimide slab gels without reduction. Gels 15 were visualized using autoradiography at -70°C and cronex lightening plus intensifying screens (Dupont). .2. CHARACTERIZATION OF THE BpSO RECEPTOR The subsections below describe the results of the experiments conducted using the methods described above. 5.2.1. IDENTIFICATION OF A B-CELL SPECIFIC SO kDA CELL SURFACE MARKER, BpSO.

A mAb to BpSO was raised by immunizing BALB/c sice with human tonsillar lymphocytes and fusing immune spleen, cells with the NS-1 myeloma. One clone, G28-5, produced an igG, mAb 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 B-cells ©r B-eell lines. A comprehensive screening of normal tissues by established methods (Clark, etjal** 1935, Proc. Mat. Acad. Sci. USA 82:1766-1770; Ledbetter et-al., 1936, Human Immunol. 15:: 30-44? Ledbetter, 1935, ir5. Perspectives in Immunogenetics and 2 1 Histocompatibility, ASHIt Hew York, 6, pp. 325-340) revealed that the G28-5 antibody reacts with E rosette negative (Er-) cells from blood sr tonsils but not with nylon wool nonadherent T cells, PHA-induced T-cell blasts,, or with 5 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 fRaji, Daudi, Namalwa), but not with four T cell lines (£EM„ KS3-2, JURKAT, and HPB-ALL)- All chronic lymphocytic leukemias 10 tested (3/3) and 90* (9/10$ ot B lymphomas tested expressed the Bp50 marker while only 28* (2/7) of non T, non 3 CALLA* acute lymphocytic leukemias expressed Bp50.

The restricted distribution of Bp50 on normal tissues was further confirmed by quantitative two-color U5 immunofluorescense (two color IF) analyses. Using an R- phycoerythrin (PE)-conjugated antibody (red) to the pan B-cell antigen 3p35 (Bl* CD20) and fluorescein-conjugated anti-Bp50 antibody (green)» we found that BpSO was expressed only on Bp35+ B-cells (Fig* 1) in blood or tonsils. Blood ,0 B-cells consistently expressed somewhat lower levels of BpSO ■than tonsillar B-cells; this is similar to HIA-DR expression, (Ledbetter et al., 1936, Human Immunol„15:30-44) and to gp54 expression (Hang, et al., 1979, J. Exp. 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 aiAb to the t cell marker, CD3(T3), and NK cell-associated marker, CDlS(Fc receptor) (Ledbetter, et al., 1979, Immunol. Rev. 47:63-82), ,0 we found that BpSO is not expressed on f cells or NK cells. Using two-color IF, we also found that CD3+ PHA blasts that, expressed high levels of IL-2 receptors did not express Bp 50» 2 2 The G28-5 antibody reacted with a single polypeptide an tonsillar lymphocytes that migrated at approximately 50 Ed under non-reducing conditions (Fig. 2A). This molecule is larger than previously reported B-cell markers in the same S molecular weight range such as Bp39 or 3»45 (Zipf, et al., 1983, J. Immunol. 131s3064-3072? Xitner, et.al., 1981, Mature 294, 458-460? Clark, et_al., 1986, in Leukocyte Typing XX, eds. Reinherz, et al., Springer Verlag, Berlin, Chap. 22 Vol. 2, 155-167? Slovin, et«l.r 1982, Proc. Hat. 10 Acad. Sci. USA 79:2649-2653; Thor 1 ey-Lawson, et al.» 1985, J. Immunol. 134:3007-3012,, and Fig. 23). The exposure tine for this gel was selected so that the molecular weights of the other B-cell markers could be readily compared with 3p50. The 3p39 marker,, unlike 3p50, is expressed a si 15 .'granulocytes and Bp4 5? unlike Bp50, is restricted to B-cell blasts. Antibodies to 3p39 (41-H16) and Bp45 (MNM6, Blast-1, Blast-2) made available through an international workshop (Clark, et al., 1986, in Leukocyte Typing II, eds. Reinherz, et .al., Springer Verlag, Berlin, Chap. 12 Vol. 2, 155-167) 2o did not block the binding of fluoresceinated anti-BpSO antibodies to B-cells.. Thus, based on tissue distribution, biochemical analysis, and blocking studies/ the G28-5 Monoclonal antibody recognises a 50-Kd structure distinct from other known B-cell antigens. 5.2.2. 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 B 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 Bp 50* 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 NK cells. Furthermore, ConA- 2 3 ■activated CD3+ T-cell blasts expressed IL-2 receptors but, did not express BpSO.

Two-color flow cytometric analyses allow the quantitative measurement of the density relationship between S two surface antigens. Me previously showed that the dense, resting B-cells in the mantle zone of secondary follicles express IgM end low0 levels of Bp35g whereas the buoyant, activated B-cells in the germinal center are XgM-negative and express elevated levels of Bp3 5 (Ledbetter, et al., 10 Hunan Immunol- 15:30). Figure 3 shows that both IgM~ positive and IgK-negative B-cell subsets expressed BpSO in equal amounts,, indicating that BpSO is expressed on both resting B-cells and 3-cells activated in vivo. .3. AUGMENTATION OF 3-CELL PROLIFERATION 1 5 'WITH ANTI-Bp50 ANTIBODY As previously explained, B-cells can be activated with low deses of anti-u chain specific antibodies. Ha recently found that the B-cell-speeific marker Bp3S (Bl), a 35-kDa polypeptide, say also function in early 3-cell activation" 20 the 1F5 mAb to Bp2S£! like low doses of anti-u antibody# activates B-cells to increase in cell volume and RNA content and to becssae responsive to BCGF (Clark., et_&l«# 3.985, Proc. Nat. Acad. Sci. USA 32:1766-1/70? Collav. at al,, 1985, J. Immunol. 135:3755-3301). Therefore, it was off interest to 25 compare the effect of anti-Bp5Q mAb in the proliferation of untreated B-cells or B-cells activated with either anti-Bp35 or anti-u antibodies (Table X). Anti-3p35 in solution or anti-u antibodies attached to SEFHAROSB (Trade Mark) beads, under appropriate conditions alone, could stisraAlate some B-cell 30 proliferation (Table 1„ line 1) ; in contrast, anti»Sp5Q antibodies alone did not stimulate proliferation (Table X, line 2). However, anti-BpSO mAb augmented proliferation cornsider&bly «her. cultured with anti-u beads or vith anti-BpsS. In this respect anti-BpSO resembled BCGF (Table X, 35 line 3) . Thus, it was important to determine whether 2 4 anti-BpSO and BCGF together could induce B-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 5 somewhat more than either stimulant a!one» BCGF over a three-log range, when used with anti-BpSO without other signals, had no effect on proliferation of dense 3-cell® even when anti-BpSO was used at doses ranging from 0.1 to 10 ug/ml.

Table 1 Augmentation of Anti-Ig or Anti™Bp3S Induced B Cell Proliferation with Anti-BpSO Antibodies Mean Proliferation + S.E- of B Cells Cultured With: Line Co-stimulant Media Anti-u-beads Anti-3p3 5 1 Hone 2 Anti-BpSO 3 BCGF 4 Anti-BpSO 4- BCGF 1,212+547 1,219+4 62 , 539+308 719+718 38,792+1,329 25,465+616 456+217 14,217+445 9,443+343 1,456+126 54,393+2 r53™ 46,488+3,387 Proliferation of (Sense Er- tonsillar 3-cells C tS3?: surface 3 as described. 3riefly, IgM' cells) was measured on day 2 x 10s cells/200 ul well were cultured in quadruplicate for 4 8 hrs with HPMI 164 0 medium containing 154 fetal bovine serum plus additives without antibody or with either 2C3 monoclonal antibody to u chains coupled to sepharose beads ("anti-u heads*.* 50 ug/ml) or free IFS anti~Bp33 antibody (5 ug/ml). Cultures containing media, "anti-u beads," or anti-Bp35 were cultured alone or with BCGF (5* final concentration,, Cytokine Technology, Buffalo, Sew York: has no detectable IL-1 or IL-2 activity), with anti-BpSO (1:1000 dilution of ascites) as co-stimulants. After 40 hrs cells were pulsed with H-thymidine, and counts incorporated were measured after 18 hrs- -3-1. ANTI-3p50 mAb AUGMENTS PROLIFERATION ONLY AFTER B-CELLS ARE ACTIVIATED BV ANTI~Bd35 OH ANTI-U-ANTIBODIES The results in Table 1 suggest that anti-BpSO mAb could 5 not induce proliferation by itself™ As shown in Fig. 4, doses of anti-BpSO ranging from 0.05 sag to 2.0 ug/al had no effect on ~H~thymidine uptake. However, in the preser.ee of optimal levels of anti-Bp3S mAb, as little as 0.1 to O.S ug/ml of anti-BpSO antibodies augmented 10 proliferation substantially™ As much as 50,000 to 70,,000 epra war® detectable at the optimal time of proliferation when highly purified B-cells were cultured only with anti-Bp3S plus anti-3p50- A consistent observation was that higher doses of anti-BpSO (greater than 2-5 ug/ml) were less 15 effective than doses in the 100-200 ng range.

These results suggested that anti-Bp50 may function only after B-cells .are activated by other signals. Data shown in Fig, S suggest that this is indeed the case. If 3-cells were first activated with anti~Bp35, anti-BpSO could 20 be added as late as 24-48 hours later and still augment proliferation at day 4. In contrast,, when cells yere first treated with anti-BpSO, anti-Bp35 was effective only if added vithin a few hours after the start of cultures.

Similar results were found when anti-u rather than anti-Bp3 5 25 was 'iised. .3.2. antx-bo50 »Ab DO POT ACTIVATE B-CELLS out OF g_ but" do induce activated b-cells to progress TBBOUGH THE CELL CYCLE Previously® we have found that anti-Bp35, like low 30 doses of anti-u antibodies, induce resting tonsillar B-cells In G0 to enlarge (Clark, jet al., 1936, Leukocyte Typing II, ®ds„if Reinherz, jet al.. Springer Verlag, Berlin, Vol. 2, 4 55-4 62) and to enter the G, phase of the cell cycle (Gollay, et al. , 1985, J. Immunol. 135:3795-3801). Thus, it 35 was of interest to compare the ability of anti-BpSO mAb to anti-Bp35 mAb for their effects on B-cell activation. As 2 6 shown in Fig. unstimulated dense tonsillar B-cells even after 3 to 4 days in culture had a uniform RNA profile-characteristic of cells in CQ (Darzynkiewicz, 1980,, Proc. Nat. Acad. Sci. USA 77:6697-6702). However, about 15-301 of 5 cells stimulated "with anti-Bp35 or anti-u had increased RNA content indicative of entry into G1. In contrast, neither anti-Bp50 (Fig. S3) nor BCGF (Fig. 6C) ©lone induced significant numbers of B-cells to enter Gn. For instance, 2 days after activation, anti~Bp35 and anti-Ig mAb induced 10 respectively 13.51 and 20.9% of tonsillar cells to enter G1, whereas cells treated with only anti-BpSO (2.7%) or BCGF (3.2%) remained at media control levels (2.2%). However, when either anti-BpSO or BCGF were added together with anti-Bp35 or anti-u antibodies, the proportion of cells 15 entering G1 increased dramatically. Similarly, anti~Bp50 and BCGF alone did not induce B-cells to enter S phase (Table 2) t 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-BpSO and BCGF on Cell Cycle Progression in Tonsillar Lymphocytes Competence Signal aedia affiti~3p3 5 anti~lg Progression Signal none 89.9 80.4 65.6 % Cells G, 7.1 14.5 27.® s/g2/m 2.5 3.7 5.7 media a^ti-3p3S anti-Ig &«ti~3p5Q * S3 .6 54.3, 4 3.6 12.0 35.S 36.2 3.3 9.7 IS.2 ciedia anti-Bp35 anti-Ig BCGF 05.4 56.6 48.4 11.7 32.6 36.1 2.2 11.6 14.X Z 7 Percentage of cells in CQI G1 , or S and G^ determined with the use of the acridine orange-staining procedure (Darzynkiewicz, et al*, 1980 Proc. Hat. Acad. Sci. USA 71: 6697-S702) ; X x 10° dense tonsillar lymphocytes with 5 anti-Bp35 (5 ug/ml), anti-u on beads (50 ug/ml)0 anti-Bpso (0.4 ug/ml), BCGF (St) or combinations as shown. .3.3. OPTIMAL CONDITIONS FOH AUGMENTING B-CELL PROLIFERATION WITH ANTI-BpSO ANTIBODIES_ Antibodies to Bp50 by themselves have little or as detectable effect on dense resting s-cells (Table 3).

However, in th® presence of agersts that can activate 3- cells, such as anti-Ig, anti-9p35 and TPA, anti-3p50 ssM> clearly augmented proliferation. ^nti-BpSO did sot costimulate with several interleukins, including purified ^ IL-1, recombinant IL-2 and BCGF (low) . a comparison of the effects of anti-BpSO with those of BCGF (low) showed that the same agents that were costimulatory with anti~Bp50 were also costimulatory with BCGF (low) (Table 3). Of particular interest was th® finding that together BCGF and anti-BpSO 20 still were not costimulatory for resting cells.

Table 3" Augmentation of B-cell Proliferation with Anti-BpSO Antibodies ox 3-Cell Growth Factor Mean Proliferation ± S. 2. of B-Cells Cultured with: Anti-BpSO " BCGF Co-stimulant Madia (200 na/ml) rs%) none 96 + 1 2S7 + IS 285 + / "5! anti-Ig ,333 + 391 41,634 + 2,103 a 094 + 61 anti~Bp35 (5 ug/al) 487 + 45 8,143 + 280 1,733 + 32 TPA (2 ng/ml) 7,361 + 537 21,163 + 871 13,064 1,030 IL-1 (10 V/nl) 264 + 2 303 £ -3 221 s IL-2 C100 o/nl) 204 + 34 3S0 £ ^ 220 11 BCGF (5*) 220 + 7 851 4- 28 270 + IS Dens® Er- tonsillar B-cells /greater than 951 slgM' cells) cultured for 48 hr at 2 x 10" cells/well followed by 24 hr pulse with H-thymidine before counting. 28 The kinetics of proliferation augmented by anti-BpSO is shown in Fig. 1. 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-Ig 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 anti-BpSO (greater than 2-5 ug/ml) were less effective than doses in the 0.1-0.5 tag/ml range.

Human B-cells are exquisitely sensitive to inhibitory effects mediated by the Fc receptors of antibodies binding to surface Ig (Parker, 1980,. Immunol. Hev. 52:115; 3ijsterbosch, et al., 1385^ J. Exp. Med» 162: 1825). Thus, it was important to compare the efficacy of whole anti-Bp50 siAb with that of anti-BpSO F(ab')j fragments. Over a 100-fold dose range ?(ab')2 fragments were clearly as effective as, or aiore effective than, whole antibody at augmenting B-cell proliferation (Table 4). Thus, the Fc domain of anti-BpSO »Ab is not required for anti-3p50 to exert its effect and, if anything, may be inhibitory. In other words, anti-BpSO, like BCGFj, apparently can act as a soluble mediator without the aid of Fc receptor-mediated accessory cell function. 2 9 Table 4 The Fc Domain of Anti-3p50 Antibodies is Not Required for Augmenting B-Cell Proliferation Mean Proliferation of 3 Cells Cultured with: Dose Anti-SpSO fuq/ml) Media ,^nti"-Bp35 none _w 295 + 16 269 ± 27 whole Ab 0.125 278 + 32 51140 ± 20 1.25 27S + 24 4 , 68 S + 342 12.5 1S3 + 15 3 f 852 + 203 F(shn2 0.125 594 + 21 ,635 + 449 1.25 531 ± 3 ,893 £ 575 12. S 279 ± 3 9,411 £ 870 Cell culture conditions were as described ir. Table 3. *3.4 DIFFERENCES BETWEEN AMTX-Bp50 AND BCGF IfLQW) ACTIVITY , Anti~3p50 and BCGF flow) had a similar effect on B-cells and were costimulatory with the same agents gTable 3).

However* several lines of evidence indicate that. anti-BpSO and the BCGF -ased ir. this study apparently operate through different signals,. Firsts BpSO molecules, unlike BCG? (low) receptors (Bijsterbosch, et, 1985, J- Exp. Med. 162: 1825), are expressed on resting blood B-cells (Fig. 3J„ Second, although both anti-BpSO and BCGF (law) function »ost effectively when added after anti-Bp35 or anti-Igf anti-BpSO clearly was optimally effective shells added 12 hours after cultures began (sig. 8A) - In contrast, BCGf (low) .could be 3 0 added as long as 24 'hours after start of cultures and still optimally augment proliferation (Fig. 33). These kinetic experiments* which are modeled after the approach of Howard and Paul (1983, Ann. Rev. Immunol. 1:307), suggest that a S Bp50-dependent signal may normally exert its effect before BCGF» Both anti-BpSO and BCG? Cl°w) augmented proliferation of B-cells activated with anti-Bp35 or anti-Ig (Table 3). However, the effect of anti-BpSO and BCGF (low) were 10 additive in many experiments (Figure 7). Figure 9 shows a titration of BCGF (low) in an experiment where anti-BpSO 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 IS or by anti-Bp35. Optimal concentrations of BCGF (low) were 5-10%, while 2SI was inhibitory. Thus, when anti-Bp50 and BCGF (low) were both used at their optimal concentrations, they still showed additive effects on B-cell proliferation.

Finally,, both normal and malignant B-cell subsets 20 differed in their responses to anti-3p50 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 swch as anti-3p35 ((Table S) or TPA (Fig. 10) was consistently necessary to allow blood B-cells to respond to anti~3p50. While dense tonsillar B-cells generally did not respond to either BCGF or anti-Bp50, buoyant B-cells did respond (Table 5}. B-cell malignancies also differed in their responsiveness to anti-BpSO versus BCGF. For example, some B-cell lymphomas responded to TPA plus BCGF Clow) but not to TPA pl^ss G28-5 anti-3p50 JFig® 103 and D). In contrast, dense tonsillar B-cells and peripheral bI©od B-cells responded to TPA plus either BCGF (low) or anti-BpSO (Fig* 10A and C)„ Table 8® B-Cell Subacta Differ In Thoir Responsivenoaa to Anti-BpSO' or DOS' Mean Proliferation £* SJLMj of Lymphocytes Prom Stimulation Blood Tonsils Hup i Exp 1 E: «p 1 Dense Buoyant none 52? + 70 - 659 i 133 foe 106 m* %J Ji/i 11 BCGF ts%) 13,918 * 1 J.0A2 37,59* * lf0|J ),W * 171 131* n ijiJU 37 ; mli* Bp JO (©. i ug/m 0 3lf * 21 1,00 i. Ii 1 # I > I i, 11 *72* i i»3«ri 10 anti-&p33 (J u^/ml) m * 50 643 g if 663 t ll,S Is'llJ* 10 i ,p:>i 6i anti-BpJiQ ♦ afili »BpJ3 . .12,271 * 316 - ass 13,667 i. 333 >6,539» 1,335 »,»»3» u& anti-BpjMJ t flCGF •3 e >s <3> 3,9*3 ♦ 111 1,3*2* if Ceil culture conditions n described In Table L Blood. Nylon wood adherent lymphocytes IR cells plui monocyte*) were depleted oi monocytes by incubation on plastic dishes overnight prior to stimulation'(Exp. 1 and Exp. 2). In B*ps J, bimd O-lymphocylei were depleted of monocytes by incubation on plastic dishes lor I hr. prior, to stimulation. Tonsillar Er* lymphocytes were fractionated using Percoli gradients into dmm (pellet} m buoyant (traction Ll subsets (32). 32 .4. USES of ANTI-Bp50 ligands and Bp50 The ligands of the present invention way be 'used in vivo or in vitro, in their unmodified or modified forms to modulate ijmnuna responses., For example» th® ligands S 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~proliterative agents are coupled to the ligands, these modified ligands may be used to decrease 10 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 the BpSO antigen whether or not the malignancy is B-cell in origin. IIS Both the ligands of the present invention and/or 3p50 itself can be used irs vitro. Such applications include in vitro assays, such as immunoassays for the detection of cells which express the BpSO antigen and/or for the detection of shed BpSO antigen, if any, in body fluids™ In 20 this instance the ligand or BpSO could he labeled with & radiolabel, fluor, enzyme, enzyme substrate, dye, etc. In addition, the ligands may be ^ssed to separate aad/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 OSES OF ligands such AS anti-3P50 TO augment B-CELL proliferation Previous studies have suggested that the factors involved in the induction of 9-cells from into the S1 phase of the cell cycle are distinct from the factors or 3 3 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-Sp35 alone have little or no effect on B-cell proliferation. Vet, 5 these same agents can drive B-cells t© a point in cell activation where they are susceptible to growth factors. In contrast,, grovth factors such ass 3CGF 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,, the results presented herein provide additional support for a modal of distinct regulation of 3-cell activation and growth steps. Here we have shown that activation and proliferation signals in human B-cells may be IS transmitted through distinct cell surface structures.

Although anti-Bp35 mAb activated B-cells to enter the G, phase of the cell cycle, alone, it induced little or no proliferation. Anti-BpSO mAb had the opposite effect: it could not activate B-calls, but when added ever, as late as 2q 12-24, hours after activation could induce B-cell growth.

The Bp50 laolecule presumably could, raormally 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 BCGFs 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, et al., 1984, Immunol. Rev. ,0 18:75-96: Kishimoto, 1985, Ann. Rev. Immunol. 3:133-157; Swain, et _al. 1983, J. 5xp. Med. 158:822-835; Howard etal., 1984, Immunol, Rev. 73:135-210; Ambrtss, jst al., J. Clin. . 3 4 Invest. 75:732-7 39; Axnbrus, 1985, J. Exp. Med, 162:1319-1335). Thus, BpSO sight be a receptor for one of these factors. with the exception of IL-2 receptors and the C3d 5 receptor,, the receptors on 8~cells for growth signals have not vet been Identified. The mAb AB-1 reacts with a 3~cell marker expressed only on activated B-cells and blocks BCGF-dependent proliferation, and thus might recognise the BCGF receptor or a related structure,, Bp50 appears to be distinct 10 from the AB-1 starker since the 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 B-cells (Jung, et al., 1984, J. Exp. Mad. 160:1919-1924). BpSO is on all B-cellss which based an absorption analysis and direct binding assays 15 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, 1979e J. Exp, Med. 149sl424-l433) previously described a 54-kDa glycoprotein* 20 9PS4» that liSee BpSO is expressed on all B-cells but at lower levels on blood B-cells than tonsillar B-cells, It is possible,,, but unlikely, that the rabbit heteroantiserum and anti-Bp50 recognise the same or related structures: unlike anti~Sp50 »Ab# the rabbit antiserum to gp54 alone was sufficient to stimulate B-cell proliferation. &nti-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. Hat. Acad. Sci. USA 82:1766-1770). Similarly, BpSO aay not strictly function only as a "growth" signal: anti-3p50 antibodies together with activation signals Canti-3p35 or 3 5 anti-u) not only augment proliferation but also augment the total number of B-cells entering G1 (Table 2}. In other words„ anti-BpSO as costimulant acts to promote the progression of both the activation (GQ to G1) and growth 5 (G to S) phases of the cell cycle. The BCGF used in these studies also had similar activity (Fig. SC) ... Thus, anti-9p35 and anti-3p50 £©r 3CGF) appear to be most analogous to the *,compatene®f"' and "progression" factors described in studies of fibroblast growth regulation. How B-cells respond to 10 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 15 purified B-cells in the absence of antigen or other known factors. The natural ligands for these structures are not yet known. However, since mAb to appropriate epitopes can mimic both soluble factors and signals mediated by cell-cell interactions, it nay be possible to use appropriate 20 combinations of aftb 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 3-cell malignancies, immunodeficiencies and certain autoimmune diseases. ,5 The new monoclonal antibody, G28-5, that reacts with a sif?,gl©"Chair» polypeptide of approximately SO Kd expressed on the surface of human B-cells is but a particular embodiment of the ligands of the present invention which can augment the proliferation of activated B-cells. Since human 3-cell proliferation can be augmented similarly by T-cell-derived *?0 8CGF& including low- and high-molecular-weight BCGF we compared the activity of anti-BpSO G23-S with that of & BCGF preparation containing predominantly low-molecular-weight 36 BCGF* Anti-BpSO G28-5 and BCGF Clow) were very similar in that they were costimulatory with the same activation agents (anti~Ig, anti-Bp35 and TPA) but were not costimulatory 'with each other or with IL-1 or IL-2. Furthermore, the activity S of anti-BpSO G23-5 was not dependent on its Fc domain since F(ab'J2 fragments of G28-5 ware functionally active. This suggest© that soluble anti~3p50, like soluble BCGF,, does rsot require Fc-receptor-bearing accessory cells to exert an effect- Furthermore, both anti-BpSO and BCGF are effective 10 only in the presence of an activation stimulus. Irs other vords, anti-Bp50 and BCGF are not "competence" factors, but rather promote the "progression" of B~Gcalls through the cell cycle.

While it is possible that Bp50 may function as receptor IS for & ligand such as a B-cell growth factor, several results suggest that BpSO is not the receptor at least for the BCGF O ow) used in this study: it is expressed on blood 3-cells while BCGF (low) receptors apparently are not. Candidate structures for the BCGF flow) receptor, unlike 3p50, are also 20 expressed only on activated B-cells. Furthermore, both normal and malignant B-cell populations differ in their responsiveness to anti-BpSO versus BCGF (low) (Table 5 and Figure 10). For instance, some 3 lymphomas proliferate in response to BCGF flow), but not in response to anti-BpSO. «,g Finally, in a number of experiments, optimal concentrations of anti-BpSO aimd BCGF together induced more proliferation than either one alone. Anti-BpSO mimics the activity of other BCGF, such as BCGF (high) that are co-stimulatory with anti-IgM (Ambrus, et al., 198S# J. Exa. Hed. £S2"1319? Ambrus, et al., 1985, J- Clin. Invest. 75:732]). This suggests that BpSO could function as the receptor for BCGF fhigh). 37 Although BpSO may be a receptor for a soluble ligand, as!ternatively, BpSO sn&y function as a receptor for a cell-call mediated signal that regulates BCGF receptor levels and/or autocrine production. Precedence for differentiation 5 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 XL-2 receptor expression on activated T cells {Ledbetter, et si. 1985, J. Immunol- 135:18195 • An analogous mechanism may 10 be operating with anti-Bp50 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 BpSO on certain leukemic cells. The BpSO molecule also shares similarities with th® Tp44 aolecwle 15 that functions to influence 2L-2 production™ We and others have shown that the 9.3 anti~Tp4 4 antibody augments proliferation of T cells activated by anti-CD3 or TPA (Ledbetter, et _alv, 1985, J. Immunol- 135:2331,? Hara, et al., 1985, J. Exp. Med- 161:1513). Similarly, anti-Bp50 augments 20 the proliferation off B-cells activated by anti-3p35 or TPA. The Tp4 4 signal functions by stimulating IL-2 production rather than by* stimulating T cell growth- The BpSO signal presumably could function ir. an analogous manner by stimulating B-cell autocrine production (Gordon, et al., 2^ !®84i7 Maturet, Loncl. 310; 145}. .4.2. MODIFIED LIGANDS USED FOH IMMUNOSUPPRESSION OH TREATMENT 'OF MALIGN AN AC! SS According to this embodiment, the ligand of the present invention can be modified by the attachment of an «%rs vl antiproliferative agent s© that the resulting molecule can be used to kill cells which express the BpSO 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 the autoimmune response. These modified ligands can also be used to immunosuppress a transplant patient to prevent rejection of & graft. Accordingly, cytotoxic agersts which are used for th© 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 be used to treat malignancies in which tumors or cells express the BpSO 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 ligarsd coupled to a cytotoxin which is more effective in killing proliferating sells 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, pp. 2249-1313, 1980. 3-Q 5U-' Table 6 Chajnotherapeutic Agents Which Can be Coupl. to Anti~Bd50 Liaands Class Alkylating Agent Antisnetabol ites Natural Products Type nitrogen Mustard Ethylenimine Derivatives Alkyl Sulfonates nitrosoureas Triazenres Folic Acid Analogs Pyrimidine Analegs Purine Anlogs Vinca Alkaloids Antibiotics Agent Mechloretham ine Cyclophosphamide Melphalan Uracil Mustard Chlorambucil Thiotepa Busulfan Camus tine Lomustine Semustine Streptozocin Dacarbazine Methotrexate Fluorouracil Cytarabine Azaribine Me r capt ©pur£ne Thioguanine vinblastine Vincristine Dactinomyciss ID&yrjorubici^ Doxorubicin Bleomycin Mithramycia Mitomycin Enzymes L-Asparaginase 4 0 Miscellaneous Agents Hormones and Antagonists IS Radioactive Isotopes Platinum Coordinated Complexes Substituted Urea Methyl HydrasiBe Derivative Adrenocortical Suppressant Adrenocorti-costeroids Progestins Estrogens Antiestrogen Androgens Phosphorous lodi rse Cisplatin Hydroxyurea Procarbasina Hitotane Prednisone Hydroxyprogesterone caproate Medroprogesterone acatata Hegestroi acetate Diethylstilbestrol Ethinyl estradiol Tamoxifen Testosterone propionate Fluoxymesterone sn?i ua phosphate So(^t£m Iodide Kt%y method knovn in th® art can be used to couple the ligand to the chemotherapeutic or antiproliferative agent-Examples of such methods have been enumerated previously (see Section 5, supra).

S.4.3. OTHER USES OF LIGANDS AND 3'P5Q In addition to the therapeutic applications the ligands and BpSO itself have other applications in both in vitro and In vtro diagnostic assays,, separation schemes, ate.

The BpSO receptor can be used to manufacture and/or design th® ligands ©£ 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, Sp50, itself may be useful as a soluble factor which mediates immunity, e.g. a lymphokine.

In addition to therapeutic treatment and diagnostic assay®, the ligands of the present invention could be used for identifying or separating cells which express the BpSO antigen. In additions, if an appropriate radiolabel or radio-opatju© compound is linked to the ligand,, the ligand could be 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.

«• DEPOSIT OF CELL LI^SS The following hybridoma has been deposited with the American Type Culture Collection, Bockville, MD(S and has been assigned the listed accession number: Hvbrldomgi ATCC Accession Number G28-S 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 of one aspect of the invention and any cell lines which are functionally 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 tcs fall within the scop® of the appended claims. 4 2

Claims (60)

CLAIMS s

1. A substantially pure ligand that binds to 8p50f a 50 kiloDalton B-cell surface antigen defined by monoclonal antibody G28-5. 5

2. A substantially pure ligand according to claim 1 which upon binding to an activated B-cell stimulates the activated B-eell 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 10 which comprises an antibody molecule or an Fv„ Fab, F( ab')0 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, F(ab')j or Fab' portion of the monoclonal antibody 15 molecule.,

5. A ligand according to claim 4 in which the monoclonal antibody molecule comprises G28-5, or any Fv, Fab, F(ab'>2 or Fab' portion thereof.

6. A ligand according to claim 4 in which the monoclonal 20 antibody molecule has been produced by a hybridoma cell line as deposited with the ATCC having accession no. HS911Q e or a mutant recombinant or genetically engineered derivative thereof.

7. A ligand according to either of claims land 2 which 25 comprises a lymphokine.

8. A ligand according to claim 7 in which the lymphokine comprises a 3-cell growth factor. 4 3

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

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

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.

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

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

14. A ligand according the compound coupled to

15. A ligand according the conpoujnd coupled to to any one of Claims the ligand ccnwrises to any one of Claims the ligand comprises 1 to 9 inclusive in %/hich an antibiotic. 1-9 inclusive in which a vines alkaloid.

16. A ligand according to any one of Cleiirs 1-9 inclusive in which the compound coupled to the ligand cororises an enzyme.

17. A ligand according to any one of Claims 1-9 inclusive in uhich the compound coupled to the ligand comprises a platinum coordinated ccxiplex.

18. - A ligand according to Claim 11 in uhich the ccnqpound coupled to the ligand comprises a radioisotope.

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

20. A substantially pure 50 kiloDalton B-cell surface antigen which is defined toy monoclonal antibody G28-S. 4 4 ■

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

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.

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 Gq to G1 stage of the cell cycle,,

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

25. A method according to either of claims 22 or 23 is perfox-rned in vitro.

26. A method according to any one of claims 22 to 25 in Mhich 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 to BpSO.

27. A method, according to any one of claims 22 to 25 in which the ligand comprises a monoclonal antibody molecule that binds to BpSO or an Fve Fab„ Flab' )5„ or 25 Fab' portion of a monoclonal antibody molecule that binds to Bp50.

28. A method according to claim 27 in which the monoclonal antibody molecule comprises G28-5, or any Fv, Fab. F(ab"),e or Fab' portion thereof. s 4S

29. 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 H39110, or a mutant, recombinant or genetically engineered 5 derivative thereof.

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

31. A method according to claim 30 in which the lymphokine comprises a B-cell growth, factor. 10

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

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. 15

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

35. A method according to claim 23 in which the activated B-cells ar© treated with the ligand that binds to BpSO within about 12 hours after activation by the 20 second ligand that, binds to Bp35.

36. A method for suppressing proliferation of cells which express 3p50» a 50 kiloDalton B-cell surface antigen defined by monoclonal antibody G28-5, comprising treating the cells which express BpSO with an effective dosa of a 25 ligand that binds to 3p50, which ligand is coupled to an antiproliferative agent.

37. „ A method according to claim 36 in which the cells which express BpSO comprise B-cells. 4 8

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

39. 3 9. 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. 5

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

41. A method according to claim 36 in which the ligand comprises an antibody molecule or an Fv„ Fab, F(ab')0 or Fab' portion of the antibody molecule. 10

42. a method according to claim 35 in which the antibody molecule comprises a monoclonal antibody molecule or an Fv, Fab, F(ab') ^ or fab"' portion of the monoclonal antibody molecule.

43. A method according to claim 42 in which the 15 monoclonal antibody comprises G28-5f or any Fv, Fab„ F( ab' ) -j, or Fab1' portion thereof.

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, 20 or any mutant, recombinant or genetically engineered derivative thereof -

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

46. A method according to claim 45 in which the 25 lymphokine comprises a B-cell growth factor.

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

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

49. A method according to any one of claims 36 to 46 ir. which the antiproliferative agent comprises an antibiotic.

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

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

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

53. A method according to any on© of claims 36 to 46 in which the antiproliferative agent comprises a radioisotope.

54. 34. A substantially pure ligand as described in claim 1 substantially as hereinbefore specifically described.

55. A substantially pure surface antigen according to claim 20 substantially as hereinbefore specifically described. ■ 4 8

56. A method according to claim 22 for augmenting proliferation of B-cells substantially as hereinbefore specifically described,

57. A method according to claim 36 for suppressing 5 proliferation of B-cells substantially as hereinbefore described.

58. Use of a ligand that binds to Bp50? a 50 kiloDalton B-cell surface antigen defined by monoclonal antibody G23-5, 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 proliferation is augmented.

59. Use of a ligand that binds to Bp50f a 50 kiloDalton B-cell surface antigen defined by monoclonal 15 antibody G28-5. which ligand is coupled to an anti-prolxferative agentt for suppressing in vivo proliferation of cells which express BpSO.

60. Use according to claim 58 or 59, substantially as hereinbefore described. f. E. kelly & co., agents FOR THE applicants. /