LU86919A1 - COORDINATES AND METHODS FOR INCREASING THE PROLIFERATION OF B LYMPHOCYTES - Google Patents

Description

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I Title issued,: ¾.¾ LUXEMBOURG

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Invention Patent Application ________________________ ... . ___________________________________________________________ (ii I. Request

The company, known as:: ........ ONCQGEN, ........ company, .in -commandite, ...... 3-005 ...- First <

Avenue, SEATTLE., ....... Washington ...... 981.21, ..... United States of America, represented by .Mister ..... Jacques ..... .of Muyser, - acting as a representative. --------------------------------------- --------------------—.................—----------- ------------------- -..... (3i depositemt) this ......... twelve .. June 19.00 eighty ... .sept .................... ......- - "+ to .15 ............... .hours, to the Ministry of the Economy and the Middle Classes, in Luxembourg: 1. the present request for obtaining a patent of invention concerning: "Coordinates and processes with a view to increasing proliferation (5i des. ..lymphocytes ..... E, "........................ ............... ............... ..... ..... -........................ -.................... ...... - 2. description in language ...... French ......... .............. of the invention in three copies: 3. 10 .............. drawing boards, in three copies: 4. the receipt of taxes paid to the Registration Office in Luxembourg on IQ June 1987; 5. the delegation of power, dated Sew York ................................... the ... 3.7 1987 '6. the pre-cause document (authorization): declaration and assuming responsibility for this declaration, that the inventor (s) is (are): (b' 1. Jeffrey A. LEDEETTER, 616N .W. 67 Street, SEATTLE,

Washington 98117., United States of America .......

2, Eoward A. CLARK, 1505 16th East, SEATTLE, Washington 98112, ................. United States of America _... ....... .................................. ................ ........

\ claims! nti for the above patent application the priority of one (or more) application (s) of (7) ............. patent .......... ......................... ................. filed (sten (8) aux. United States of America on (9) June 13, 1986 .......- ............................. ............... ................................... ....... .........

under N ° (10) 873.884 ................... ..........

on behalf of (11) s inventors __ ........

elilféliscnt) domicile for him (her) and. if appointed, for its representative, in Luxembourg ....

.......... 35, boulevard Royal. _ ...... ......... (12i requests (s) the grant of a patent for the invention for the subject described and represented in the abovementioned appendices.

ayeiuqpumement of this issue at 18 ................... ......................... .......... months. (131

The depositor / agent: ......,. ............... .............. (14) ’l 1 t i Î Π. Deposit Minutes

The aforementioned patent application has been filed with the Ministry of the Economy and the Middle Classes. Intellectual Property Service in Luxembourg. dated: 2 June 1987 /. fy \ / y. '' 'Vi% \ P T. the Minister of Economy and the Middle Classes, μ / ^. fit -.- .. ¾%. \ to .. ..hours / J:, \. p. d.

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CLAIM OF PRIORITY

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of the patent application / fe in the United States of America • of June 13, 1986 (No. 873.884); Brief Description filed in support of a request for

PATENT

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Luxembourg on behalf of: ONCOGEN, limited partnership SEATTLE, Washington 98121 (United States of America) for: "Coordinates and procedures for increasing the proliferation of B lymphocytes" • \ r, ”*

Coordinates and methods for increasing the proliferation of B cells.

1. INTRODUCTION

The present invention relates to ligands such as antibody molecules or fragments of antibody molecules or other ligands such as lymphokines which bind to a surface marker of 50kDa B-cell lynphocytes, designated in the present specification by the name Bp50, which inter-10 comes in the proliferation of B lymphocytes, but not in the early activation of B lymphocytes; The present invention also relates to the Bp50 antigen itself, B lymphocytes. In a particular embodiment of the present invention, there is described a monoclonal antibody, G28-5, which defines Bp50 and appears to play a role in proliferation of activated B lymphocytes ^ without however having any detectable effect on the proliferation of B lymphocytes at rest.

The ligands such as antibodies, lymphokines and their fragments according to the present invention can be used to direct and control the proliferation and / or differentiation of human B lymphocytes. In addition, the ligands of the present invention can be modified by the binding of other compounds which can be used in the treatment of 2 t and / or the detection of malignant cells which express the Bp50 antigen.

2. FOUNDATION OF THE INVENTION

The activation of resting B cells from the GQ phase to the cell cycle phase and the subsequent initiation of the proliferation of activated B cells are distinct steps requiring equally distinct regulatory mechanisms. Certain agents, in particular factor-p1 stimulating murine B lymphocytes (BSF-p1) (Rabin et al., 1985, Proc. Nat.

Acad. Sei. EUA 82, 2935-2939) or low doses of anti-immunoglobulin (anti-Ig) (DeFranco, et al., 1985, J. Immunol. 135: 87-94; Wetzel et al., 1984, J. Immunol 133: 2327-2332; DeFranco et al., 1982, 15 J. Exp. Med. 155: 1523-1536; Muraguchi et al., 1984, J. Immunol. 132: 176-180) are factors in " activation "or" skill ". In other words, these agents cause the B lymphocytes to grow, to synthesize more ribonucleic acid and to enter 20 G ^, but on their own, they do not cause the synthesis of deoxyribonucleic acid in B cells. Other "growth" factors such as human B cell growth factor (BCGF) and interleucin-2 (IL-2) cause activated B cells. 25 through the cell cycle and entering the S phase, but they do not trigger resting B cells (Kehrl et al., 1984, Immunol. Rev. 18: 75-96; Muraguchi et al., 1984, J Immunol 132: 176-180;

Zubler et al. 1984, J. Exp. Med. 160: 1170-1183; 30 Jung et al., 1984, J. Exp. Med. 160: 1597-1604).

A number of factors that promote the growth of B cells have now been described by researchers from both murine and human systems. These factors include B cell growth factors (BCGF) derived from several different sources, including hybridomas or T cell lines, B cell lines or dendritic cells. Although both interleucin-1 (IL-1) and interleucin-2 (IL-2) have been shown to increase the growth of B cells, they are apparently distinct from certain BCGFs. For example, monoclonal antibodies (mAb) against a murine BCGF (O'Hara et al., 1985, Nature (Lond.) 315: 333) or a human BCGF (Ambrus et al., 1985, J. Exp. Med. 162: 1319) block the activity of BCGF, but not the activity of IL-1 or IL-2. Although distinct from IL-1 or IL-2, the BCGFs themselves appear to be heterogeneous based on biochemical data and differential activity on different subsets of B cells or different dosages co-stimulation. For example, a high molecular weight human BCGF at 60 kilodaltons (kDa), BCGF (high), which is distinct from a low molecular weight form at 12 kDa, BCGF (low) has been identified (Ambrus et al. , 1985, J. Clin. Invest. 75: 732).

The c-deoxyribonucleic acid encoding a murine 20 kDa BCGF, called, with some hesitation, "B-cell stimulating factor pl" (BSF-pl), has recently been cloned and sequenced (Noma et al., 1986, Nature 319: 640). The recombinant lymphokine not only exerts BCGF activity, but it can also activate resting B cells and cause differentiation of IgG-producing cells. therefore, it differs from human BCGF (high) and BCGF (low) in both its molecular weight and its range of activities.

These activation and growth signals probably ensure regulation of the cells by interaction with specific surface structures 4, 4, which are specific for B lymphocytes. In addition to the specific signal for the antigen passing through surface Ig, it has been identified several other surface B-cell candidate polypeptides which in some way can act in the activation or growth of B-cells. For example, 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: 1126) have been characterized and recently functional receptors for IL- have been identified. 2 on B lymphocytes (Zub 1er et al., 1984, J.

Exp. Med. 160: 1170; Jung, et al., 1984, J. Exp. Med. 160: 1597; Muraguchi et al., 1985, J. Exp. Med. 161: 181). However, receptors for B cell activation and growth factors have yet to be fully characterized. Several candidate superficial B cell polypeptides have been identified which, in some way, can act 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) directed against the murine B lymphocyte Lyb2 antigen activated B lymphocytes and, recently, evidence has been given suggesting that Lyb2 may be the receptor for BSF-p1 (Yakura, 1985, Fed. Proc. 44: 1532). Likewise, the Applicant has found that an appropriate mAb (1F5) directed against a 35 kDa polypeptide, Bp35, activates human B lymphocytes from the GQ phase to the G1 phase (Clark et al., 1985, Proc. Nat Acad. Sci. EUA 82: 1766-1770; Gollay et al., 1985, J. Immunol. 135: 3795-3801). Aggregates of C3d or antibodies directed against the 140 kDa C3d receptor, Bpl40, cause the proliferation of B lymphocytes which are dependent on T lymphocytes (Melchers et al., 1985, 5 t

Nature 317: 264-267; Nemerow et al., 1985, J. Immunol. 135: 3068-3073; Frade et al., 1985, Eur. J. Immunol. 15: 73-76). Although BCGFs have been identified in both mice and humans, the receptors for these factors have not yet been isolated.

Wang et al. (Wang et al., 1979, J.

Exp. Med. 149: 1424-1433) prepared polyclonal antisera which identified a 54 kDa polypeptide (gp54) on human B lymphocytes and demonstrated that rabbit antisera directed against gp54 caused the division of tonsill B lymphocytes. Recently, Jung and Fu (Jung et al., 1984, J. Exp.

Med. 160: 1919-1924) isolated an mAb (AB-1) directed against an antigen at 55 kDa, restricted to activated B lymphocytes and blocking proliferation dependent on BCGF. However, it is not yet known whether or not anti-gp54 or AB-1 recognizes a BCGF receptor.

3. SUMMARY OF THE INVENTION The present invention relates to ligands which (a) bind to Bp50, namely, a 50 kDa B cell specific surface polypeptide described herein, and which (b) increase the proliferation of activated B cells . The invention also relates to the Bp50 antigen itself which is defined by the monoclonal antibody G28-5 and which acts in the proliferation of activated B cells. Furthermore, the invention relates to ligands which bind to Bp50, but which have no function or biological effect such as an increase in 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 binding site of the antigen or antibodies and Μ 6 si * / fragments chemically modified; these fragments include, without any limitation, Fv, Fab, F (ab ') 2, Fab' and the like. In addition, the ligands of the present invention include lymphokines which may include, without limitation, 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 with the ligand. These compounds include, without limitation, cytotoxic agents, therapeutic agents, chemotherapeutic agents, markers such as radiolabellers, dyes, enzymes, X-ray opaque compounds and the like.

In their modified or unmodified form, the ligands of the present invention can be used to direct, regulate and modify the proliferation and / or differentiation of human B lymphocytes.

The present invention is based on the discovery that two human B cell differentiation antigens,. namely Bp35 and the B cell antigen described herein, namely Bp50, apparently play distinctive roles as signal receptors in the activation of B cells. Monoclonal antibodies (mAb) directed against Bp35 and Bp50 all emit two of the positive signals to B cells, thereby stimulating their transition through the cell cycle. Like anti-Ig antibodies, mAb directed against Bp35 acts primarily to activate resting B cells so that they become competent to enter the cell cycle phase. In contrast, a monoclonal antibody of the type described here or its F (ab ') 2 fragment directed against Bp50, namely a poly- 7 ί.

J

50 kDa peptide expressed on all B lymphocytes, acts to stimulate activated B lynphocytes so that they cross the cell cycle, while increasing the proliferation of activated B lymphocytes.

Like anti-Ig antibodies, monoclonal antibodies against Bp 35 activate tonsill B cells and cause B cells to proliferate at low levels. In contrast, the anti-Bp50 monoclonal antibody alone does not activate B cells, nor does it cause B cells to proliferate but, together with anti-Bp35 or anti-Ig antibodies, increases the proliferation of B lymphocytes. In this respect, the action of the anti-Bp50 antibody resembles the activity of growth factors for B lymphocytes (BCGF). As little as 0.05 µg / ml anti-Bp50 is needed to increase proliferation and, like BCGF, anti-Bp50 is effective even when added 12 to 24 hours after B cells have been activated with anti-Ig or anti-Bp35. Without additional exogenous signals, the anti-Bp35 and anti-Bp50 antibodies together cause a strong proliferation · of purified B lymphocytes at rest. These results suggest that the surface molecules of Bp35 and Bp50 are involved in controlling the regulation of activation and progression of B lymphocytes through the cell cycle. Given the importance that anti-Bp35 molecules and the like have on the effect and action of the ligands of the present invention, the article by Clark et al., 1985, Proc. Natl. Acad. Sei. (EUA) 82: 1766-1770 is mentioned here for reference.

Although the activity of anti-Bp50 resembles that of BCGF (weak), since anti-Bp50 35 and BCGF (weak) are both co-stimulants with 8 1 r - "" l the same agents, but not with each other and since both anti-Bp50 and BCGF (weak) only influence activated B cells and act in a soluble form, the activity of anti-Bp50 can be distinguished from that BCGF (low), since both the proliferation of B cells stimulated with optimal amounts of anti-Bp50 and anti-Bp35 (or anti-Ig) can be increased further with BCGF (low) and only both B cells in the blood and certain B cell lymphomas react differently to anti-Bp50 vis-à-vis BCGF. To exercise optimal activity, anti-Bp50 must be added within 12 hours of activation of B cells, while BCGF (weak) retains optimal activity even when added 24 hours after activation. In addition, Bp50 is expressed on all B cells, while the receptors or BCGF (weak) are restricted to activated B cells. Therefore, in coordination, anti-Bp50 and BCGF (weak) 20 can regulate the growth of B lymphocytes, but apparently they behave in this way through the intervention of distinct signals.

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

In another embodiment, the ligands of the invention can be chemically modified so that the cells in which 9, i! ! ί i bind the coordinates, be killed. Since all B cells express the Bp50 antigen, this method should result in the suppression of the immune response. For example, a cytotoxic drug related to a ligand of the present invention can be used in vivo to induce immunosuppression in order to cross histocompatibility barriers in patients who have received a transplant; alternatively, these modified ligands can be used to combat autoimmune diseases.

In another embodiment of the present invention, malignancies such as Bp50-expressing tumor cells can be treated using a ligand of the invention linked to a chemotherapeutic agent useful for the treatment of such a neoplastic disease. These modified ligands can be used in vivo to direct the chemotherapeutic agent to any type of malignant cell which expresses the Bp 50 antigen, including cells which are not B cells, but which express Bp50. When using the ligands of the invention, which increase the proliferation of B cells, there should be a particular advantage when treating B cell malignancies when the chemotherapeutic agent linked to the ligand is a more effective agent for kill proliferating cells; in this case, potentiation of the effect of the drug should be obtained.

Alternatively, the ligands of the invention can be used in vitro to identify or separate cells which express the Bp50 antigen and / or to determine, in the humors of the organism, the presence of the Bp50 antigen which can be lost 10 ί a ί ί or not. In addition, the ligands of the invention can be used in vivo to visualize cells or tumors which express the Bp 50 antigen.

The purified Bp50 antigen of the present invention can be used to form antibodies, as well as to form or design other ligands of the invention. In addition, the Bp50 antigen could be used in assays such as diagnostic immunoassays. In addition, Bp50 itself can be used as a mediator of cellular immunity in vivo or in vitro.

3.1. DEFINITIONS

As used in this specification, the following abbreviations will have the meanings indicated: AO = acridine orange BCGF = B cell growth factor BCGF (high) = human BCGF at 60 kDa 20 BCGF (low) = 12 kDa human BCGF

Bp35 = Superficial polypeptide (CD20) specific for B kyphocytes at 35 kDa, defined by mAb 1F5 Bp50 = Superficial polypeptide specific for B lymphocytes at 50 kDa, defined by mAb G28-5 25 Fv = Variable region or antigen binding site of an antibody molecule.

It can be any fragment which contains the idiom of the molecule, including without limitation Fab, F (ab ') g, Fab' and the like.

IF = Immunofluorescence

Ig = Immunoglobulin IL-1 = Interleucin 1 IL-2 = Interleucin 2 35 kDa = Kilodalton * »1 <11 mAb = Monoclonal antibody SDS-PAGE = Electrophoresis on polyacrylamide gel / sodium dodecyl sulfate TPA = 12-0 acetate -tetradecanoylphorbol-13.

5 4. DESCRIPTION OF THE FIGURES

Figure 1. The expression of Bp50 is restricted to Bp35 + lyrrphocytes B. Cytometric analysis with two-color flow of 50,000 lymphocytes was carried out as described (Clark et al., 1985, Proc. Nat.

10 Acad. Sei. EUA 82: 1766-1770). The data diagram is plotted by the number of lymphocytes with respect to the logarithm of green fluorescence and the logarithm of red fluorescence where 4-5 points represent approximately a doubling of fluorescence. Data is presented to show autofluorescent negative cells. The PE (red) -anti-Bp35 (1F5) staining against FITC (green) -anti-Bp50 (G28-5) shows that all the Bp50 + cells are also Bp35 +.

Figure 2. Biochemical comparison of the Bp50 poly peptide with other surface antigens of B lymphocytes. Bp50 immunoprecipitation of T-labeled tonsil cells at the surface was performed as described. Isolated antigens were subjected to electrophoresis on 10% sodium dodecyl sulfate / polyacrylamide plate gels without reduction. The gels were visualized by autoradiography and with reinforcing screens. Table A: column 1, anti-Bp50 (G28-5); column 2, anti-Bp95 (G28-8); column 3, Sepharose-goat anti-mouse Ig only. Exposure time: 4 days. Table B: column 1, anti-Bp50 (G28-5); column 2, anti-Bp45 (BLASTE-2); column 3, anti-Bp39 (G28-1); column 4, anti-Bp39 (41-H16); column 5, 35 Sepharose-goat anti-mouse Ig only. On i * »* 12 f has chosen an exposure time of two days so that the bands of columns 2 to 4 are not overexposed and that they can be clearly distinguished from Bp50. One in five experiences.

Figure 3. Analysis of Bp50 expression by two-color immunofluorescence. Tonsillary or peripheral blood mononuclear cells were isolated by centrifugation on "Ficoll" and they were stained with G28-5 (anti-Bp50) conjugated to PE (red) in combination with reference antibodies conjugated to fluorescein ( green), including 2C3 (anti-IgM); 1F5 (anti-Bp35); HBlOa (anti-DR); and 9.6 (anti-CD2, receptor E). The cells were analyzed with a FACS IV equipped with logarithmic amplifiers with four decades in both the dimensions of red and green. A forward and right angle moving spot detector was used to monitor the monocytes. The uncolored cells are placed on the back of the grid, the red fluorescence is on the right and the green fluorescence on the left.

Figure 4. Dose response curves for increased proliferation of dense Ermygdal B cells by anti-Bp50 antibodies as shown: media only; anti-Bp50 only plus anti-Bp35 (5yug / ml) only; BCGF only; anti-Bp35 plus BCGF; anti-Bp35 plus graduated doses of anti-Bp50. The average proliferation + standard error of four samples was measured on day 3.

Figure 5. Anti-Bp50 mAbs are most effective in increasing proliferation if added after a B-cell activation signal. Er-amygda-35 dense link B-cells were incubated for 4 days with only media - "'" * 13 anti-Bp50 (0.5yag / ml) was added at different times after incubation, anti-Bp35 (5yjg / ml) was added at different times after l 'incubation ; anti-Bp50 was maintained at a constant value then, later, at different times, anti-Bp50 was added

Bp35; anti-Bp35 was kept constant and then, at different times, anti-Bp50 was added to cultures. Over the past 10 hours, <3 H-thymidine was added and its incorporation was measured.

Figure 6. Comparison of the suitability of anti-Bp35. and anti-Bp50 to bring about resting tonsillary B lymphocytes to leave the Gq stage of the cell cycle. Day 3 after treatment, media only 15 (_), anti-Bp35 only (-----); and anti-Ig only (.....), A, no additional additives; B, anti-Bp50 (0.5 µg / ml) was added to each group; C, 5% BCGF was added to each group. The data are plotted in the form of the relative number of cells with respect to the logarithm of red fluorescence of acridine orange (ribonucleic acid).

Figure 7. K-cell proliferation kinetics after stimulation with anti-Bp50 vis-à-vis BCGF. Dense E-tonsilloid B-cells were stimulated with media only; 10% BCGF only; anti-Bp35 only; anti-Bp50 only; anti-Bp35 + 10% BCGF; anti-Bp35 + anti-Bp50; and anti-Bp35 + anti-Bp50 + 10% BCGF.

Proliferation was measured on the days indicated by an 18 hour pulse of H-thymidine. The proliferation in quadruplication was measured and the standard errors are indicated. One in three experiences.

14 ί. »*

Figure 8. Time elapsing after stimulation with anti-Bp35 when anti-Bp50 (A) or BCGF (B) increases proliferation optimally. Dense T-cell E-lymphocytes were stimulated as indicated 5 and proliferation was measured by an 18 hour pulse 3 of H-thymidine on day 3. Media; anti-Bp35 only added at the times indicated; anti-Bp50 or BCGF only; anti-Bp35 added at the start of the culture, then addition of anti-Bp50 or BCGF 10 at the times indicated; anti-Bp50 or BCGF added at the start of the culture, then addition of anti-Bp35. One in two experiences. The proliferation in quadruplication was measured and the standard errors are indicated. Doses used: anti-Bp35, 5 g / ml; anti-Bp50, 0.2 µg / ml; BCGF (weak) 5%. The concentrations used were as follows: anti-Bp35, 5 µg / ml; anti-Bp50, 0.2yag / ml; BCGF, 5%.

Figure 9. Anti-Bp50 and BCGF exert additive effects on the proliferation of B lymphocytes.

Dense tonsillar E-B cells were stimulated with graduated doses of BCGF (low) together with anti-Bp50 only; anti-Bp35 only; anti-Ig beads only; anti-Bp35 + anti-Bp50; or anti-Bp50 + anti-Ig. Proliferation was measured on day 3 after stimulation with an 18 hour 3 H-thymidine pulse. The proliferation in quadruplication was measured and the standard errors are indicated. One in four experiences.

6

Doses used for 10 ° cells .: anti-Bp35, 5 ^ ug / ml; Anti-Bp50, 0.2 µg / ml; anti-Ig beads, 50 ^ ig / ml.

Figure 10. Comparative effects of anti-Bp50 and BCGF on normal and malignant B lymphocytes.

Peripheral blood E-B lymphocytes (A) or dense tonsil E-B lymphocytes (C) were stimulated with or without TPA (75 ng / ml) in the presence of 10% of 15 c * K ** BCGF or 1 yug / ml of anti-Bp50. Two separate lymphomas of B cells (Tables B and D) were stimulated in the same way. Proliferation was measured on day 3 by incorporation of H-thymidine in a 12 hour pulse. The proliferation in quadruplication was measured and the standard errors are indicated.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to ligands which (a) bind to Bp50, which is a surface polypeptide specific for 50 kDa B cells, and which (b) increase the proliferation of activated B cells. The invention also relates to the Bp50 antigen itself which is defined by mAb G28-5 and which acts in the proliferation of B lymphocytes. Furthermore, the invention relates to ligands which bind to Bp50, but which do not exercise neither a function nor a biological effect such as the increase in the proliferation of activated B lymphocytes.

The ligands of the present invention include antibody molecules, monoclonal antibody molecules and fragments of these antibody molecules, which contain the site of binding of the antigen binding to the Bp50 receptor, including including antibodies and chemically modified fragments; these fragments include, without any limitation, Fv, Fab, F (ab ') 2, Fab' and the like. In addition, the ligands of the present invention include lymphokines that bind to the Bp50 receptor. These ligands can include, without limitation, BCGFs, as well as chemically modified lymphokines and the like. The ligands of the invention can be used in their modified or unmodified forms for modulating and regulating immune responses, as well as in the therapy of malignancies expressing the Bp50 antigen.

These uses are described in more detail in section 5.4 below.

When the ligand is a mono-clonal antibody or a fragment of this antibody, the monoclonal antibody against Bp50 can be prepared by adopting any technique ensuring the production of antibody molecules by continuous cell lines in culture. For example, the 10 hybridoma technique originally developed by Köhler and Milstein (1975, Nature 256: 495-497), as well as other techniques that have been made more recently available such as the human B cell hybridoma technique ( Kozbor et al., 1983, Immunology Today 4:72) 15 and the EBV-hybridoma technique for the production of human monoclonal antibodies (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan. R.

Liss, Inc., pages 77-96) and the like fall within the scope of the present invention.

Antibody fragments containing the idiotype of the molecule could be generated by known techniques. For example, such fragments include, without limitation: the F (ab ') g 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 (ab ') 2 fragment which can be generated by treating the antibody molecule with papain; and the 2Fab or Fab 30 fragments which can be generated by treating the antibody molecule with papain and a reducing agent to reduce the disulfide bridges.

When the ligand which binds to Bp50 is a lymphokine, this can be obtained from natural sources or, if its amino acid sequence ί '' 4 * r 17 is known or deduced, the lymphokine can be synthesized by chemical synthesis methods. Alternatively, if the lymphokine gene sequence is known, recombinant deoxyribonucleic acid techniques can be adopted to clone the gene into an expression vector for transcription and translation of the gene sequence in a cell appropriate host.

Depending on its intended use, the coor-dinat or suitable fragments of the ligand can be chemically modified by attaching one or other compound belonging to a variety to the ligand by adopting known coupling techniques. Such techniques can include, without any limitation, the use of carbodiimide, cyanogen bromide, difunctional reagents such as glutaraldehyde, N-succinimidyl 3- (2-pyridyldithio) propionate, Schiff base reactions, attachment to sulhydryl moieties, use of sodium isothiocyanate or an enzymatic linkage, to name a few. When a radioisotope is to be attached to the ligand, enzymatic means, oxidative substitution, chelation, etc. can also be used. For an overview of the chemical reagents that can be used for protein modification, see Lundblad and Noyes,

Chemical Reagents for Protein Modification, volume II, CRC Press, Inc., Boca Raton, Florida, chapter 30 5, pages 123-139, 1984.

The coupling or chemical bonding of a compound to the ligand could be directed to a site of the ligand which does not participate in the binding to Bp50. To this end, the ligand binding site could be protected before carrying out the coupling reaction. For example, the ligand can first be linked to Bp50 in order to protect the Bp50 binding site, after which the coupling reaction can be carried out to link the desired compound to the reactive sites available on the coordinating complex / Bp50. As soon as the coupling reaction is completed, the complex can be disrupted, thereby generating a modified ligand to which the desired compound is attached, thus exerting minimal influence on the site of the molecule, on which Bp50 binds.

When the ligand comprises a monoclonal antibody such as G28-5, in which the Fc domain of the molecule is not required for the ligand to exert its effect (see paragraph 5.3.3 below), it may be advantageous to direct the coupling of the desired compounds to the Fc domain of the molecule.

The following subclauses describe the new surface marker for 50 kDa B cells, namely Bp50, which apparently acts in the proliferation of B cells, as well as ligands which bind to the new 50 kDa receptor, as well as their uses. To give an example of the ligands of the present invention, there is also described a monoclonal antibody which defines Bp50, as well as its FCab ') ^ fragments which, like BCGF, increase the proliferation of B cells. Unlike anti-Bp35 mAb which can cause B cells to rest in the Gq phase, to enter the G phase, anti-Bp50 mAb does not activate B cells at rest. Anti-Bp35 and anti-Bp50 mAbs together, without any additional exogenous signal, cause strong activation and proliferation of purified B lymphocytes.

The experiments described below also demonstrate that the anti-Bp50 activity resembles the activity

4 X

'i 19 BCGF, but that anti-Bp50 is distinct from BCGF, since anti-Bp50 and low molecular weight BCGF are clearly additive and act differently on different subsets or malignancies of B cells. Bp50 can be a receptor for a distinct BCGF or for a transmembrane signal which modulates the production of BCGF or the expression of the BCGF receptor.

5.1. METHODS ADOPTED TO CHARACTERIZE THE Bp50 RECEPTOR 10 Cell preparations. Mononuclear normal or leukemic heparinized blood mononuclear cells were isolated by Ficoll-Hypaque gradients (Pharmacia, Piscataway, NJ). Mononuclear cells were obtained from tonsil tissue as described by Clark et al., 1985, Proc. Nat. Acad. Sei. EUA 82: 1766-1770. T cells were depleted with formation of rosettes from sheep erythrocytes treated with AET (amino-ethyl isothiuronium bromide hydrobromide) and by Ficoll-Hypaque gradient separation. In some experiments, blood B cells were enriched by isolating cells adhering to nylon wool. Monocytes were removed by incubation on plastic petri dishes once or twice at 37 ° C for 45 minutes, unless otherwise indicated. Fractions of floating or dense tonsillary B cells were isolated by graded Percoll gradients as described by Clark et al., 1985, Proc. Nat. Acad. Sei. EUA 82: 1766-1770. Dense tonsillar B cell preparations logically contained more than 95% slg + Bp35 + cells. Preparations enriched with blood B cells had 60-85% slg + cells.

Lymphoid cells from B cells were isolated by moderately dissociating 20 - lymphoid cells; * in the middle, then carrying out a Ficoll-Hypaque gradient centrifugation.

Monoclonal antibodies. The antibody G28-5 directed against Bp50 was generated by immunizing BALB / c mice with human E-tonsillar lymphocytes and by fusing immune spleen cells with NS-1 myeloma (Köhler et al., 1975, Nature 256 : 495-497; Ledbetter et al., 1979, Immunol. Rev.

47: 63-82). Cultures of hybrid cells secreting an antibody reactive with tonsill B lymphocytes and not with T lymphocytes were identified using indirect immunofluorescence (IF) and analysis with a FACS IV cell selector; cultures containing an antibody giving histogram plots similar to those of known mAbs directed against pan-markers of B lymphocytes (eg, Bp35) were cloned and selected for further studies. The G28-5 clone produced an IgG1 mAb which reacted only with B or normal or malignant B cell lines. Other mAbs used in this study have been described in detail by Clark et al., 1985, Proc. Nat. Acad. Sei. EUA 82: 1766-1770;

Clark et al., 1986, Human Immunol. 16: 100; Ledbetter 25 et al., 1986, Human Immunol. 15: 30-44; Ledbetter et al., 1985, in Perspectives in Immunogènetics and Histocompatibility, ASHI, New York, 6, pages 325-340. These monoclonal antibodies include 1F5 (lgG2a) anti-Bp35, HBlOa (lgG2a), anti-HLA-DR, 2C3 (lgG1) anti-yu chain, G19-4 (IgG ^) anti-CD3, FC-2 (IgG2a) anti-receptor for Fc CD16 and 9.6 (IgG2 &) anti-CD2 (receptor E) supplied by Doctor Paul Martin (Martin et al., 1983, J. Immunol. 131: 180).

The IgG 2 mAbs were purified by precipitation using 45 or 50% saturated ammonium sulfate Λ 1221 'J. *

J

and DEAE-Sephacryl column chromatography and the IgG2a mAbs were purified using Sepharose columns coated with Protein A. The F (ab ') 2 fragments of G28-5 were prepared by the method of 5 Parham (Parham, et al., 1983, J. Immunol. 131: 2895), they were purified in a column of Sephacryl S200 2 meters long and their purity was determined by SDS-PAGE (Ledbetter et al. ., 1985, J. Immunol. 135: 1819). MAb 2C3 directed against yj 10 chains was conjugated with Sepharose 4B beads

(Pharmacia Fine Chemicals, Uppsala, Sweden) using cyanogen bromide coupling.

Conjugations to fluorescein and phycoerythrin. Purified mAbs were either directly conjugated to fluorescein using fluorescein isothiocyanate (FITC; Molecular Probes) (green) by the Goding method (Goding, et al., 1976, J. Immunol. Meth. 13: 215-226), either conjugated to R-phycoerythrin (PE) (red) using SPDP 20 (Pharmacia) according to a method that the Applicant has described in detail in Ledbetter et al., 1985, in Perspectives in Immunogenetics and Histocompatibility, ASHI, 'New York, 6, 119-129. Lymphoid cells were incubated in round bottom microtiter plates for 30 minutes with an appropriate dilution of green and / or red mAb, washed twice, and then analyzed in a FACS IV cell selector.

Two-color immunofluorescence. Two-color studies were performed with a fluorescent activated cell selector (FACS IV: Becton-Dickinson, Mountain View, CA) using a dichroic mirror at 560 nm to split the beam and a long pass filter 580 and a filter short pass 540 35 (Ditric Optics, Hudson, MA) in front of the photo tubes 1 • i 22 »multipliers of red and green respectively.

In addition, a two-color compensator (T. Nozaki, Stanford University) was used to correct minor overflows of the green and red signals.

5 For each two-color staining, data from 40,000 cells were collected and stored on floppy disks. The data are presented in the form of a number of cells (vertical) with respect to the logarithm of green fluorescence with respect to 10 logarithm of red fluorescence on a grid at 64 x 64 points. A value of approximately 4.5 points represents a doubling of fluorescence. Colorless cells are located at the back corner of the grid; red fluorescence is on the right and green fluorescence on the left. The flow cytometry system for two-color immunofluorescence with fluorescein and phycoerythrin is described in more detail by Ledbetter et al., 1985, in Perspectives in Immunogènetics and Histocompatibility, 20 ASHI, New York, 6, 119-129 and 325 -340.

Culture of cells. Tonsillary lymphoid cells or blood 5-5 10 x 10 ml were cultured in quadruplication in 96-reservoir microtiter plates containing 200 yal of RPM1-1640 medium supplemented with 15% fetal bovine serum, antibiotics, glutamine and pyruvate (R15). After 1 to 7 days, 3 cells were pulsed with 0.5yaCi of H-thymidine per reservoir (New England Nuclear, 6.7 Ci / millimole; 1 Ci = 37) for 18 hours. The cells were then harvested on glass fiber filters with a harvester and the radioactivity was measured in a scintillation counter. In some experiments, antibodies or factors were added at different times after the start of cultures; during these experiments, * i 23 'i * / proliferation was measured on day 3.

Co-stimulating factors. A purified BCGF was obtained from "Cytokine Technology" (Buffalo, New York), containing no detectable activity of IL-1, IL-2 or interferon. This BCGF was prepared by the method of Maizel et al. (Maizel et al., 1982, Proc. Nat. Acad. Sci. EUA 79: 5998) which demonstrated that the major activity of BCGF in this material resides in a 12 kDa species hereinafter called "BCGF (weak)" (Mehta et al., 1985, J. Immunol. 135: 3298). The purification steps included DEAE affinity chromatography at the preparation scale, followed by hydroxylapatite column chromatography. IL-1 15 in a pure state until homogeneous was the generous gift of Doctor Steven Dower (Dower, et al., 1985, J. Exp. Med. 162: 501). Recombinant IL-2 has been kindly supplied by "Cetus Corporation". TPA (12-0-tetradecanoyl-phorbol-13 acetate) was supplied by 20 "Sigma".

Detection of cell activation.

Caused changes in cell volume by mAb and / or factors were measured using a cell selector and a forward angle moving spot detector. Cell cycle changes were measured in cellular ribonucleic acid and deoxyribonucleic acid contents by staining activated cells with acridine orange and measuring the relative ribonucleic acid (red) and acid contents. deoxyribonucleic (green) cells with a cell selector according to the method of Darzynkiewicz et al. (Darzynkiewicz, et al., 1980, Proc. Nat. Acad. Sci.

EUA 77: 6697-6702). Changes in the relative contents of surface antigens

L X

24 '*' *

J

cells were checked using an mAb directly conjugated with fluorescein, then they were quantified by levels of direct immunofluorescence with an Epies V cell selector.

5 Biochemical characterization of Bp50. The immunoprecipitation of Bp50 out of tonsil cells marked on the surface was carried out as described by

Ledbetter et al., 1985, J. Immunol. 134: 4250-4254.

Isolated antigens were electrophoresed on 10% SDS / polyacrylamide plate gels without reduction. The gels were visualized using autoradiography at -70 ° C and lightening screens plus reinforcement Cronex (Dupont).

5.2. CHARACTERIZATION OF THE Bp50 RECEPTOR 15 The following subclauses describe the results of the experiments carried out using the methods described above.

5.2.1. IDENTIFICATION OF A SURFACE CELL MARKER at 50 kDa, SPECIFIC TO 2 ° B, Bp50 LYMPHOCYTES

An mAb antibody against Bp50 was raised by immunizing BALB / c mice with human tonsillar lymphocytes and fusing immune spleen cells with NS-1 myeloma. One clone, G28-5, produced an IgG ^ mAb which did not contain the NS-1 light chain. Upon careful examination by immunofluorescence analysis, it was found that G28-5 reacted only with normal or malignant B-cell lines or B-cells. A very wide selection of normal tissues by established methods (Clark, et al., 1985, Proc. Nat. Acad. Sci. EUA 82: 1766-1770; Ledbetter et al., 1986, Human Immunol. 15: 30-44 ; Ledbetter, 1985, in Perspectives in Immunogènetics and Histocompatibility, 35 ASHI, New York, 6, pages 325-340) revealed that the anti-body G28-5 reacted with negative cells forming rosettes E (Er -) from blood or tonsils, but not with T lymphocytes not adhering to nylon wool, blasts of 5 T lymphocytes caused by PHA (= phytohemagglutinin) or with blood granulocytes, monocytes, globules red or platelets. It reacted strongly with all seven B lymphoblastoid cell lines tested and with three 10 Burkitt lymphoma lines (Raji, Daudi, Namalwa), but not with four T cell lines (CEM, HSB-2, JURKAT and HPB-ALL). All chronic lymphocytic leukemias tested (3/3) and 90% (9/10) B lymphomas tested expressed the Bp50 marker, while only 28% (2/7) leukemias non-T, non-B CALLA + acute lymphocytic expressed Bp50.

The restricted distribution of Bp50 on normal tissues was further confirmed by quantitative analyzes of two-color immunofluorescence (two-color IF). Using an antibody (red) conjugated to R-phycoerythrin (PE) and directed against the pan-antigen Bp35 of B lymphocytes (B1, CD20) and the antibody (green) anti-Bp50 conjugated to fluorescein, the Applicant found that Bp50 was expressed only on Bp35 + B cells (Figure 1) in the blood or tonsils. Blood B cells expressed Bp50 logically at somewhat lower concentrations than tonsill B cells, which is similar to the expression of HLA-DR (Ledbetter et al., 1986, Human Immunol. 15: 30- 44) and to the expression of gp54 (Wang, et al., 1979, J. Exp. Med. 149: 1424-1433), which are also weaker on the B lymphocytes of the blood. Bp50 35 was expressed at similar concentrations in i> 26 * of the sub-populations of tonsill B cells separated on Percoll gradients in floating and dense fractions. Using the mAb conjugated to PE and directed against the marker of T lymphocytes, CD3 (T3) and the marker associated with the NK cells, CD16 (Fc receptor) (Ledbetter, et al., 1979,

Immunol. Rev. 47: 63-82), the Applicant has found that Bp50 is not expressed on T lymphocytes or NK cells. By using two-color immuno-fluorescence, the Applicant has also found that CD3 + PHA blasts which expressed high concentrations of IL-2 receptors do not express Bp50.

The G28-5 antibody reacted with a single polypeptide on tonsil lymphocytes which migrated at about 50 Kd under non-reducing conditions (Figure 2A). This molecule is larger than that of the markers of B lymphocytes mentioned above, in the same range of molecular weights-20, such as Bp39 or Bp45 (Zipf, et al. 1983, J. Immunol. 131: 3064-3072; Kitner , et al., 1981,

Nature 294, 458-460; Clark, et al., 1986, in Leukocyte Typing. II, eds. Reinherz, et al., Springer Verlag, Berlin, chapter 12, volume 2, 155-167;

25 Slovin, et al., 1982, Proc. Nat. Acad. Sei. USA

79: 2649-2653; Thorley-Lawson, et al., 1985, J. Immunol. 134: 3007-3012, and Figure 2B). The exposure time for this gel was selected so that the molecular weights of the other 30 B cell markers can be easily compared with Bp50. Unlike Bp50, the marker Bp39 is expressed on granulocytes and, unlike Bp50, Bp45 is restricted to blast cells of B lymphocytes. Antibodies directed against Bp39 (41-H16) and Bp45 (MNM6, blaste-1, 35 blaste- 2) provided by an international working group

Jl * 27. * (Clark, et al., 1986, in Leukocyte Typing II, eds. Reinherz, et al., Springer Verlag, Berlin, chapter 12, volume 2, 155-167) did not block the binding of anti- Bp50 treated with fluorescein, 5 to B lymphocytes. Therefore, on the basis of tissue distribution, biochemical analysis and blocking studies, the monoclonal antibody G28-5 recognizes a distinct 50 Kd structure that of other known antigens of B lymphocytes.

10 5.2.2. Bp50 EXPRESSION IS RESTRICTED

TO LYMPHOCYTES B.

Distribution studies of hematopoietic cell and tissue lines, as well as detailed two-color flow cytometric analyzes revealed that Bp50 was expressed only on B cells. As illustrated in Figure 3, Bp50 is expressed on a small subset of blood lymphocytes and on a large population of tonsill lymphocytes. Virtually all Bp50 + lymphocytes in both blood and tonsils also expressed Bp35 and HLA-DR, but they did not express T cell molecules, CD2 (Figure 1) or CD3, nor did Fc receptors IgG found on NK cells. In addition, blasts of CD3 + T lymphocytes activated by ConA expressed IL-2 receptors, but not Bp50,

Two-color flow cytometric analyzes allow the quantitative measurement of the density relationship between two surface antigens.

The Applicant has previously indicated that dense resting B cells located in the peripheral region of secondary follicles expressed IgM and low concentrations of Bp35, while activated floating B cells located in the germinal center 35 are IgM negative and express concentrations 28

J

'l * elevated from Bp35 (Ledbetter, et al., Human Immunol. 15:30). Figure 3 shows that both subsets of IgM-positive and IgM-negative B cells expressed Bp50 in equal amounts, thus indicating that Bp50 is expressed both on resting B lymphocytes and B cells activated in vivo.

5.3. INCREASED PROLIFERATION OF B LYMPHOCYTES WITH ANTI-Bp50 ANTIBODY.

As discussed above, B cells can be activated with low doses of anti-ya chain specific antibodies. Recently, the Applicant has discovered that the marker Bp35 (B1) specific for B lymphocytes, namely a 35 kDa polypeptide, could also fulfill its function during the early activation of B lymphocytes: just like low doses of antibodies anti-ya, mAb 1F5 directed against Bp35 activates B lymphocytes so that their volume and their ribonucleic acid content increase and so that they become sensitive to BCGF (Clark, et al., 1985, Proc.

Nat. Acad. Sei. EUA 82: 1766-1770; Gollay, et al., 1985, J. Immunol. 135: 3795-3801). Consequently, it has been interesting to compare the effect of the anti-Bp50 mAb in the proliferation of untreated B lymphocytes or of B lymphocytes activated either with anti-Bp35 antibodies or with anti-ya antibodies (Table 1). . Under appropriate conditions only, anti-Bp35 antibodies in solution or anti-γ antibodies attached to Sepharose beads could stimulate some B cell proliferation (Table 1, line 1); on the other hand, anti-Bp50 antibodies alone did not stimulate proliferation (Table 1, line 2). However, anti-Bp50 mAb significantly increased proliferation when cultured with anti-ya beads or with anti-Bp35. In this respect, - * Λ ι 29 anti-Bp50 resembled BCGF (table 1, line 3).

Therefore, it was important to determine whether anti-Bp50 and BCGF together could cause the proliferation of B lymphocytes. As illustrated in Table 1, line 4, anti-Bp50 and BCGF together did not cause any proliferation, but they increased the proliferation of cells activated by anti-yj or anti-Bp35 to a degree somewhat greater than either stimulant alone. In a three-log interval and when used with anti-Bp50 without other signals, BCGF had no effect on the proliferation of dense B cells, even when anti-Bp50 was used at low doses. lying in the range of 0.1 to 10 µg / ml.

15 TABLE 1

Increased proliferation of B lymphocytes, caused by anti-Ig or anti-Bp35, with anti-Bp50 antibodies 20 Average proliferation + standard error of B lymphocytes cultured with: _

Line Co-sti- Pearls Anti-Bp35 mulant Anti-yu media 25 1 None 1,212 + 547 10,219 + 462 5,539 + 308 2 Anti-Bp50 719 + 718 38,792 + 1,329 25,465 + 616 3 BCGF 456 + 217 14,217 + 445 9,443 + 343 4 Anti-Bp50 + BCGF 1.456 + 126 54.393 + 2.537 46.488 + 3.387 30 _

The proliferation of Er-dense tonsillary B lymphocytes (95% of superficial IgM cells) was measured on day 3, as described. In summary, 2 x 105 cells / 200 yal reservoir were cultured in quadruplication for 48 hours with 30 µ t RPMI 1640 medium containing 15% fetal bovine serum plus additives without antibody, or with 2C3 monoclonal antibody directed against ya chains, coupled to Sepharose beads ("anti-yj beads", 50yug / ml) or the free anti-Bp35 1F5 antibody (5yjg / ml). Cultures containing media, "anti-ya pearls" or anti-Bp35 were grown alone or with BCGF (final concentration: 5%, Cytokine Technology, Buffalo, New York; no detectable activity of IL-10 or IL -2), with anti-Bp50 (dilution 1: 1,000 of ascites) as co-stimulants. After 40 hours, 3 cells were pulsed with H-thymidine and the incorporated counts were measured after 18 hours.

15 5.3.1. mAb ANTI-Bp50 INCREASES PROLIFERA

ONLY AFTER B LYMPHOCYTES HAVE BEEN ACTIVATED BY ANTI-Bp35 or ANTI-yu ANTIBODIES.

The results in Table 1 suggest that anti-Bp50 mAb could not, by itself, cause proliferation. As shown in Figure 4, doses of anti-Bp50 between 0.05 yug and 2.0 yag / ml had no effect on uptake of H-thymidine. However, in the presence of optimal anti-Bp35 mAb concentrations, a concentration as low as 0.1 to 0.5 µg / ml anti-Bp50 antibody significantly increased proliferation. Values as high as 50,000 to 70,000 counts per minute could be detected at the optimum time of proliferation when highly purified B cells were grown only with anti-Bp35 plus anti-Bp50. As has been observed logically, higher doses of anti-Bp50 (more than 2-5 µg / ml) have been less effective than doses in the range of 100-200 ng.

These results suggested that anti-Bp50 i t 31 - '' could perform its function only after activation of B cells by other signals. The data in Figure 5 suggests that this is, in fact, the case. If B cells were first activated with anti-Bp35, anti-Bp50 could be added up to 24-48 hours later still increasing proliferation on day 4. In contrast, when cells have all first treated with anti-Bp50, anti-Bp35 was only effective when it was added within a few hours of starting the cultures. Similar results were found when using anti-yu rather than anti-Bp35.

5.3.2. ANTI-Bp50 mAbs DO NOT ACTIVATE GQ PHASE B LYMPHOCYTES, BUT THEY ACTIVATE B LYMPHOCYTES TO PROGRESS THROUGH

CELL CYCLE._

Previously, the Applicant has found that, just like low doses of anti-ya antibodies, anti-Bp35 causes tonsillary B lymphocytes at rest in the GQ phase to grow (Clark, et al., 1986, Leukocyte Typing II, eds., Reinherz, et al., Springer Verlag, Berlin, volume 2, 455-462) and to enter the G ^ phase of the cell cycle (Gollay et al., 1985, 25 J. Immunol. 135: 3795 -3801). Therefore, it was interesting to compare the faculties of anti-Bp50 mAb and anti-Bp35 mAb concerning their effects on the activation of B lymphocytes. As indicated in FIG. 6A, even after 3 to 4 days in culture, 30 Unstimulated dense tonsill B cells had a uniform ribonucleic acid profile, characteristic of G-phase cells (Darzynkiewicz, 1980, Proc. Nat. Acad. Sci. EUA 77: 6697-6702). However, about 15-30% of cells stimulated with anti-Bp35 or anti-ya had a higher content of ribonucleic acid * 32, thus indicating their entry into the G1 phase. In contrast, neither anti-Bp50 (Figure 6B) nor BCGF (Figure 6C) alone did not cause large numbers of B cells to enter the G ^ phase. For example, 2 days after activation, anti-Bp35 and anti-Ig mAb caused 13.5% and 20.9% of tonsillar cells, respectively, to enter the phase, while cells treated only with anti-Bp50 ( 2.7%) or BCGF 10 (3.2%) remained at concentrations of control media (2.2%). However, when anti-Bp50 or BCGF was added together with anti-Bp35 or antiyi antibodies, the proportion of cells entering the G ^ phase increased considerably. Similarly, anti-Bp50 and BCGF alone did not cause B cells to enter the S phase (Table 2) but, together with anti-Bp35 or anti-u, they increased the number two to three times cells in phase S.

<. t i 4 * 33 TABLE 2

Effect of anti-Bp50 and BCGF on the progression of the cell cycle in tonsillar lymphocytes.

5 Cell Signal Signal,%

GQ S / G2 / M progression skill

Media None 89.9 7.1 2.5 anti-Bp35 "80.4 14.5 3.7 10 anti-Ig" 65.6 27.6 5.7

Anti-Bp50 media 83.6 12.0 3.3 anti-Bp35 "54.1 35.5 9.7 anti-Ig" 43.6 36.2 16.2

BCGF media 85.4 11.7 2.2 15 anti-Bp35 "56.6 32.6 11.6 anti-Ig" 48.4 36.1 14.1

Percentage of cells in the Gq, G ^ or S and G2 phases, determined by adopting the acridine orange staining method (Darzynkiewicz, et al., 1980, Proc. Nat. Acad. Sci. USA 77: 6697-6702); 0 1 x 10 dense tonsil lymphocytes with anti-Bp35 (5yug / ml), anti-yu on pearls (50 ^ ag / ml), anti-Bp50 (0.4 ^ ig / ml), BCGF (5%) or their combinations, as shown.

5.3.3. OPTIMAL CONDITIONS TO INCREASE THE PROLIFERATION OF B LYMPHOCYTES WITH ANTIBODIES

ANTI-Bp50_ 30 In themselves, antibodies to

Bp50 have little or no detectable effect on dense B cells at rest (Table 3). However, in the presence of agents that can activate B cells, for example, anti-Ig, anti-Bp35 "and TPA, anti-35 Bp50 mAb markedly increased proliferation. Anti-Bp50 1 * 34 did not cause co-stimulation with different interleucins, including purified IL-1, recombinant IL-2 and BCGF (weak). A comparison of the effects of anti-Bp50 with those of BCGF (weak) revealed that 5 the same agents which were co-stimulants with anti-Bp50, were also co-stimulants with BCGF (weak) (Table 3). It was particularly interesting to discover that BCGF and anti-Bp50 together were still not co-stimulants for eellu-10 them at rest.

TABLE 3

Increased proliferation of B cells with anti-Bp50 antibodies or a B cell growth factor

15 _

Average proliferation + standard error of B lymphocytes cultured with:

Co-stimulant Anti-Bp50 BCGF media

20 _ (200 ng / ml) _ (5%) _ None 96 + 1,267 + 15,285 + 74

Anti-Ig 5.833 + 391 41.634 + 2.103 25.094 + 61

Anti-Bp35 (5 yjg / ml) 457 + 45 8,143 + 280 1,733 + 32 25 TPA (2 ng / ml) 7,361 + 537 21,163 + 871 13,064 + 1,030 IL-1 (10 U / ml) 264 + 2,308 + 23 221 + 8.

IL-2 (100 U / ml) 204 + 34 350 + 7 220 + 11 BCGF (5%) 220 + 7 851 + 28 270 + 18 20 Dense E-tonsill B lymphocytes (more than 95% of sIgM + cells) cultivated during 48 hours at 2 x 10 cells / reservoir, then by pulse 3 for 24 hours with H-thymidine before counting.

35 35 * κχ *

The proliferation kinetics increased by anti-Bp50 is illustrated in FIG. 7. The proliferation peak appeared on day 4, then it declined, whether or not the cells were activated with 5 anti-Bp35 or other activators such as anti-Ig or TPA. The proliferation kinetics increased by BCGF or by anti-Bp50 were similar.

As little as 0.05 µg anti-Bp50 antibody increased proliferation.

In subsequent studies, an optimal dose of 0.3 µg / ml was used. It has been logically observed that when using whole antibody molecules, higher doses of anti-Bp50 (more than 2-5 µg / ml) were less effective than doses in the range of 0.1 to 0.5 ^ ag / ml.

Human B lymphocytes are extremely sensitive to the inhibitory effects caused by Fc receptors of antibodies which bind to superficial Ig (Parker, 1980, Immunol. Rev. 52: 115; 20 Bijsterbosch, et al., 1985, J. Exp. Med. 162: 1825).

Therefore, it was important to compare the efficacy of anti-Bp50 whole mAb with that of F (ab ') 2 antl-Bp50 fragments. In a hundredfold assay interval, F (ab ') 2 fragments were clearly as effective, if not more effective, than a whole antibody in increasing the proliferation of B lymphocytes (Table 4). Therefore, the Fc domain of anti-Bp50 mAb is not required for anti-Bp50 to exert its effect and it can even be an inhibitor. In other words, just like BCGF, anti ~ Bp50 can apparently act as a soluble mediator without calling on accessory cellular function with the Fc receptor as mediator.

-Λ 4 '36 ‘ι TABLE 4

The Fc domain of anti-Bp5Q antibodies is not required to increase the proliferation of B lymphocytes

5 Average proliferation of B lymphocytes cultured with: Anti-Bp50 Medium dose Anti-Bp35 (yjg / ml) 10 None - 295 + 16 269 + 27

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

The cell culture conditions were those described in Table 3.

20 _________

5.3.4. DIFFERENCES BETWEEN ACTIVITY

D1ANTI-Bp50 AND BCGF (LOW) ._

Anti-Bp50 and BCGF (weak) exerted a similar effect on B lymphocytes and they were co-stimulants with the same agents (Table 3). However, several orders of evidence indicate that anti-Bp50 and the BCGF used in this study clearly come into play when different signals are used. First, unlike BCGF receptors (weak) (Bijsterbosch, et al., 1985, J. Exp. Med. 162: 1825), Bp50 molecules are expressed on B cells at rest in the blood (Figure 3 ).

Second, although both anti-Bp50 and BCGF (weak) most effectively exercise their function when added after anti-Bp35 or anti-Ig,

V

37 d h * anti-Bp50 clearly exerted its optimum effect when it was added 12 hours after the cultures started (FIG. 8A). On the other hand, BCGF (weak) could be added after a period as long as 24 hours after the start of the cultures, while still optimally increasing the proliferation (FIG. 8B). These kinetic experiments, which are modeled on the method of Howard and Paul (1983, Ann. Rev. Immunol. 1: 307), suggest that a Bp50-dependent signal can normally exert its effect before BCGF.

Anti-Bp50 and BCGF (low) both increased the proliferation of B cells activated with anti-Bp35 or anti-Ig (Table 3). However, the effects of anti-Bp50 and BCGF (weak) were additive in many experiments (Figure 7).

FIG. 9 illustrates a titration of BCGF (low) during an experiment in which anti-Bp50 was used at its optimal concentration (0.2 μg / ml).

BCGF (weak) was able to further increase the proliferation of B lymphocytes at rest in the presence of anti-Bp50 after activation either by anti-Ig or by anti-Bp35. Optimal BCGF concentrations (low) were 5-10%, while concentrations of 25% were inhibitory. Therefore, when both anti-Bp50 and BCGF (low) were used at their optimal concentrations, they always exerted additive effects on the proliferation of B lymphocytes.

Finally, both the subsets of normal B cells and malignant B cells differ in their response to anti-Bp50 and to BCGF (weak). For example, some blood B cells were sensitive to BCGF (weak), but they did not respond to anti-Bp50 (Table 5). An additional activation signal such as anti-Bp35 (Table 5) or TPA (Figure 10) was logically necessary for B lymphocytes in the blood to respond to anti-Bp50. Although, in general, dense tonsill B lymphocytes did not respond to either BCGF or anti-Bp50, floating B cells responded (Table 5). The malignancies of B lymphocytes were also differentiated by their sensitivity to anti-Bp50 vis-à-vis BCGF. For example, some B cell lymphomas responded to TPA plus BCGF (weak), but not to TPA plus anti-Bp50 G28-5 (Figures 10B and D). In contrast, dense tonsill B cells and peripheral blood B cells responded to TPA plus BCGF 15 (weak) or anti-Bp50 (Figures 10A and C).

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p ε -Br .3 2 H H H H H Ό H O a 3 30 + + + + c 0 2a S S S i 5 i 5 sssslgg t 40 * * 5.4. USES OF ANTI-Bp50 AND Bp50 COORDINATES.

The ligands of the present invention can be used in vivo or in vitro both in their unmodified forms and in their modified forms, to modulate immune responses. For example, the ligands themselves can be used as "adjuvants" to increase an immune response to a vaccine or to increase the immune response of an individual with immunosuppression. Alternatively, if cytotoxins or anti-proliferative agents are coupled to the ligands, these modified ligands can be used to decrease the immune response, for example, in an autoimmune disease or in patients who have received a transplant in order to avoid rejection of the graft. These modified ligands could also be used to treat malignancies where cells or tumors express the Bp50 antigen, whether or not the malignancy originates in B lymphocytes.

The ligands of the present invention and / or Bp50 itself can be used in vitro.

Such applications include in vitro assays such as immunoassays for the detection of cells which express the Bp50 antigen and / or for the detection of the Bp50 antigen possibly lost in the moods of the body. In this case, the ligand or Bp50 could be labeled with a radio-marker, fluorine, an enzyme, an enzyme substrate, a dye, etc. In addition, the ligands can be used to separate and / or identify cells that express the Bp50 antigen, in which case the ligand can be coupled to a stationary support or to a fluorine that can be used in a FACS (35 cell selector activated by fluorescence).

4 - 41

The different applications and uses of the ligands and Bp50 of the present invention are described in more detail below.

5.4.1. Bp50 RECEPTOR AND USES OF COORDINATES SUCH AS ANTI-Bp50 TO INCREASE

PROLIFERATION OF LYMPHOCYTES B .__

Previous studies have suggested that the factors involved in the induction of Gq phase B cells in the cell cycle phase 10 are distinct from the factors or conditions required for transit in the S phase. This model is based, in principle , studies showing that agents such as low doses of anti-Ig or anti-Bp35 B-cell activating factors alone had little or no effect on the proliferation of B-cells. However, these same agents can lead B cells to an activation point 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 they do increase the growth of activated B cells.

Although the present invention is not limited to any theory or explanation, the results presented here further support a distinct regulatory model for the stages of growth and activation of B cells. In this case, the Applicant has demonstrated that activation and proliferation signals appearing in human B cells could be transmitted by distinct cellular surface structures. Although anti-Bp35 mAb has activated B lymphocytes to make them enter the G ^ phase of the cell cycle, on its own, it causes little or no proliferation. mAb anti-35 Bp50 had the opposite effect: it could not activate:. -i t 42 B cells but, when added even after as long as 12-24 hours after activation, it may have caused the growth of B cells.

5 As assumed, the Bp50 molecule would normally behave as a receptor for a ligand such as a soluble growth factor or for a signal mediated by cell-cell contact (i.e. a ligand lying on the surface of another cell). Previous studies have identified several BCGFs derived from T cells which, like anti-Bp50, increase the proliferation of B cells. Both high molecular and low molecular forms of growth factors B cells have been identified and different types have been shown to exert additive effects (Kehrl, et al., 1984, Immunol.

Rev. 18: 75-96; Kishimoto, 1985, Ann. Rev. Immunol.

20 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., J. Clin. Invest.

75: 732-739; Ambrus, 1985, J. Exp. Med. 162: 1319-1335). Therefore, Bp50 could be a receptor for one of these factors.

With the exception of the IL-2 receptor and the C3d receptor, the receptors found on B lymphocytes for growth signals have not yet been identified. MAb AB-1 reacts with a B cell marker expressed only on activated B cells and blocks BCGF-dependent proliferation, so that it could recognize the BCGF receptor or a related structure.

Bp50 appears to be distinct from the AB-1 marker, since mAb AB-1 does not block the binding of 4 - * 1 43 the anti-Bp50 G28-5 antibody and, unlike mAb G28-5 , it reacts only with activated B lymphocytes (Jung et al. 1984, J. Exp. Med. 160: 1919-1924). Bp50 is found on all B 5 lymphocytes which, on the basis of absorption analysis and direct binding assays, does not seem to be the case for BCGF receptors.

The data currently available to the Applicant indicate that Bp50 and the receptor for low molecular weight BCGF are distinct structures.

Using a rabbit heteroantiserum, Wang et al. (Wang, 1979, J. Exp. Med. 149: 1424-1433) previously described a 54 kDa glycoprotein, gp54, which, like Bp50, is expressed on all B cells, but at lower concentrations on blood B cells than on tonsill B cells. It is possible, but improbable, that the rabbit heteroantiser and anti-Bp50 recognize the same structures or related structures: in contrast to anti-Bp50 mAb, the rabbit antiserum directed against gp54 alone was sufficient to stimulate the proliferation of B cells.

Unlike anti-Bp50, anti-Bp35 alone can activate B cells from the Gq phase to the G1 phase and, therefore, it can be called "activation signal". It is not yet clear whether or not Bp35 comes into play only during early activation of B lymphocytes, since anti-Bp35 antibodies can stimulate the division of certain B lymphocytes (Clark et al., 1985, Proc. Nat Acad. Sci. EUA 82: 1766-1770). Likewise, Bp50 cannot strictly come into play only as a "growth signal": together with activation signals (anti-Bp35 or anti-ya), antibodies; 4 »44 i anti-Bp50 not only increase proliferation, but also the total number of B lymphocytes entering the G1 phase (Table 2). In other words, as a co-stimulant, anti-Bp50 acts to activate 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 exerted a similar activity (Figure 6C).

Therefore, anti-Bp35 and anti-Bp50 (or BCGF) appear to be essentially analogous to the "competence" and "progression" factors described in studies carried out on the regulation of fibroblast growth. How B cells respond to anti-Bp35 or anti-Bp50 may clearly depend on their state of differentiation or activation.

It has been shown here that two mAbs, namely anti-Bp35 ("competence" signal) and anti-Bp50 ("progress" signal) together can cause a large proliferation of highly purified B lymphocytes in the absence of antigen or other known factors. The natural coordinates for these structures are not yet known. However, since mAb directed against determinant antigenic groups can mimic both soluble factors and signals mediated by cell-cell interactions, it may be possible to use appropriate combinations of mAb to direct and regulate the proliferation or differentiation of human B lymphocytes which in turn will help to design in vivo strategies to combat human diseases such as B cell malignancies, immune deficiencies and certain autoimmune diseases .

35 The new monoclonal antibody, G28-5, which

7 '* J

45 reacts with a single chain polypeptide of about 50 Kd, expressed on the surface of human B lymphocytes, is only one particular embodiment of the ligands of the present invention, which can increase the proliferation of B lymphocytes activated.

Since the proliferation of human B cells can be increased in the same way by BCGF derived from T cells, including low molecular weight and high molecular weight BCGF, we have compared the activity of G28-5 anti-Bp50 with that of a BCGF preparation containing mainly a low molecular weight BCGF.

Anti-Bp50 G28-5 and BCGF (weak) were very similar in that they were co-stimulants with the same activating agents (anti-Ig, anti-Bp35 and TPA), however they did not were not co-stimulating with each other or with IL-1 or even IL-2. Furthermore, the activity of anti-Bp50 G28-5 did not depend on its Fc domain, since F (ab ') 2 fragments of G28-5 20 were functionally active, which suggests that, just as BCGF soluble, anti-Bp50 soluble does not require accessory cells carrying an Fc receptor to exert an effect. In addition, anti-Bp50 and BCGF are both effective only in the presence of an activating stimulus. In other words, anti-Bp50 and BCGF are not factors of "competence", but rather favor the "progression" of B lymphocytes through the cell cycle.

Although in all probability Bp50 may come into play as a receptor for a ligand such as a B cell growth factor, several results suggest that Bp50 is not the receptor, at least for BCGF (weak ) used in this study: it is expressed on 35 B lymphocytes in the blood, while BCGF 4 46 receptors (weak) are apparently not. In contrast to Bp50, candidate structures for the (weak) BCGF receptor are also expressed only on activated B cells. In addition, 5 of the normal and malignant B lymphocyte populations differ in their sensitivity to anti-Bp50 vis-à-vis BCGF (low) (Table 5 and Figure 10).

For example, some B lymphomas proliferate in response to BCGF (weak), but not in response to anti-10 Bp50. Finally, in a number of experiments, optimal concentrations of anti-Bp50 and BCGF, together, caused greater proliferation than individually. Anti-Bp50 mimics the activity of other BCGFs such as BCGFs (high) which are co-stimulants with anti-IgM (Ambrus, et al., 1985, J.

Exp. Med. 162: 1319; Ambrus, et al., 1985, J. Clin. Invest. 75: 732), suggesting that Bp50 may come into play as a receiver for BCGF (high).

Although Bp50 may be a receptor for a soluble ligand, alternatively, Bp50 may act as a receptor for a signal mediated by cell-cell contact which regulates BCGF 25 receptor concentrations and / or autocrine production. The precedence of differentiation antigens serving as amplifiers of an autocrine receptor pathway comes from studies carried out with T lymphocytes. MAb directed against the common leukocyte antigen Lp220 increases the pro-liferation by raising the expression of the receptor of IL-2 on activated T lymphocytes (Ledbetter, et al., 1985, J. Immunol. 135: 1819). An analogous mechanism can come into play with anti-Bp50 and the expression of certain BCGF receptors. Apparently, Bp50 35 and BCGF (weak) are under some coordinated control A *; 47 because, like the IL-1 and IL-2 receptors, BCGF increases the expression of Bp50 in certain leukemic cells. The Bp50 molecule also shares similarities with the Tp44 5 molecule which comes into play to influence the production of IL-2. The Applicant and other researchers have demonstrated that the anti-Tp44 antibody 9.3 increases the proliferation of T lymphocytes activated by anti-CD3 or TPA (Ledbetter, et al., 1985, J. Immunol.

10,135: 2331; Hara, et al., 1985, J. Exp. Med. 161: 1513).

Similarly, anti-Bp50 increases the proliferation of B lymphocytes activated by anti-Bp35 or TPA. The Tp44 signal works by stimulating the production of IL-2 rather than by stimulating the growth of T cells.

15 Probably, the Bp50 signal could act in an analogous way by stimulating the autocrine production of B lymphocytes (Gordon, et al., 1984, Nature, Lond. 310: 145) 7 5.4.2. MODIFIED C00RDINATES USED FOR IMMUNOSUPPRESSION OR TREATMENT OF MALIGNANCIES.

According to this embodiment, the co-dinat of the present invention can be modified by the attachment of an antiproliferative agent, so that the molecule obtained can be used to kill cells which express the Bp50 antigen. Such modified ligands can be used in the treatment of autoimmune diseases, in order to suppress the proliferation of B lymphocytes and thus suppress the autoimmune response. These modified ligands can also be used to provide immunosuppression in 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 K * ι 48 the invention. When using ligands which increase the proliferation of B cells, this should have an increased effect, since both the drug will be directed to the 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 Bp50 antigen. The attachment of these chemotherapeutic agents to the ligands of the invention must ensure greater specificity of the drug for malignant cells. In addition, a particular advantage should be obtained when treating 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 ensure potentiation of the action of the cytotoxin.

Consequently, the chemotherapeutic agents or the antiproliferative agents which can be coupled to the ligands of the present invention include, without any limitation, the agents listed in Table 6 below which comes from Goodman and Gilman, The Parmacological Basis of Therapeutics, 6th edition, MacMillan Publishing Co., Inc., New York, pages 1249-1313, 1980, referred to here for reference.

- * »49 TABLE 6

Chemotherapeutic agents which can be coupled to anti-BpSO ligands Class Type Agent 5 Al-Mustard agent Chlormethine nitrogen kylation Cyclophosphamide

Melphalan Uracil mustard Chlorambucil 10 Ethy derivatives- Thiotepa lene-imine

Busulfan alkyl sulfonates

Nitrosoureas Carmustine 15 Lomustine Semustine Streptozotocin Triazenres Dacarbazine

Antimeta- Analogues of Methotrexate 20 bolites folic acid

Fluorouracil pyrimidine Cytarabine analogues

Azaribine

Mercaptopurine analogs 25 purine Thioguanine

Natural Vinblastine Alkaloids Products Periwinkle Vincristine (Pervinca) 30 Antibiotics Dactinomycin

Daunorubicin

Doxorubicin

Bleomycin

Mithramycin 35 Mitomycin - A. ♦ 50 TABLE 6 (continued)

Class Type Agent

L-asparaginase enzymes

Cisplatin 5 Complex Agents various platinum coordinates

Urea substi- Hydroxy-urea killed Derivative of Procarbazine 10 methyl-hydrazine

Mitotane adrenal cortex suppressor 15 Hormones and Cortico- Prednisone steroid antagonists

Progestins Hydroxy-progesterone caproate Medro-progesterone acetate

Megestrol acetate Estrogens Diethyl-stilbestrol

Ethinyl-estradiol Anti-estrogen Tamoxifen 25 Androgens Testosterone propionate

Fluoxymesterone

Isotopes Phosphorus Sodium phosphate 32P

radioactive Iodine Sodium iodide 131I

30__

Any method known in the art can be adopted to couple the ligand to the chemotherapeutic or antiproliferative agent.

Examples of such methods have been listed previously (see paragraph 5, above).

A * 51 "

5.4.3. OTHER USES OF COORDINATES

AND Bp50

In addition to therapeutic applications, the ligands and Bp50 itself allow other applications in diagnostic assays both in vitro and in vivo, separation schemes, etc.

The Bp50 receptor can be used for the preparation and / or the design of the ligands of the invention. Bp50 can also be used with the ligands of the invention in in vitro assays which require a standard to quantify the amount of Bp50 detected in a test portion. Finally, Bp50 itself may be useful as a soluble factor constituting a mediator of immunity, for example, a lymphokine.

In addition to therapeutic treatment and diagnostic assays, the ligands of the present invention could be used to identify or separate cells that express the Bp50 antigen. In addition, if an X-ray opaque compound

or an appropriate radiolabel is linked to the ligand, this could be used for the in vivo visualization of tumors expressing the Bp50 antigen. From the above description, other uses should be apparent to those of skill in the art.

6. DEPOSIT OF CELL LINES

The following hybridoma has been deposited in the American Type Culture Collection ", Rockville, MD, and has been assigned the following serial number:

30 Hybridome ATCC order number

G28-5 HB9110

The scope of the present invention is in no way limited by the deposited hybridoma, since the deposited type is given simply in order to illustrate an aspect of the invention and that any - + * 52 lines cells which are functionally equivalent fall within the scope of the present invention. In fact, on reading the foregoing description and in view of the appended figures, those skilled in the art will recognize various modifications of the invention in addition to those illustrated and described in the present specification. It is understood that these modifications fall within the scope of the claims below.

i * 4 4 53

TABLE OF CONTENTS

Page 1. Introduction ........................... 1 2. Basis of the invention ........... .... 2 3. Summary of the invention ................ 5 5 3.1. Definitions ...................... 10 4. Description of the figures ................ 11 5. Detailed description of the invention ... 15 5.1. Methods adopted to characterize the Bp50 receptor ....... 19 10 5.2. Receiver characterization

Bp50 .......................... 24 5.2.1. Identification of a surface marker of 50 kDa cells specific to B cells, Bp50 ............ 24 5.2.2. The expression of Bp50 is restricted to B lymphocytes .................. 27 20 5.3. Increase in the proliferation of B lymphocytes with the anti-Bp50 antibody .................. 28 5.3.1. anti-Bp50 mAb increases proliferation only after the B lymphocytes have been activated by anti-Bp35 or anti-^ μ antibodies ................... 30 5.3. 2. The anti-Bp50 30 mAbs do not activate the Gq phase B lymphocytes, but they cause the activated B lymphocytes to progress through the cell cycle ......... 31 54 * *

Page 5.3.3. Optimal conditions to increase the proliferation of 5 B lymphocytes with anti-Bp50 antibodies ................ 33 5.3.4. Differences between anti-Bp50 and BCGF activity (weak) ...... 36 10 5.4. Use of anti-coordinates

Bp50 and Bp50 .................. 40 5.4.1. Bp50 receptor and uses of ligands such as anti-Bp50 to increase proliferation of B lymphocytes ........ 41 5.4.2. Modified coordinates used for immunosuppression or treatment of malignancies ...... 47 5.4.3. Other uses of ligands and Bp50 ..... 51 6. Deposit of cell lines ............ 51

Claims (67)

1. Virtually pure coordinate which (a) binds Bp50, namely a surface antigen of B lymphocytes at 50 kilodaltons, defined by the monoclonal antibody G28-5, and which (b) upon binding to a B lymphocyte activated, stimulates the activated B lymphocyte to bring it to cross the cell cycle in order to increase the proliferation of this B lymphocyte 2. Coordinate according to claim 1, 10 characterized in that it comprises an antibody molecule or an Fv, Fab, F (ab ') g or Fab' fraction of the antibody molecule. 3. The coordinate system of claim 2, wherein the antibody molecule comprises a monoclonal antibody molecule or an Fv, Fab, F (ab ') 2 or Fab' fraction of the monoclonal antibody molecule. 4. A monoclonal antibody molecule according to claim 3, characterized in that it comprises G28-5 or any Fv, Fab, F (ab ') 2 or Fab' fraction of G28-5. 5. A monoclonal antibody molecule according to claim 3, produced by a hybridoma cell line, deposited at ATCC under the order number HB9110, or one of its mutants, one of its recombinants * "56 or one of its derivatives belonging to genetic engineering. 6. Coordinate according to claim 1, characterized in that it comprises a lymphokine. 7. Lymphokine according to claim 6, 5 characterized in that it comprises a growth factor for B lymphocytes. 8. The coordinate system of claim 6, wherein the lymphokine is located on the surface of a cell. 9. Coordinate according to claim 1, 2 or 6, characterized in that it also comprises a compound coupled to be coordinat. 10. The ligand of claim 9, wherein the compound which is coupled thereto comprises an antiproliferative agent. 11. The coordinate of claim 9, wherein the compound which is coupled thereto comprises an alkylating agent. 12. A coordinate according to claim 9, wherein the compound which is coupled thereto comprises an antimetabolite. 13. Coordinate according to claim 9, in which the compound which is coupled thereto comprises an antibiotic. 14. Coordinate according to claim 9, in which the compound which is coupled thereto, comprises an alkaloid extracted from the periwinkle (Pervinca). 15. The coordinateate of claim 9, wherein the compound which is coupled thereto comprises an enzyme. 16. The coordinate according to claim 9, wherein the compound which is coupled thereto comprises a coordinated complex of platinum. 17. Coordinate according to claim 9, in which the compound which is coupled thereto comprises a * * 57 / radioisotope. 18. The coordinate of claim 9, wherein the compound which is coupled thereto comprises a fluorescent compound. 19. Virtually pure coordinate which binds to Bp50, namely a surface antigen of B lymphocytes at 50 kilodaltons, defined by the monoclonal antibody G28-5. 20. Coordinate according to claim 19, 10 characterized in that it comprises an antibody molecule or an Fv, Fab, F (ab ') 2 or Fab' fraction of the antibody molecule. 21. The coordinate of claim 20, wherein the antibody molecule comprises a monoclonal antibody molecule or an Fv, Fab, F (ab ') 2 or Fab ’fraction of the monoclonal antibody molecule. 22. Coordinate according to claim 19, characterized in that it comprises a lymphokine. 23. Lymphokine according to claim 22, characterized in that it comprises a growth factor for B lymphocytes. 24. Coordinate according to claim 19, 20 or 22, characterized in that it also comprises a compound coupled to be ligand. 25. Coordinate according to claim 24, in which the compound which is coupled thereto, comprises an antiproliferative agent, 26. The coordinate of claim 24, wherein the compound which is coupled thereto comprises an alkylating agent. 27. The coordinate of claim 24, wherein the compound which is coupled thereto comprises an antimetabolite. 28. Coordinate according to claim 24, * * 58 in which the compound which is coupled thereto, comprises an antibiotic. 29. The coordinate of claim 24, wherein the compound which is coupled thereto comprises an alkaloid extracted from periwinkle (Pervinca). 30 G28-5, such that activated B cells cross the cell cycle and proliferate. 30. The coordinate of claim 24, wherein the compound which is coupled thereto comprises an enzyme. 31. The coordinate of claim 24, wherein the compound which is coupled thereto comprises a coordinated complex of platinum. 32. Coordinate according to claim 24, in which the compound which is coupled thereto, comprises a radioisotope. 33. The coordinate of claim 24, wherein the compound which is coupled thereto comprises a fluorescent compound. 34. Superficial B cell antigen at 50 kilodaltons, which is defined by the monoclonal antibody G28-5. 35. A practically pure antigen at 50 kilodaltons according to claim 34, characterized in that it comprises a polypeptide. 36. Method for increasing the proliferation of B lymphocytes, characterized in that it consists in treating activated B lymphocytes, with an effective dose of a ligand which binds to Bp50, namely a surface antigen B cells at 50 kilodaltons, defined by the monoclonal antibody 37. The method of claim 36, wherein the B lymphocytes have been activated by treatment with an effective dose of a second co-ligand which binds to Bp35, namely a 35 kilodalton B cell surface antigen, of such that the B lymphocyte progresses from the GQ phase to the cell cycle phase. 38. Method according to claim 36 or 37, characterized in that it is carried out in vivo. 39. Method according to claim 36 or 37, characterized in that it is carried out in vitro. 40. The method of claim 36 or 10 37, wherein the ligand comprises an antibody molecule which binds to Bp50, or an Fv, Fab, F (ab ') 2 or Fab' fraction of the antibody molecule, which binds to Bp50. 41. The method of claim 36 or 15, wherein the ligand comprises a monoclonal antibody molecule which binds to Bp50, or an Fv, Fab, F (ab ') 2 or Fab' fraction of a molecule of monoclonal antibody, which binds to Bp50. 42. The method of claim 41, wherein the monoclonal antibody molecule comprises G28-5 or any Fv, Fab, F (ab ') 2 or Fab' fraction of G28-5. 43. The method according to claim 41, in which the monoclonal antibody is produced by a hybridoma cell line, deposited at ATGG under the order number HB9110, or one of its mutants, one of its recombinants or one of its derivatives belonging to genetic engineering. 44. The method of claim 36 or 30, wherein the ligand comprises a lymphokine. 45. The method of claim 44, wherein the lymphokine comprises a B cell growth factor. 46. The method of claim 44, wherein the lymphokine is located on the surface of a cell. 47. The method of claim 37, wherein the second ligand comprises an antibody molecule which binds to Bp35. 48. The method of claim 47, wherein the antibody which binds to Bp35, further comprises a monoclonal antibody. 49. The method of claim 37, wherein the activated B cells are treated with the ligand which binds to Bp50, approximately within 12 hours of activation by the second ligand which binds to Bp35. 50. A method for suppressing the proliferation of cells which express Bp50, namely a surface antigen of B lymphocytes at 50 kilo-daltons, defined by the monoclonal antibody G28-5, characterized in that it consists in treating cells which express Bp50, with an effective dose of a ligand which binds to Bp50, this ligand being coupled to an antiproliferative agent. 51. The method of claim 50, wherein the cells which express Bp5Q comprise -B lymphocytes. 52. The method of claim 50, wherein the cells which express Bp50 include malignant cells. 53. Method according to claim 50, characterized in that it is carried out in vivo. 54. The method of claim 50, 30 characterized in that it is carried out in vitro. 55. The method of claim 50, wherein the ligand comprises an antibody molecule or an Fv, Fab, F (ab ') 2 or Fab' fraction of the antibody molecule. Λ * 61 56. The method of claim 55, wherein the antibody molecule comprises a monoclonal antibody molecule or an Fv, Fab, FCab'ig or Fab 'fraction of the monoclonal antibody molecule. 57. The method of claim 56, wherein the monoclonal antibody comprises G28-5 or any Fv, Fab, Fiab ') ^ or Fab' fraction of G28-5. 58. The method of claim 56, wherein the monoclonal antibody is produced by a hybridoma cell line, deposited at ATCC under the order number HB9110, or one or other of its mutants , its recombinants or its derivatives belonging to genetic engineering. 59. The method of claim 50, wherein the ligand comprises a lymphokine. 60. The method of claim 59, wherein the lymphokine comprises a B cell growth factor. 61. The method of claim 50, 55 or 59, wherein the antiproliferative agent comprises an alkylating agent. 62. The method of claim 50, 55 55 or 59, wherein the antiproliferative agent com takes an antimetabolite. 63. The method of claim 50, 55 or 59, wherein the antiproliferative agent comprises an antibiotic. 64. The method of claim 50, 55 or 59, wherein the antiproliferative agent comprises an alkaloid extracted from periwinkle (Pervinca). 65. The method of claim 50, 55 or 59, wherein the antiproliferative agent comprises an enzyme. \ 62 A * ( 66. The method of claim 50, 55 or 59, wherein the antiproliferative agent comprises a coordinated complex of platinum. 67. The method of claim 50, 555 or 59, wherein the antiproliferative agent comprises a radioisotope.