BE1000587A4 - Ligands AND METHODS TO INCREASE THE PROLIFERATION OF CELLS B. - Google Patents

Abstract

New B cell receptor, namely Bp50, which is a 50 kilodalton polypeptide and which is involved in the proliferation of B cells. It is possible to use ligands such as lymphokines, antibody molecules or Fv fragments of molecules antibodies, which bind to Bp50 and which increase the proliferation of activated B lymphocytes, in order to regulate the proliferation or differentiation of B lymphocytes

Description

       

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  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 lymphocytes, designated in this specification by l Bp50, which is involved 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 of 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 the proliferation of active B cells. 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 regulate the proliferation and / or differentiation of human B lymphocytes. Furthermore, the ligands of the present invention can be modified by the attachment of other compounds which can be used in the treatment

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 and / or the detection of malignant cells which express the Bp50 antigen.



  2. OF THE INVENTION
The activation of resting B cells from the Go phase to the G1 phase of the cell cycle and the subsequent initiation of the proliferation of activated B cells are distinct steps requiring equally distinct regulatory mechanisms. Certain agents, in particular factor-pl stimulating murine B lymphocytes (BSF-pl) (Rabin et al., 1985, Proc. Nat.



  Acad. Sei. USA 82.2935-2939) or low doses of anti-immunoglobulin (anti-Ig) (DeFranco, et al., 1985, J. Immunol. 135: 87-94; Wetzel et al., 1984,
 EMI2.1
 J. Immunol. 133 et al., 1982, J. et al., 1984, J. Immunol. 132 are factors of "activa-: 2327-2332; DeFrancotion" or "competence". In other words, these agents cause the B cells to grow, & synthesize more ribonucleic acid and enter G1, 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. 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; 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

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 different sources, in particular hybridomas or T lymphocyte lines, Bou lymphocyte lines of dendritic cells. Although both interleucin-1 (IL-1) and interleucin-2 (IL-2) have been shown to increase growth of B cells, they are apparently distinct from some BCGFs.

   For example, monoclonal antibodies (mAbs) to a murine BCGF (O'Hara et al., 1985, Nature (Lond.) 315: 333) or a BCGF
 EMI3.1
 human (Ambrus et al., 1985, J. Exp. Med. 162 block the activity of BCGF, but not the activity of IL-1 or IL-2. Although they are distinct: 1319) of IL -1 or IL-2, the BCGFs themselves seem to be heterogeneous on the basis of biochemical data and of differential activity on different subsets of Bou Lymphocytes with different co-stimulation dosages. 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 lymphocytes and cause the differentiation of IgG-producing cells. ; therefore, it differs from human BCGF (high) and BCGF (low) in both its molecular weight
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 and its range of activities.



  These activation and growth signals probably ensure the regulation of cells by interaction with specific surface structures.

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 EMI4.1
 several B cell lymphocytes In addition to the specific signal for the antigen passing through superficial Ig, several other superficial B polypeptide candidate polypeptides have been identified which, in some way, can act in the activation or growth of B lymphocytes. 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 IL-2 receptors have been identified on B lymphocytes (Zubler 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) have found that monoclonal antibodies (mAb) directed against the Lyb2 antigen. murine B cells activated B cells and, recently, there has been evidence 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 phase G0 to phase G1 (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 proliferation of B lymphocytes which are dependent on T lymphocytes (Melchers et al., 1985,

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 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, 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 antiserum 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 antigp54 or AB-1 recognizes a BCGF receptor.



    3. SUMMARY OF THE INVENTION
 EMI5.1
 The present invention relates to ligands which (a) bind to Bp50, namely a 50 kDa lymphocyte 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 active B lymphocytes. Furthermore, the invention relates to ligands which bind to Bp50, but which do not exercise any function or any biological effect such as an 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 binding site of the antigen or antibodies and fragments

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 chemically modified; these fragments include, without any limitation, Fv, Fab, F (ab '), 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 ensure the direction, regulation and modification of the proliferation and / or differentiation of human B lymphocytes.



   The present invention is based on the discovery that two differentiating antigens for human B cells, namely Bp35 and the B cell antigen described herein, namely Bp50, apparently play distinctive roles as signal receptors in the activation of lymphocytes. B. Monoclonal antibodies (mAbs) against Bp35 and Bp50 both send positive signals to B cells, thereby stimulating their transition through the cell cycle. Like anti-Ig antibodies, mAb directs against Bp35 works primarily to activate resting B cells so that they become competent to enter the G phase of the cell cycle.

   In contrast, a monoclonal antibody of the type described here or its F (ab ') - fragment directs against Bp50, namely a poly-

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 50 kDa AL peptide expressed on all B lymphocytes, acts to stimulate active lymphocytes 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 B lymphocytes
 EMI7.1
 Tons of lymphocytes and cause proliferation of lymphocytes On the other hand, by itself, the anti-Bp50 monoclonal antibody does not activate B lymphocytes, any more than it causes proliferation of B lymphocytes But, together with anti-Bp35 or anti -Ig, it increases the proliferation of B lymphocytes. In this regard, the action of the anti-Bp50 antibody resembles the activity of growth factors of B lymphocytes (BCGF). As little as 0.05 fg / 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 antiBp35 and anti-Bp50 antibodies together cause a strong proliferation of Bpurified delyhocytes at rest. These results suggest that the surface molecules of Bp35 and Bp50 are involved in controlling the regulation of the activation and progression of 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 and BCGF (weak) are both co-stimulants with

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 the same agents, but not with each other and since anti-Bp50 and BCGF (weak) both influence only activated B cells and act in a soluble form, the activity of anti-Bp50 can be distinguished that of BCGF (weak), since both the proliferation of B lymphocytes stimulated with optimal amounts of anti-Bp50 and anti-Bp35 (or anti-Ig) can be increased further with BCGF (weak) and qu both blood B cells and
 EMI8.1
 certain lymphomas of anti-Bp50 lymphocytes with respect to 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) can regulate the growth of B lymphocytes but, apparently, they behave in this way AT 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" to increase an immune response to a vaccine. Alternatively, these ligands can be used to increase the immune response of an individual with immunosuppression.



   In another embodiment, the ligands of the invention can be chemically altered such that the cells to which

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 bind the coordinates, be killed. Since all B cells express 11 Bp50 antigen, this method should result in the suppression of the immune response. For example, a cytotoxic drug linked to a ligand of the present invention can be used in vivo to induce immunosuppression in order to cross the histocompatibility barriers in patients having received a transplant; alternatively, these modified ligands can be used to combat autoimmune diseases.



   In another embodiment of the present invention, malignancies such as tumor cells expressing Bp50, 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 lymphocytes, there should be a particular advantage when treating malignancies of the lymphocytes. When the chemotherapeutic agent linked to the ligand is a more effective agent for killing the cells which proliferate; 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 that express the Bp50 antigen and / or to determine, in the humors of the organism, the presence of the Bp50 antigen which can be lost

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 or not. In addition, the ligands of the invention can be used in vivo to visualize cells or tumors which express the Bp50 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 = Lyrnphocyte B Growth Factor
 EMI10.1
 BCGF (high) human at 60 kDa = BCGFBCGF (weak) = human BCGF at 12 kDa Bp35 = Superficial polypeptide (CD20) specific for 35 kDa lyrnphocytes B, defined by mAb 1F5 Bp50 = Superficial polypeptide specific for 50 kDa B lymphocytes, defined by mAb G28-5 Fv = Variable region or antigen binding site of an antibody molecule.
 EMI10.2
 



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



  IF = Immunofluorescence Ig = Immunoglobulin
 EMI10.3
 IL-1 =
Interleucine lIL-2 = Interleucine 2 kDa Kilodalton

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 mAb = Monoclonal antibody SDS-PAGE = Electrophoresis on polyacrylamide gel / sodium dodecyl sulfate TPA = 12-0-tetradecanoylphorbol-13 acetate.



    4. DESCRIPTION OF THE FIGURES
Figure 1. The expression of Bp50 is restricted to Bp35 B lymphocytes. The two-color flow cytometric analysis of 50,000 lynphocytes was carried out as described (Clark et al., 1985, Proc. Nat.



  Acad. Sci. 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 (lF5) staining against FITC (green) -anti-Bp50 (G28-5) shows that all the Bp50 + cells are also Bp35 +.



   Figure 2. Biochemical comparison of the Bp50 polypeptide with other surface antigens of B lymphocytes. Bp50 immunoprecipitation of 125 labeled 1 surface tonsil cells 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.
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  Table A: column 1, (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, Sepharose-goat anti-mouse Ig only. We

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 chose 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 A Bp50. One in three 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); HB10a (anti-DR); and 9, 6 (anti-CD2, E receptor). 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.

   Unstained cells are placed on the back of the grid; red fluorescence is on the right and green fluorescence on the left.



   Figure 4. Dose response curves for increased proliferation of dense Er-tonsill B lymphocytes by anti-
 EMI12.1
 Bp50 as indicated: media only; anti-Bp50 only plus anti-Bp35 (Spg / ml) only; BCGF only; anti-Bp35 plus BCGF; anti-Bp35 plus graduated doses of anti-Bp50. The average standard error proliferation of four samples was measured on day 3.



   Figure 5. Anti-Bp50 mAbs are most effective in increasing proliferation if they are added after a B-cell activation signal. Dense Er-tonsil B cells are incubated for 4 days with unique backgrounds

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 ment, anti-Bp50 (0.5 g / ml) was added at different times after incubation, anti-Bp35 (5 rg / ml) was added at different times after incubation; anti-Bp50 was maintained at a constant value then, later, at different times, antiBp35 was added thereto; anti-Bp35 was kept constant and then, at different times, anti-Bp50 was added to cultures. In the past 10 hours,
 EMI13.1
 Q 3H-thymidine was added and its incorporation was measured.



   Figure 6. Comparison of anti-aging ability
 EMI13.2
 bring resting tonsil lymphocytes out of the Go stage of the cell cycle.



  Day 3 after treatment, media only. Bp35 and, from anti-Bp50 to (), anti-Bp35 only (-----); and anti-Ig only (.....), A, no additional additives; B, anti-Bp50 (0.5 pglml) was added to each group C, 5% of BCGF was added to each group. The data are shown in the form of diagrams 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 against BCGF. Dense E-tonsil 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.



  The proliferation was measured on the days indicated, by
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 g an 18-hour pulse of 3H-thymidine. The proliferation in quadruplication was measured and the standard errors are indicated. One in three experiences.

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  Figure 8. Time elapsing with anti-Bp35 when (A) or BCGF (B) increases proliferation optimally. Dense E-tonsill B cells were stimulated as indicated and pulse proliferation was measured
 EMI14.2
 3 of 6 p.m. of 3H-thymidine on day 3. Anti-Bp35 media only added at the times indicated; antiBp50 or BCGF only; anti-Bp35 added at the start of the culture, then addition of anti-Bp50 or BCGF at the times indicated; anti-Bp50 or BCGF adds at the start of the culture, then addition of anti-Bp35. One in two experiences. The proliferation was measured in quadruplication and the standard errors are indicated.



  Doses used: anti-Bp35, 5 ug / ml; anti-Bp50,
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 Ot2 BCGF (weak) 5%. The concentrations used were as follows: anti-Bp35, 5 μg / ml anti-Bp50, 0,; 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 + antiBp50; or anti-Bp50 + anti-Ig. The proliferation was measured on day 3 after stimulation with a
 EMI14.4
 g 18 hour pulse of 3H-thymidine. The proliferation in quadruplication was measured and the standard errors are indicated. One in four experiences.
 EMI14.5
 Doses used for cells. : anti-Bp35,5? ; anti-Bp50, 0,; anti-Ig beads, 50 Figure 10. Comparative effects of anti-Bp50 and BCGF on normal and malignant B lymphocytes.



  Peripheral blood E-B lymphocytes (A) or dense tonsillary E-B lymphocytes (C) were stimulated with or without TPA (75 ng / ml) in the presence of 10%

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 BCGF or 1 pglml of anti-Bp50. Two separate delymphocyte B lymphomas (Tables B and D) were stimulated in the same way. The proliferation was measured on day 3 by incorporation of 3H-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
 EMI15.1
 which (a) bind to Bp50, which is a surface polypeptide for 50 kDa lymphocytes, and which (b) increase the proliferation of active delymphocytesB.



  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. In addition, the invention relates to ligands which bind to Bp50, but which neither exert a nor a biological effect such as an increase in the proliferation of activated B lymphocytes.



   The ligands of the present invention include antibody molecules, monoclonal antibody molecules and fragments of these d-molecules! antibodies, which contain the binding site of the antigen binding to the Bp50 receptor, including antibodies and chemically modified fragments; these fragments include, without any limitation, Fv, Fab, F (ab '), 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, BCGF, as well as chemically modified lymphokines and the like. The ligands of the invention can be used in their modified or unmodified forms to ensure the modulation and regulation of immune responses, as well as in the

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 therapy for malignancies expressing the Bp50 antigen.



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



   When the ligand is a monoclonal 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 hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256: 495-497), as well as other techniques that have been made more recently available such as the lymphoma hybridoma technique
Human B (Kozbor et al., 1983, Immunology Today 4:72) 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 idiocy 'of the molecule could be generated by known techniques. For example, such fragments include, without any limitation: the F (ab ') fragment which can be generated by treating the antibody molecule with pepsin; Fab 'fragments which can be generated by reducing the disulfide bridges of the F (ab') p fragment; the F (ab ') 2 fragment which can be generated by treating the antibody molecule with papain; and the 2Fab or Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent to reduce the disulfide bridges.



   When the ligand which binds to Bp50, is a lymphokine, this can be obtained from natural sources or from an amino acid sequence.

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 is known or inferred, 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 ensuring transcription and translation of the gene sequence in a host cell appropriate.



   Depending on its intended use, the ligand or appropriate 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 limitation, the use of carbodiimide, cyanogen bromide, difunctional reagents such as glutaraldehyde, N-sueeinimidy 3- (2-pyridyldithio) propionate (SPDP), base reactions of Schiff, the binding to sulhydryl fractions, the use of sodium isothiocyanate or an enzymatic bond, to name but a few.

   When a radioisotope must be attached to the ligand, it is also possible to use enzymatic means, oxidative substitution, chelation, etc. 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 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 AL Bp50 bond. To this end, the ligand binding site could be protected before carrying out the reaction.

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 of coupling. 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 ligand / Bp50 complex. 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 includes 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 section 5.3.3 below), it may be advantageous to direct coupling of the desired compounds to the Fc domain of the molecule.



   The following subclauses describe the new superficial marker for 50 kDa B cells, namely Bp50, which apparently acts in the proliferation of B cells, as well as the ligands which bind to the new 50 kDa receptor, as well as their
 EMI18.1
 uses. To give an example of the ligands of the present invention, a monoclonal antibody which defines Bp50 is also described, as well as its F (ab ') 2 fragments which, like BCGF, increase the proliferation of B lymphocytes. Unlike anti-Bp35 mAb which can cause B lymphocytes at rest in the GO phase, to enter the G1 phase, anti-Bp50 mAb does not activate the B lymphocytes 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

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 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 may be a receptor for a separate 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 RECEPTOR
Bp50
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 of 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.

   The monocytes were removed by incubation on plastic Petri dishes once or twice at 370C for 45 minutes. unless otherwise stated. Fractions of floating or dense tonsillary B cells were isolated by step gradients of Percoll 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% sIg + Bp35 + cells. Preparations enriched with blood B cells had 60-85% sIg + cells.



  B lymphocyte cells were isolated by moderately dissociating lymphoid cells

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 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 (Kohler 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 comprising an antibody giving histogram plots similar to those of known mAbs directed against pan-markers of B lymphocytes (for example, Bp35) were cloned and selected for further studies. The G28-5 clone produced a GI 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 et al., 1986, Human Immunol. 15: 30-44; Ledbetter et al., 1985, in Perspective in Immunogenetics
 EMI20.1
 and HistOcOm2atibiLLtand Histocompatibility, ASHI, New York, 6, pages 325-340. These monoclonal antibodies include 1F5) anti-Bp35, HbIlOa), 2C3 (igG) anti-chalne f'G19-4 FC-2 (igG) of Fc CD16 and aa (IgG-anti-CD2 (receptor E) supplied by the Doctor Paul Martin (Martin et al., 1983, J. Immunol. 131: 180).



  The IgG1 mAbs were purified by precipitation using 45 or 50% saturated ammonium sulfate

  <Desc / Clms Page number 21>

 
 EMI21.1
 and column chromatography of DEAE-Sephacryl and the mAbs of IgG-were purified using columns of Sepharose coated with Protein A. The fragments F (ab ') 2 of G28-5 were prepared by Parham's method (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 and al., 1985, J. Immunol.



    135: 1819). MAb 2C3 directed against 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 (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 active 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 an AL filter short passage 540 (Ditric Optics, Hudson, MA) in front of the photo tubes

  <Desc / Clms Page number 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.



  For each two-color staining, data from 40,000 cells were collected and stored on floppy disks. The data is presented in the form of a number of cells (vertical) with respect to the logarithm of green fluorescence with respect to the logarithm of red fluorescence on a grid at 64 x 64 points. A value of about 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 Immunogenetics and Histocompatibility, ASHI, New York, 6, 119-129 and 325-340 .



    Cell Culture: Anygdalian lymphoid cells or blood were grown at 5-
 EMI22.1
 5 10 x ml in quadruplication in 96-reservoir microtiter plates containing 200 μl of RPMl-1640 medium supplemented with 15% fetal bovine serum, antibiotics, glutamine and pyruvate (R15). After 1 to 7 days, cells were pulsed
 EMI22.2
 Q with 0, 3H-thymidine per reservoir (New England Nuclear, 6, 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 experiences:

  <Desc / Clms Page number 23>

 
 EMI23.1
 proliferation was measured on day 3.



  Factors We got a purified BCGF 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 resided 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 a
 EMI23.2
 hydroxylapatite column chromatography. IL-1 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 was kindly provided by "Cetus Corporation". TPA (12-0-tetradecanoyl-phorbol-13 acetate) was supplied by "Sigma".



   Detection of cell activation.



  Caused changes in cell volume were measured by mAb and / or factors using. a cell selector and a movable spot detector at a forward angle. Cell cycle changes were measured in ribonucleic acid and deoxyribonucleic acid contents by staining activated cells with acridine orange and measuring the relative ribonucleic acid (red) and deoxyribo- acid contents. nucleic (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

  <Desc / Clms Page number 24>

 cells were checked using mAb directly conjugated with fluorescein, then quantified by direct immunofluorescence levels with an Epics V cell selector.
 EMI24.1
 



  Munoprecipitation characterization of Bp50 out of surface labeled 125I tonsillar cells was performed 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 -70oC and lightening screens plus Cronex reinforcement (Dupont).



     5. 2. CHARACTERIZATION OF THE Bp50 RECEIVER
The following sub-paragraphs describe the results of the experiments carried out by adopting the methods described above.



     5. 2. 1. IDENTIFICATION OF A SURFACE CELL MARKER at 50 kDa. SPECIFIC TO LYMPHOCYTES
 EMI24.2
 B, Bp50
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 IgGl mAb that did not contain the NS-1 light chain. Upon careful examination by immunofluorescence analysis, it was found that G28-5 reacted only with B or normal or malignant B lymphocyte lines. A very wide selection of normal tissues by established methods (Clark, et al., 1985, Proc.



  Nat. Acad. Sei. EUA 82: 1766-1770; Ledbetter et al., 1986, Human Immunol. 15: 30-44; Ledbetter, 1985, in Perspectives in Immunogenetics and Histocompatibility, ASHI, New York, 6, pages 325-340) revealed that the anti-

  <Desc / Clms Page number 25>

 body G28-5 reacted with negative cells forming E (Er-) rosettes from blood or tonsils, but not with T lymphocytes not adhering to nylon wool, T cell blasts caused by PHA (= phytohemagglutinin) or with blood granulocytes, monocytes, red blood cells or platelets.

     11 reacted strongly with all seven B lymphoblastold cell lines tested and with three Burkitt lymphoma lines (Raji, Daudi, Namalwa), but not with four T lymphocyte 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) lymphocytic leukemias acute non T, non B CALLA + expressed Bp50.



   The restricted distribution of Bp50 on normal tissues was further confirmed by quantitative two-color immunofluorescence (two-color IF) analyzes. By using an antibody (red) conjugated to R-phycoerythrin (PE) and directed against the pan-antigen Bp35 of B lymphocytes (bol, CD20) and the antibody (green) anti-Bp50 conjugated by fluorescein, the Applicant has finds that Bp50 was expressed only on Bp35 + B cells (Figure 1) in the blood or tonsils. B cells in the blood logically expressed Bp50 at somewhat lower concentrations than those in tonsill B cells, which is similar to the expression of HLA-DR (Ledbetter et al., 1986, Human Immunol.



  15: 30-44) and the expression of gp54 (Wang, et al., 1979, J. Exp. Med. 149: 1424-1433), which are also weaker on blood B lymphocytes. Bp50 has been expressed at similar concentrations in

  <Desc / Clms Page number 26>

 sub-populations of tonsill B lymphocytes separated on Percoll gradients in floating and dense fractions. Using the mAb conjugates to PE and directed against the marker for T lymphocytes, CD3 (T3) and the marker associated with NK cells,
CD16 (Fc receptor) (Ledbetter, et al., 1979,
Immunol. Rev. 47: 63-82), the Applicant has found that Bp50 was not expressed on T lymphocytes or NK cells.

   Using bi-color immunofluorescence, the Applicant has also found that CD3 + PHA blasts which have 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 around 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, 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 - Leukocy. Te Typing II, eds. Reinherz, et al., Springer Verlag, Berlin, chapter 12, volume 2, 155-167;
 EMI26.1
 Slovin, et al., 1982, Proc. Nat. Acad. Sei. EUA 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 B cell markers can be easily compared with Bp50. Contrary to Bp50, the marker Bp39 is expressed on granulocytes and, unlike Bp50, Bp45 is
 EMI26.2
 restricted to B cell blasts. Antibodies to Bp39 (41-H16) and Bp45 (MNM6, baste-1, blaste-2) supplied by an international working group

  <Desc / Clms Page number 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 fluorescein-treated anti-Bp50 antibodies to B cells. Therefore, based on the distribution of tissues, biochemical analysis and blocking studies, the monoclonal antibody G28-5 recognizes a structure at 50 Kd distinct from that of other known antigens of B lymphocytes.



   5. 2. 2. THE EXPRESSION OF Bp50 IS RESTRICTED TO LYMPHOCYTES B.



   Distribution studies of lineages of
 EMI27.1
 hematopoletonic cells and tissues, as well as detailed two-color flow cytometric analyzes found that Bp50 was expressed only on B cells. As shown in Figure 3, Bp50 is expressed on a small subset of blood lymphocytes and a large population tonsillar lymphocytes. Virtually all Bp50 + lymphocytes in both blood and tonsils also expressed Bp35 and HLA-DR, but they did not express the T-cell molecules, CD2 (Figure 1) or CD3, nor did the Fc d receptors 'IgG found on NK cells. In addition, CD3 + T cell blast blasts activated by ConA expressed IL-2 receptors, but not Bp50.



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



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

  <Desc / Clms Page number 28>

 
 EMI28.1
 Bp35 elevations (Ledbetter, et al., Human Immunol. Figure 3 shows that both subsets of IgM-positive and IgM-negative B cells expressed Bp50 in equal amounts, indicating that Bp50 is expressed both on resting B cells and activated B cells 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 antibodies specific to anti-u chains.



  Recently, the Applicant has discovered that the marker Bp35 (B1) specific for B lymphocytes, AL, namely a polypeptide at 35 kDa, could also fulfill its function during the early activation of B lymphocytes: just like low doses of anti -, mAb 1F5 directs 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 was 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-u antibodies (Table 1).



  Under appropriate conditions only, anti-Bp35 antibodies in solution or anti-pu antibodies attached to Sepharose beads could stimulate some proliferation of B lymphocytes (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 cultivated
 EMI28.2
 with anti-u beads or with anti-Bp35. In this regard, I

  <Desc / Clms Page number 29>

 anti-Bp50 looked like BCGF (Table 1, line 3).



  Therefore, it was important to determine whether antiBp50 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 anti-activated cells? or anti-Bp35 to a degree somewhat higher 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 doses in the range in the range of 0.1 to 10 pglml.
 EMI29.1
 TABLE 1 Increase in proliferation of B lymphocytes, caused by anti-Ig or anti-Bp35, with anti-Bp50 antibodies
 EMI29.2
 
 <tb>
 <tb> Proliferation <SEP> medium <SEP> error <SEP> type <SEP> from
 <tb> lymphocytes <SEP> B <SEP> cultivated <SEP> with <SEP>:

    <SEP>
 <tb> Lineage <SEP> Co-sti-Pearls <SEP> Anti-Bp35 <SEP>
 <tb> mulating <SEP> Backgrounds <SEP> anti
 <tb> 1 <SEP> None <SEP> 1. <SEP> 212 + 547 <SEP> 10. <SEP> 219 + 462 <SEP> 5. <SEP> 539 + 308 <SEP>
 <tb> 2 <SEP> Anti-Bp50 <SEP> 719 + 718 <SEP> 38. <SEP> 792 + 1. <SEP> 329 <SEP> 25. <SEP> 465 + 616 <SEP>
 <tb> 3 <SEP> BCGF <SEP> 456 + 217 <SEP> 14. <SEP> 217 + 445 <SEP> 9. <SEP> 443 + 343 <SEP>
 <tb> 4 <SEP> Anti-Bp50
 <tb> + <SEP> BCGF <SEP> 1. <SEP> 456 + 126 <SEP> 54. <SEP> 393 + 2. <SEP> 537 <SEP> 46. <SEP> 488 + 3. <SEP> 387 <SEP>
 <tb>
 
 EMI29.3
 The proliferation of Erdenses tonsillary B lymphocytes (95% of superficial IgM + cells) was measured on day 3, as described.

   In summary, we have
 EMI29.4
 5 cultivates 2 x cells / 200 PI reservoir in quadruplication for 48 hours with the medium

  <Desc / Clms Page number 30>

 RPMI 1640 containing 15% fetal bovine serum plus additives without antibody, or with the monoclonal antibody 2C3 directed against u chains, coupled
 EMI30.1
 Sepharose pearls ("anti-u pearls", 50 pg / ml) or the free anti-Bp35 1F5 antibody (5 pglml). Cultures containing media, “anti-u” or anti-Bp35 beads were grown alone or with BCGF (final concentration: 5%, Cytokine Technology, Buffalo, New York; no detectable activity of IL-1 or IL- 2), with anti-Bp50 (dilution 1: 1,000 of ascites) as co-stimulants.

   After 40 hours, we pulsed
 EMI30.2
 g cells with 3H-thymidine and the incorporated counts were measured after 18 hours.



     5. 3. 1. mAb ANTI-Bp50 INCREASES PROLIFERATION ONLY AFTER B LYMPHOCYTES HAVE BEEN ACTIVATED BY ANTI-Bp35 or ANTI-u 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 µg and 2.0 µt / ml had no effect on the binding of 3H-thymidine. However, in the presence of optimal anti-Bp35 mAb concentrations, a concentration as low as 0.1 to 0.5 rg / ml of 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 pglml) were less effective than doses in the range of 100-200 ng.



   These results suggested that anti-Bp50

  <Desc / Clms Page number 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. However, when cells 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 anti-u was used rather than antiBp35.



     5. 3. 2. THE ANTI-Bp50 mAbs DO NOT ACTIVATE GOt B-LYMPHOCYTES BUT THEY ACTIVATE B-ACTIVE LYMPHOCYTES TO PROGRESS THROUGH THE CELL CYCLE.



   Previously, the Applicant has found that, just like low doses of anti-u, anti-Bp35 antibodies causes tonsil B lymphocytes at rest in the GO phase to enlarge (Clark, et al., 1986, Leukocyte. Typing 11 eds. Reinherz, et al., Springer Verlag, Berlin, volume 2,455-462) and to enter phase G. of the cell cycle (Gollay et al., 1985, J. Immunol. 135: 3795-3801). Therefore, it was interesting to compare the faculties of mAb antiBp50 and mAb anti-Bp35 concerning their effects on the activation of B lymphocytes.

   As indicated in FIG. 6A, even after 3 to 4 days in culture, dense unstimulated tonsill B lymphocytes had a uniform profile of ribonucleic acid, characteristic of cells in the Go phase (Darzynkiewicz, 1980, Proc. Nat. Acad. Sci. EUA 77: 6697-6702). However, approximately 15-30% of cells stimulated with anti-Bp35 or anti? had a higher content

  <Desc / Clms Page number 32>

 in ribonucleic acid, thus indicating their entry into phase G On the other hand, neither anti-Bp50 (FIG. 6B), nor BCGF (FIG. 60 alone did not cause significant numbers of B lymphocytes to enter into phase G. By example, 2 days after activation, anti-Bp35 and anti-Ig mAbs caused 13.5 and 20.9% of tonsil cells respectively to enter the G1 phase,

   while cells treated only with anti-Bp50 (2.7%) or BCGF (3.2%) remained at concentrations of control media (2.2%). However, when anti-Bp50 or BCGF was added together with antiBp35 or anti-n antibodies, the proportion of cells entering the G phase increased considerably. Similarly, anti-Bp50 and BCGF alone did not cause B lymphocytes to enter the S phase (Table 2) but, together with anti-Bp35 or antiJP, i15 increased two to three times the number of cells in phase S.

  <Desc / Clms Page number 33>

 



    TABLE 2 Effect of anti-Bp50 and BCGF on the progression of the cell cycle in tonsil lymphocytes.
 EMI33.1
 
 <tb>
 <tb>



  Signal <SEP> from <SEP> Signal <SEP> from <SEP> Cells, <SEP>%
 <tb> competence <SEP> progression <SEP> G0 <SEP> G1 <SEP> S / G2 / M
 <tb> Backgrounds <SEP> None <SEP> 89, <SEP> 9. <SEP> 7, <SEP> 1 <SEP> 2, <SEP> 5 <SEP>
 <tb> anti-Bp35 <SEP> " <SEP> 80.4 <SEP> 14.5 <SEP> 3.7
 <tb> anti-Ig "65, <SEP> 6 <SEP> 27, <SEP> 6 <SEP> 5, <SEP> 7 <SEP>
 <tb> Backgrounds <SEP> anti-Bp50 <SEP> 83, <SEP> 6 <SEP> 12, <SEP> 0 <SEP> 3, <SEP> 3 <SEP>
 <tb> anti-Bp35 <SEP> " <SEP> 54.1 <SEP> 35.5 <SEP> 9.7
 <tb> anti-Ig <SEP> " <SEP> 43.6 <SEP> 36.2 <SEP> 16.2
 <tb> Backgrounds <SEP> BCGF <SEP> 85, <SEP> 4 <SEP> 11, <SEP> 7 <SEP> 2, <SEP> 2 <SEP>
 <tb> anti-Bp35 <SEP> " <SEP> 56.6 <SEP> 32.6 <SEP> 11.6
 <tb> anti-Ig <SEP> " <SEP> 48.4 <SEP> 36.1 <SEP> 14.1
 <tb>
 
 EMI33.2
 Percentage of cells in phases G, or S and G,

   determined by adopting the acridine orange staining method (Darzynkiewicz, et al., 1980, Proc. Nat. Acad. Sci. EUA 77: 6697-6702) i 1 × 10 6 dense tonsillar lymphocytes with anti-Bp35 ( 5 fg / ml), anti? on pearls (50 pg / ml), anti-Bp50 (0.4 g / ml), BCGF (5%) or their combinations, as indicated.



   5. 3. 3. OPTIMAL CONDITIONS TO INCREASE THE PROLIFERATION OF B LYMPHOCYTES WITH ANTI-Bp50 ANTIBODIES
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-Bp50 mAb markedly increased proliferation. Anti-Bp50

  <Desc / Clms Page number 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 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 resting cells.



    TABLE 3 Increased proliferation of B cells with anti-Bp50 antibodies or a B cell growth factor
 EMI34.1
 
 <tb>
 <tb> Proliferation <SEP> medium <SEP>! <SEP> error <SEP> type <SEP> from
 <tb> lymphocytes <SEP> B <SEP> cultivated <SEP> with <SEP>:

    <SEP>
 <tb> Co-stimulant <SEP> Backgrounds <SEP> Anti-Bp50 <SEP> BCGF
 <tb> (200 <SEP> ng / ml) <SEP> (5%)
 <tb> None <SEP> 96 <SEP> + <SEP> 1 <SEP> 267 <SEP> + <SEP> 15 <SEP> 285 <SEP> + <SEP> 74
 <tb> Anti-Ig <SEP> 5. <SEP> 833 <SEP> + <SEP> 391 <SEP> 41. <SEP> 634 <SEP> + <SEP> 2. <SEP> 103 <SEP> 25. <SEP> 094 <SEP> + <SEP> 61
 <tb> Anti-Bp35
 <tb> (5 g / ml) <SEP> 457 <SEP> # <SEP> 45 <SEP> 8.143 <SEP> # <SEP> 280 <SEP> 1,733 <SEP> # <SEP> 32
 <tb> TPA <SEP> (2 <SEP> ng / ml) <SEP> 7. <SEP> 361 <SEP> + <SEP> 537 <SEP> 21. <SEP> 163 <SEP> + <SEP> 871 <SEP> 13. <SEP> 064 <SEP> + <SEP> 1. <SEP> 030 <SEP>
 <tb> 11. <SEP> -1 <SEP> (10 <SEP> V / ml) <SEP> 264 <SEP> + <SEP> 2 <SEP> 308 <SEP> + <SEP> 23 <SEP> 221 <SEP> + <SEP> 8
 <tb> IL-2 <SEP> (100 <SEP> U / ml) <SEP> 204 <SEP> + <SEP> 34 <SEP> 350 <SEP> + <SEP> 7 <SEP> 220 <SEP> + <SEP> 11.
 <tb>



  BCGF <SEP> (5%) <SEP> 220 <SEP> + <SEP> 7 <SEP> 851 <SEP> + <SEP> 28 <SEP> 270 <SEP> + <SEP> 18
 <tb>
 
 EMI34.2
 Dense Er-tonsillar B cells (more than 95% of sIgM cells) for 48 hours at 2 x cells / reservoir, then by pulse 3 for 24 hours with 3H-thymidine before counting.

  <Desc / Clms Page number 35>

 



   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 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 fg anti-Bp50 antibody increased proliferation.



  In subsequent studies, an optimal dose of 0.3 fg / ml was used. Logically, it was observed that 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 fg / 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; 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 anti-Bp50 F (ab ') 2 fragments. In a hundredfold assay interval, F (ab ') fragments were clearly as effective, if not more effective, than a whole antibody in increasing the proliferation of B lymphocytes (Table 4). Therefore, the Fe 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, like BCGF, anti-Bp50 can apparently act as a soluble mediator without calling on accessory cellular function with the Fc receptor as mediator.

  <Desc / Clms Page number 36>

 
TABLE 4 The Fc domain of anti-Bp50 antibodies is not required to increase the proliferation of B lymphocytes
 EMI36.1
 
 <tb>
 <tb> Proliferation <SEP> medium <SEP> of
 <tb> lymphocytes <SEP> B <SEP> cultivated <SEP> with <SEP>:

    <SEP>
 <tb> Anti-Bp50 <SEP> Dose <SEP> Backgrounds <SEP> Anti-Bp35
 <tb> (ug / ml)
 <tb> None <SEP> - <SEP> 295 <SEP> + <SEP> 16 <SEP> 269 <SEP> + <SEP> 27
 <tb> Ab <SEP> integer <SEP> 0, <SEP> 125 <SEP> 278 <SEP> + <SEP> 32 <SEP> 5. <SEP> 140 <SEP> + <SEP> 20
 <tb> 1, <SEP> 25 <SEP> 275 <SEP> + <SEP> 24 <SEP> 4. <SEP> 686 <SEP> + <SEP> 342
 <tb> 12, <SEP> 5 <SEP> 163 <SEP> + <SEP> 15 <SEP> 3. <SEP> 852 <SEP> + <SEP> 203
 <tb> F <SEP> (ab ') <SEP> 2 <SEP> 0, <SEP> 125 <SEP> 594 <SEP> + <SEP> 21 <SEP> 10. <SEP> 635 <SEP> + <SEP> 449
 <tb> 1, <SEP> 25 <SEP> 531 <SEP> + <SEP> 3 <SEP> 10. <SEP> 893 <SEP> + <SEP> 575 <SEP>
 <tb> 12, <SEP> 5 <SEP> 279 <SEP> + <SEP> 8 <SEP> 9.411 <SEP> + <SEP> 870
 <tb>
 
 EMI36.2
 The cell culture conditions were those described in Table 3.



  5. DIFFERENCES BETWEEN THE ACTIVITY OF ANTI-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 with the intervention of different signals. First, unlike the (weak) BCGF receptors (Bijsterbosch, et al., 1985, J. Exp.



  Med. 162: 1825), the Bp50 molecules are expressed on resting B lymphocytes from the blood (FIG. 3).



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

  <Desc / Clms Page number 37>

 anti-Bp50 clearly exerted its optimum effect when it was added 12 hours after the cultures started (Figure 8A). However, BCGF (weak) could be added after 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 signal dependent on Bp50 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 ug / ml).



  BCGF (weak) was able to further increase the proliferation of B lymphocytes at rest in the presence of antiBp50 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 BCGF.
 EMI37.1
 (low). For example, some blood B lymphocytes were sensitive to BCGF (weak), but they did not respond to anti-Bp50 (Table 5). A signal

  <Desc / Clms Page number 38>

   . additional activation such as anti-Bp35 (Table 5) or TPA (Figure 10) was logically necessary for blood B cells to respond to anti-Bp50. Although, in general, dense tonsil B cells did not respond either
 EMI38.1
 a BCGF, ie anti-Bp50, the floating B lymphocytes 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 (low) or anti-Bp50 (Figures 10A and C).

  <Desc / Clms Page number 39>

 



   TABLE 5 The subsets of B lymphocytes differ in their sensitivity to anti-Bp50 or BCGF
 EMI39.1
 
 <tb>
 <tb> Proliferation <SEP> medium <SEP> (+ <SEP> error <SEP> type <SEP> from <SEP> the <SEP> average) <SEP> from
 <tb> lymphocytes <SEP> from
 <tb> of <SEP> blood <SEP> of <SEP> tonsils
 <tb> Stimulation <SEP> Experience <SEP> Experience <SEP> Experience <SEP> dense <SEP> floating <SEP>
 <tb> 1 <SEP> 2 <SEP> 3 <SEP>
 <tb> None <SEP> 527 <SEP> # <SEP> 70 <SEP> 659 <SEP> # <SEP> 133 <SEP> 906 <SEP> # <SEP> 106 <SEP> 385 <SEP> # <SEP> 43 <SEP> 387 <SEP> # <SEP> 12
 <tb> BCGF <SEP> (5%) <SEP> 13.918 <SEP> # <SEP> 37.584 <SEP> # <SEP> 3.347 <SEP> # <SEP> 174 <SEP> 332 <SEP> # <SEP> 33 <SEP> 1.451 <SEP> # <SEP> 57
 <tb> 1. <SEP> 082 <SEP> 2.

    <SEP> 023 <SEP>
 <tb> Anti-Bp50 <SEP> (0.5 g / ml) <SEP> 519 <SEP> # <SEP> 28 <SEP> 1.013 <SEP> # <SEP> 81 <SEP> 1.151 <SEP> # <SEP> 28 <SEP> 472 <SEP> # <SEP> 8 <SEP> 1.543 <SEP> # <SEP> 20
 <tb> Anti-Bp35 <SEP> (5 <SEP> g / ml) <SEP> 554 <SEP> # <SEP> 90 <SEP> 645 <SEP> # <SEP> 89 <SEP> 665 <SEP> # <SEP> 115 <SEP> 2.415 <SEP> # <SEP> 80 <SEP> 1.129 <SEP> # <SEP> 68
 <tb> Anti-Bp50 <SEP> + <SEP> anti-Bp35 <SEP> - <SEP> 12. <SEP> 274 <SEP> + <SEP> 546 <SEP> 15. <SEP> 667 <SEP> + <SEP> 333 <SEP> 36. <SEP> 589 <SEP> + <SEP> 1. <SEP> 335 <SEP> 4. <SEP> 843 <SEP> + <SEP> 136
 <tb> Anti-Bp50 <SEP> + <SEP> BCGF - 3. <SEP> 943 <SEP> + <SEP> 115 <SEP> 1. <SEP> 342 <SEP> + <SEP> 19 <SEP> 2. <SEP> 899 <SEP> + <SEP> 91
 <tb>
 Cell culture conditions as described in Table 1.

   Blood lymphocytes adhering to nylon wool (B lymphocytes plus monocytes) were depleted of their monocytes by incubation on plastic dishes overnight before stimulation (experiment 1 and experiment 2).



  In Experiment 3, blood E lymphocytes were depleted of their monocytes by incubation on plastic dishes for 1 hour before stimulation. Er-tonsillar lymphocytes were fractionated, using Percoll gradients, into dense (pastilles) or floating (fraction 1) subsets (32).

  <Desc / Clms Page number 40>

 
 EMI40.1
 



  5. USES OF ANTI-Bp50 IL- COORDINATES
4. AND Bp50.



   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 innarian responses. For example, the ligands themselves can be used as "adjuvants" to boost an immune response to a vaccine or to boost 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 involving cells or tumors which express the Bp50 antigen, whether or not the malignancy originates in B lymphocytes.



   The ligands of the present invention and / or BpSO 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 body's moods. In this case, the ligand or Bp50 could be labeled with a radiolabel, 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 an immobile support or to a fluorine that can be used in a FACS (activated cell selector by fluorescence).

  <Desc / Clms Page number 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 THE PROLIFERATION OF B LYMPHOCYTES



   Previous studies have suggested that the factors involved in the induction of Go phase B cells in the G phase of the cell cycle were distinct from the factors or conditions required for transit in the S phase. This model is based, in principle, in studies showing that agents such as low doses of anti-Bp35 anti-Igou B cell activation factors alone had little or no effect on the proliferation of B lymphocytes. 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 model of distinct regulation of the stages of growth and activation of B lymphocytes. In this case, the Applicant has demonstrated that Activation and proliferation signals appearing in human B lymphocytes could be transmitted by distinct cellular surface structures.

   Although antiBp35 mAb has activated B cells to enter the G phase of the cell cycle, it alone causes little or no proliferation. mAb antiBp50 had the opposite effect: it could not activate

  <Desc / Clms Page number 42>

 B cells but, when added even after as long as 12-24 hours after activation, may have caused B cells to grow.



   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. coordinate found 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 and low molecular weight forms of growth factor 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.



    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 it could recognize the BCGF receptor or a related structure.



  Bp50 appears to be distinct from the AB-1 marker, given that the mAb AB-1 does not block the binding of

  <Desc / Clms Page number 43>

 anti-Bp50 G28-5 and that, unlike mAb G28-5, it only reacts with lymphocytes
B activated (Jung et al., 1984, J. Exp. Med. 160: 1919-
1924). Bp50 is found on all B 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 hetero-antiserum, Wang et al. (Wang, 1979, J. Exp. Med. 149: 1424-
1433) have previously described a 54 kDa glycoprotein, gp54, which, like Bp50, is expressed on all B lymphocytes, but at lower concentrations on blood B lymphocytes than on tonsill B lymphocytes. It is possible, but improbable, that the rabbit heteroantiser and anti-Bp50 recognize the same structures or related structures: unlike anti-Bp50 mAb, rabbit antiserum directed against gp54 alone has been sufficient to stimulate the proliferation of B lymphocytes.



   Unlike anti-Bp50, anti-Bp35 alone can activate B cells from the GO phase to the G1 phase and, therefore, it can be called "activation signal". It is not yet clear if
Bp35 comes into play or not only during the early activation of B lymphocytes, since anti-Bp35 antibodies can stimulate the division of certain B lymphocytes (Clark et al., 1985, Proc. Nat. Acad.



  Sei. EUA 82: 1766-1770). Likewise, Bp50 cannot strictly come into play solely as a "growth signal": together with activation signals (anti-Bp35 or anti-u), antibodies

  <Desc / Clms Page number 44>

 anti-Bp50 not only increase proliferation, but also the total number of B cells entering phase G. (Table 2). In other words, as a co-stimulant, anti-Bp50 acts to activate the progression of both the activation (from Go to mistletoe) 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) seem to be essentially analogous to the factors of "competence" and "progression" described during 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 demonstrated here that two mAbs, aL namely anti-Bp35 ("competence" signal) and anti-Bp50 ("progression" signal) together can cause a large proliferation of highly purified B lymphocytes in the absence of antigen or d 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, i. l it may be possible to use appropriate combinations of mAbs to direct and regulate the proliferation or differentiation of human B lymphocytes which, in turn, will help to design in vivo strategies for combating human diseases such as B cell malignancies, immune deficiencies and certain autoimmune diseases.



   The new monoclonal antibody, G28-5, which

  <Desc / Clms Page number 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 activated B lymphocytes.



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



  G28-5 anti-Bp50 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 were not not co-stimulating with each other or with IL-1 or even IL-2. In addition, the activity of anti-Bp50 G28-5 did not depend on its Fc domain, since F (ab ') fragments of G28-5 were functionally active, which suggests that, just like soluble BCGF , soluble anti-Bp50 does not require accessory cells carrying an Fe receptor to exert an effect. In addition, antiBp50 and BCGF are both effective only in the presence of an activation 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 blood B lymphocytes, while receptors

  <Desc / Clms Page number 46>

 BCGF (low) apparently are not. In contrast to Bp50, candidate structures for the (weak) BCGF receptor are also expressed only on activated B cells. In addition, both 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 antiBp50. 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 costimulating 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 can be a receptor for a soluble ligand, alternatively, Bp50 can act as a receptor for a signal which is mediated by cell-cell contact and which regulates BCGF and / or receptor concentrations. 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 proliferation by elevating the expression of the IL receptor -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 and BCGF (weak) are under some coordinated control

  <Desc / Clms Page number 47>

 because, like the IL-1 and IL-2 receptors, BCGF increases the expression of Bp50 on certain leukemic cells. The Bp50 molecule also shares similarities with the Tp44 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 antiCD3 or TPA (Ledbetter, et al., 1985, J. Immunol.



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



  Likewise, 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.



  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).



   5. 4. 2. MODIFIED COORDINATES USED FOR IMMUNOSUPPRESSION OR TREATMENT OF MALIGNANCIES.



   According to this embodiment, the ligand 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 patient who has received a transplant to prevent rejection of a graft. Consequently, cytotoxic agents which are used for the suppression of immune responses, can be attached to the ligands of

  <Desc / Clms Page number 48>

 the invention. When using ligands which increase the proliferation of B cells, i1 should result in 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
 EMI48.1
 by fixing 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 proliferative cells than non-proliferative 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, referenced here for reference.

  <Desc / Clms Page number 49>

 
 EMI49.1
 



  TABLE 6 Ag ¯ Agents has anti-Bp50 coordinates
 EMI49.2
 
 <tb>
 <tb> chemotherapeutic <SEP> who <SEP> can <SEP> @tre <SEP> couplesClass <SEP> Type <SEP> Agent <SEP>
 <tb> Agent <SEP> al- <SEP> Mustard <SEP> Chlormethine
 <tb> kylation <SEP> nitrogen <SEP> Cyclophosphamide
 <tb> Melphalan
 <tb> Mustard <SEP> uracil
 <tb> Chlorambucil
 <tb> Derivatives <SEP> of ethy- <SEP> Thiotépa <SEP>
 <tb> lene-imine
 <tb> Sulfonates <SEP> Busulfan
 alkyl <tb>
 <tb> Nitrosoureas <SEP> Carmustine
 <tb> Lomustine
 <tb> Semustine
 <tb> Streptozotocin
 <tb> Triazenres <SEP> Dacarbazine
 <tb> Antiméta- <SEP> Analogues <SEP> from <SEP> Methotrexate
 <tb> bolites <SEP> acid <SEP> folic
 <tb> Analogs <SEP> from <SEP> the <SEP> Fluorouracil
 <tb> pyrimidine <SEP> Cytarabine
 <tb> Azaribine
 <tb> Analogs <SEP> from <SEP> Mercaptopurine
 <tb> the <SEP> purine <SEP> Thioguanine
 <tb> Products <SEP>

  Alkaloids <SEP> Vinblastine
 <tb> natural <SEP> extracts <SEP> from <SEP> the <SEP> Vincristine
 <tb> periwinkle
 <tb> (Pervinca)
 <tb> Antibiotics <SEP> Dactinomycin
 <tb> Daunorubicin
 <tb> Doxorubicin
 <tb> Bleomycin
 <tb> Mi <SEP> thramyc <SEP> ine <SEP>
 <tb> Mitomycin
 <tb>
 

  <Desc / Clms Page number 50>

 
 EMI50.1
 TABLE 6 (continued)

   
 EMI50.2
 
 <tb>
 <tb> Class <SEP> Type <SEP> Agent
 <tb> Enzymes <SEP> L-asparaginase
 <tb> Agents <SEP> Complexes <SEP> Cisplatin
 <tb> miscellaneous <SEP> coordinated <SEP> from
 <tb> platinum
 <tb> Urea <SEP> substi-Hydroxy-urea
 <tb> killed
 <tb> Déri <SEP> vé <SEP> from <SEP> Procarbazine
 <tb> methyl-hydrazine
 <tb> Suppressor <SEP> Mitotane
 <tb> adrenal cortex
 <tb> Hormones <SEP> and <SEP> Cortico-Prednisone
 <tb> antagonists <SEP> steroids
 <tb> Progestins <SEP> Caproate <SEP> of hydroxyprogesterone
 <tb> Acetate <SEP> from <SEP> medroprogesterone
 <tb> Acetate <SEP> from <SEP> megestrol
 <tb> Estrogens <SEP> Diethyl-stilbestrol
 <tb> Ethinyl-estradiol
 <tb> Anti-estrogen <SEP> Tamoxifen
 <tb> Androgens <SEP> Propionate <SEP> from <SEP> testosterone
 <tb> Fluoxymesterone
 <tb> Isotopes <SEP> Phosphorus <SEP> Phosphate <SEP> from <SEP> sodium <SEP> 32p
 <tb> radioactive <SEP> lode

   <SEP> Iodide <SEP> from <SEP> sodium <SEP> 131 <SEP> 1 <SEP>
 <tb>
 
Any method known in the art can be used to couple the ligand.
 EMI50.3
 to the chemotherapeutic or antiproliferative agent. Examples of such methods have been listed above (see paragraph 5, above).

  <Desc / Clms Page number 51>

 



    5. 4. 3. OTHER USES OF COORDINATES AND Bp50
In addition to the 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 sample.



  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-opaque compound or an appropriate radio-tag 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 obvious to those skilled in the art.
 EMI51.1
 



  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 sequence number: Hybridome ATCC sequence number
 EMI51.2
 G28-5
The scope of the present invention is in no way bounded by the deposited hybridoma, since the deposited type is given simply in order to illustrate an aspect of the invention and that any

  <Desc / Clms Page number 52>

 cell lines which are functionally equivalent are within the scope of the present invention.

   In fact, on reading the preceding description and in view of the appended figures, the skilled person will recognize various modifications of the invention in addition to those illustrated and described in this specification. It is understood that these modifications fall within the scope of the claims below.

  <Desc / Clms Page number 53>

   TABLE OF CONTENTS
 EMI53.1
 
 <tb>
 <tb> Page
 <tb> 1. <SEP> Introduction ........................... <SEP> 1
 <tb> 2. <SEP> Basis <SEP> from <SEP> the invention ............... <SEP> 2
 <tb> 3. <SEP> Summary <SEP> from <SEP> the invention <SEP> 5
 <tb> 3.1. <SEP> Definitions <SEP> 10 <SEP>
 <tb> 4. <SEP> Description <SEP> of <SEP> figures <SEP> 11
 <tb> 5. <SEP> Description <SEP> detailed <SEP> from <SEP> the invention ... <SEP> 15
 <tb> 5. <SEP> 1.

    <SEP> Methods <SEP> adopted <SEP> for <SEP> characterize <SEP> on <SEP> receiver <SEP> Bp50 ....... <SEP> 19
 <tb> 5. <SEP> 2. <SEP> Characterization <SEP> from <SEP> receiver
 <tb> Bp50 <SEP> 24
 <tb> 5. <SEP> 2. <SEP> 1. <SEP> Identification <SEP> of a
 <tb> marker <SEP> superficial <SEP> from
 <tb> cells <SEP> to <SEP> 50 <SEP> kDa
 <tb> specific <SEP> to <SEP> lymphocytes <SEP> B, <SEP> Bp50 <SEP> 24 <SEP>
 <tb> 5. <SEP> 2. <SEP> 2. <SEP> The expression <SEP> from <SEP> Bp50 <SEP> is
 <tb> restricted <SEP> to <SEP> lymphocytes <SEP> B <SEP> 27
 <tb> 5. <SEP> 3. <SEP> Increase <SEP> from <SEP> the <SEP> proliferation
 <tb> of <SEP> lymphocytes <SEP> B <SEP> with <SEP> the antibody <SEP> anti-Bp50 <SEP> 28
 <tb> 5. <SEP> 3. <SEP> 1.

    <SEP> mAb <SEP> anti-Bp50 <SEP> increases <SEP> the
 <tb> proliferation <SEP> only
 <tb> after <SEP> that <SEP> them <SEP> lymphocytes
 <tb> B <SEP> have <SEP> summer <SEP> enabled <SEP> by <SEP> of
 <tb> antibody <SEP> anti-Bp35 <SEP> or
 <tb> anti- ................. <SEP> 30
 <tb> 5. <SEP> 3. <SEP> 2. <SEP> The <SEP> mAb <SEP> anti-Bp50
 <tb> don't activate <SEP> no <SEP> them <SEP> lymphocytes <SEP> B <SEP> from <SEP> the <SEP> phase
 <tb> GO, <SEP> but <SEP> 11s <SEP> bring <SEP> them
 <tb> lymphocytes <SEP> B <SEP> enabled <SEP> to
 <tb> progress <SEP> a <SEP> through <SEP> on
 <tb> cycle <SEP> cell ......... <SEP> 31
 <tb>
 

  <Desc / Clms Page number 54>

 
 EMI54.1
 
 <tb>
 <tb> Page
 <tb> 5. <SEP> 3. <SEP> 3.

    <SEP> Conditions <SEP> optimal
 <tb> for <SEP> increase <SEP> the <SEP> proliferation <SEP> of <SEP> lymphocytes
 <tb> B <SEP> with <SEP> of <SEP> antibody
 <tb> anti-Bp50 ................ <SEP> 33
 <tb> 5. <SEP> 3. <SEP> 4. <SEP> Differences <SEP> between
 <tb> activity <SEP> of anti-Bp50
 <tb> and <SEP> from <SEP> BCGF <SEP> (weak) ...... <SEP> 36
 <tb> 5. <SEP> 4. <SEP> Uses <SEP> from <SEP> coordinates <SEP> antiBp50 <SEP> and <SEP> from <SEP> Bp50 .................. <SEP> 40
 <tb> 5. <SEP> 4. <SEP> 1. <SEP> Receiver <SEP> Bp50 <SEP> and
 <tb> uses <SEP> from <SEP> coordinates
 <tb> such <SEP> that <SEP> anti-Bp50 <SEP> for
 <tb> increase <SEP> the <SEP> proliferation
 <tb> of <SEP> lymphocytes <SEP> B ........ <SEP> 41
 <tb> 5. <SEP> 4. <SEP> 2.

    <SEP> Coordinates <SEP> modified
 <tb> use <SEP> for <SEP> immunosuppression <SEP> or <SEP> on <SEP> treatment <SEP> from <SEP> malignancies <SEP> 47
 <tb> 5. <SEP> 4. <SEP> 3. <SEP> Others <SEP> uses <SEP> from
 <tb> coordinates <SEP> and <SEP> from <SEP> Bp50 ..... <SEP> 51
 <tb> 6. <SEP> Deposit <SEP> from <SEP> lines <SEP> from <SEP> cells <SEP> 51
 <tb>



    

Claims (67)

 CLAIMS 1. Virtually pure coordinate which (a) binds to Bp50, namely a surface antigen of B lymphocytes at 50 kilodaltons, defined by the monoclonal antibody G28-5, and which (b) upon binding to an active B lymphocyte , 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, characterized in that it comprises an antibody molecule or an Fv, Fab, F (ab ') p or Fab'de 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 ') or Fab'fraction of the monoclonal antibody molecule.  4. Monoclonal antibody molecule according to claim 3, characterized in that it comprises G28-5 or any fraction Fv, Fab, F (ab ') 2 or Fab'de 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.  <Desc / Clms Page number 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, 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 couple compound to be coordinat.  10. Coordinate according to claim 9, in which the compound which is coupled thereto, comprises an antiproliferative agent.  11. Coordinate according to claim 9, in which the compound which is coupled thereto, comprises an alkylating agent.  12. Coordinate according to claim 9, in which the compound which is coupled thereto comprises an antimetabolite.  13. Coordinate according to claim 9, wherein 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. Coordinate according to 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  <Desc / Clms Page number 57>  radioisotope.  18. The coordinate of claim 9, wherein the compound which is coupled thereto comprises a fluorescent compound.  19. Practically 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, characterized in that it comprises an antibody molecule or a fraction Fv, Fab, F (ab ')? or Fab '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 coupled compound for coordinating body.  25. The coordinate of claim 24, wherein 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,  <Desc / Clms Page number 58>  in which the compound which is coupled therein, comprises an antibiotic.  29. Coordinate according to claim 24, in which the compound which is coupled thereto, comprises an alkaloid extracted from the periwinkle (Pervinca).   30. The coordinate of claim 24, wherein the compound which is coupled thereto comprises an enzyme.  31. The coordinate according to 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 active B lymphocytes with an effective dose of a ligand which binds to Bp50, namely a surface antigen of B lymphocytes at 50 kilodaltons, defined by the monoclonal antibody G28-5, such that activated B cells pass through the cell cycle and their proliferation increases.  37. The method of claim 36, wherein the B cells have been. activated by treatment with an effective dose of a second  <Desc / Clms Page number 59>  dinat which binds to Bp35, namely a surface antigen of B lymphocytes at 35 kilodaltons, so that the B lymphocyte progresses from the Go phase to the GI phase of the cell cycle.  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 37, wherein the ligand comprises a molecule  EMI59.1  of antibody which binds to Bp50, or an Fv, Fab, F or Fab 'fraction of the antibody molecule, binds to Bp50.  41. The method of claim 36 or 37, wherein the ligand comprises a monoclonal antibody molecule which binds to Bp50, or an Fv, Fab, F (ab ') 2 or Fab' fraction of an antibody molecule monoclonal, which binds to AL Bp50.  42. The method of claim 41, wherein the monoclonal antibody molecule comprises G28-5 or any Fv, Fab, F (ab ') or Fab'de G28-5 fraction.  43. The method according to claim 41, in which the monoclonal antibody is produced by a hybridoma cell line, deposited at ATCC under the order number HB9110, or one of its mutants, one of its recombinants or a of its derivatives belonging to genetic engineering.  44. The method of claim 36 or 37, 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 on the surface  <Desc / Clms Page number 60>  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  EMI60.1  with the ligand which binds to Bp50, approximately within 12 hours of activation by the second ligand which binds to Bp35.  50. Method for suppressing the proliferation of cells which express Bp50, namely a surface antigen of B lymphocytes at 50 kilodaltons, defined by the monoclonal antibody G28-5, characterized in that it consists in treating the cells which express Bp50, with an effective dose of a ligand which binds to Bp50, this ligand being coupl  EMI60.2  to an antiproliferative agent.  51. The method of claim 50, wherein the cells which express Bp50 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. Method according to claim 50, 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 ') or Fab'fraction of the antibody molecule.  <Desc / Clms Page number 61>    56. The method of claim 55, wherein the antibody molecule comprises a monoclonal antibody molecule or an Fv, Fab, F (ab ')? or Fab 'of the monoclonal antibody molecule.  57. The method of claim 56, wherein the monoclonal antibody comprises G28-5 or any Fv, Fab, F (ab ') p or Fab'de G28-5 fraction.  58. The method according to 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, of 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 lymphocyte 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 or 59, wherein the antiproliferative agent comprises 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. Method according to claim 50,  EMI61.1  55 or 59. which the antiproliferative agent comprises an enzyme.  <Desc / Clms Page number 62>    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, 55 or 59, wherein the antiproliferative agent comprises a radioisotope.