PT85073B - PROCESS FOR INCREASING PROLIFERATION OF B CELLS BY USING LIGANDS - Google Patents

Abstract

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

Description

PROCESS TO INCREASE B-CELL PROLIFERATION BY USING LIGANDS

1. INTRODUCTION

The present invention relates to ligands, such as antibody molecules or antibody fragment molecules or to other ligands such as lymphokines that bind to a 50 Kda cell surface marker, hereinafter referred to as Bp50, which functions in the proliferation of B cells but not in the early activation of células cells. The present invention also relates to the Bp50 antigen of B cells. In a special aspect of the present invention, a monoclonal antibody G28-5 is described, which defines Bp50 and appears to play a role in the proliferation of non-activated B cells but which has no detectable effect on the proliferation of the remaining B cells.

The ligands of the present invention, such as antibodies, lymphokines and their fragments, can be used to command and regulate the proliferation and / or differentiation of B cells. In addition, the ligands of the present invention can be modified by binding other composites. -

that can be used in the treatment and / or detection of malignant cells that express the Bp5O antigen.

2. BACKGROUND OF THE INVENTION

The activation of the remaining B cells from the GO phase to the G1 phase of the cell cycle and the subsequent induction of B cells activated to proliferate are different steps that require different regulatory mechanisms. Some agents, including the pl factor that stimulates murine-cells (BSF-pl) (Rabin et al., 1985, Proc. Nat, Acad. Sci. USA 82, 2935 - 2939) and low doses of immunoglobulin (anti-Ig ) (DeFranco et al., 1985, J. Immunol. 135: 87 - 94; Wetzel et al., 1984, J. Immunol. 133: 2327 -

- 2332; DeFranco et al., 1982, J. Exp. Med. 155: 1523 -

- 1536; Muraguchi et al., 1984, J. Immunol. 132: 176 -

- 180), are activation or “competence” factors, that is, they induce B cells to increase their volume, to synthesize more RNA and, the entry of Gl, but they do not induce by themselves the synthesis of DNA in B cells. Other growth factors, such as B cell growth factor (BCGF) and interleukin-2 (IL-2) cause activated B cells to cross the cell cycle and enter the B phase, but not the make to the remaining 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).

Numerous factors are now described by researchers in the murine and human systems

which promote B cell growth. They include B cell growth factors (BCGF) derived from different sources including T cell lines, B cell lines or dendritic cells.

Although interleukin-1 (IL-1) and interleukin-2 (IL-2) have been shown to increase the growth of B cells, they are apparently distinct from certain BGGFs. However, monoclonal antibodies (mAb) to murine BGGF (O'Hara et al., 1985, Nature (Lond.) 315: 333) or to human BCGF (Ambrus et al., 1985, J. Exp. Med. 162: 1319 ), blocked BGGF activity but not IL-1 or IL-2 activity. Despite being different from IL-1 or IL-2, BCGFs themselves appear to be heterologous based on their different biochemical data and activity on different sub-groups of B cells or joint stimulation assays. However, a high molecular weight 60 kilodalton (Kda) human BCGF form, BCGF (high), has been identified which is distinct from a low molecular weight 12 Kda form, BGGF (low) (Ambrus et al., 1985, J. Glin, Invest. 75: 732). The 20 kda murine BCGF encoding cDNA, designated by attempts as the B-cell stimulating factor (BSF-pl), was streaked and sequenced (Noma et al., 1986, Nature 319) 640). The recombinant lymphokine not only had BCGF activity, it could also activate the remaining B cells and induce the differentiation of IgGl-producing cells; therefore, it differs from human BCGF (upper) and BCGF (lower) in its molecular weight and scale of activity.

These activation and growth signals probably guide the cells, interacting with the B cell surface structures. In addition to the specific antigen signal along the Ig surface, several other candidate polypeptides on the cell surface have been identified. which may play a role in the activation or growth of B cells. However, cell surface receptors have been characterized for IL-1 (Dower et al., 1985, J. Exp. Med. 160: 501) and for IL-2 (Robb et al., 1984, J. Exp. Med. 160: 1126), and, recently, B-cells have been identified as functional receptors for IL-2 (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 growth and activation factors are not yet fully characterized. Several candidate polypeptides on the surface of B cells have been identified that may, in a way, play a role in the activation or growth of B cells. Subbarao and Mosier (Subbarao et al., 1983, Immunol * Rev. 69: 81 - 97), for example, they found that monoclonal antibodies (mAb) to murine B cell antigen Lyb2 activates B cells, and recently this fact has been evident, suggesting that Lyb2 may be the reactor for BSF- pl (Yakura, 1985, Fed. Proc. 44: 1532). Similarly, mAbs (1F5) suitable for a 35 Kda polypeptide, Bp35, have been found to activate human B cells from GO to G1 (Clark et al., 1985, Proc. Nat. Acad. Sci. USA 82 : 1766-1770; Gollay et al., 1985, J. Immunol. 135: 3795-3801). C3d or antibody aggregates for the receptor

- 5 of 140 Kda C3d, Bpl40, originate the proliferation of B cells that are dependent on T cells (Melchers, et al., 1985, 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 rats and humans, receptors for these factors have not yet been isolated. Wang et al. (Wang, et al., 1979, J. Exp. Med. 149: 1424 - 1433) performed polyclonal antisera that identified a 54 Kda polypeptide (gp54) in human B cells and demonstrated that human antiserum mouse for gp54 induced the division of tonsillar B cells. Recently, Jung and Fu (Jung, et al., 1984, J. Exp. Med. 160: 1919 - 1924), isolated an mAb (AB-1) for the 55 Kda antigen that restricted itself to activating B cells that blocked BCGF-dependent proliferation. However, it is still unknown whether anti-gp54 or AB-1 recognized a BCGF receptor or not.

3. SUMMARY OF THE PRESENT INVENTION

The present invention relates to ligands that

a) bind to Bp50, a specific polypeptide on the surface of the B cell surface described herein, and, b) increase the proliferation of activated B cells. The present invention also relates to the Bp50 antigen itself, which is defined by the monoclonal antibody G28-5 and plays a role in the proliferation of activated B cells. In addition, the present invention relates to ligands that bind to Bp50, but do not have a biological effect or function such as increased proliferation of activated B cells

*

The ligands of the present invention include antibody molecules, monoclonal antibody molecules and fragments of these antibody molecules that contain the antigen or antibody-combining site and chemically modified fragments; such fragments include but are not limited to Fv, Fab, Fíab '^ » ^I ' ^ab ' ^{and the} like. In addition, the ligands of the present invention comprise lymphokines, which may include, but are not limited to, human B cell growth factors as well as chemically modified lymphokines. The ligands of the present invention can be chemically modified by ligating or coupling, for example, a compound to the ligand.

Such compounds include but are not limited to cytotoxic agents, therapeutic agents, chemotherapeutic agents, labels, such as radioactive labels, dyes, enzymes, radio-opaque compounds, and the like. The ligands of the present invention, in their modified or unmodified form, can be used to control, regulate and modify the proliferation and / or differentiation of human B cells.

The present invention is based on the discovery that two human B cell differentiation antigens, Bp35 and the human B cell antigen described here, Bp50, apparently function differently as signal receptors in the activation of B cells. Antibodies monoclonal (mAb) for Bp35 θ Bp5O emit positive signals to B cells that stimulate their transition through the cell cycle. 0 mAb for Bp35, such as antibodies

- 7 anti-Ig, function mainly, activating the remaining B cells, in order to make them competent to enter the G1 phase of the cell cycle. In contrast, a monoclonal antibody described herein or its FíabOg fragment, P ^{for Β} Ρ5θ, a 50 Kda polypeptide expressed in all B cells, stimulates activated B cells to cross the cell cycle and increases the proliferation of activated B cells. Monoclonal antibodies to Bp35, like anti-Ig antibodies, activate tonsillar B cells and induce low levels of B cell proliferation. In contrast, the anti-Bp50 monoclonal antibody alone neither activates B cells nor induces B cells B to proliferate, but together with anti-Bp35 or anti-Ig antibodies, B cell proliferation increases. In this respect, the action of the anti-Bp50 antibody resembles the activity of B cell growth factors (BCGF). Only 0.05 µg / ml an.ti-Bp50 is needed to increase proliferation and, like BCGF, anti-Bp50 is effective even when added 12 to 14 hours after activation of B cells with anti-Ig or anti -Bp35. Without additional exogenous signals, the anti-Bp35 and anti-Bp50 antibodies together induce a strong proliferation of the remaining purified B cells. These results suggest that the surface molecules Bp35 and Bp50 play a role in regulating B cell activation and their progression through the cell cycle. Due to their scope, anti-Bp35 and identical molecules, have the effect and action of the ligands of the present invention, Clark, et al., 1985, Proc. Líatl. Acad. (USA) 82: 1766-1770.

Despite the anti-Bp50 activity being similar to that of BCGF (lower) since anti-pB5O and BCGF (lower) are stimulated by the same agents, but not by each other, and anti-Bp50 and BCGF (lower) ) only affect activated B cells and function in a soluble form, anti-Bp5O activity can be distinguished from BCGF (lower) activity, since the proliferation of B cells stimulated with optimal amounts of anti-Bp50 and anti -Bp35 (or anti-Ig) can later be increased with BCGF (lower) and blood B cells and certain B cell lymphomas respond differently to anti-Bp5O versus BCGF. For optimal activity, anti-Bp50 should be added 12 hours after B cell activation, while BCGF (lower) retains optimal activity even when added 24 hours after activation. In addition, Bp5O is expressed in all B cells while receptors or BCGF (lower) is limited to activated cells. In this way, anti-Bp5O and BCGF (lower) can regulate the growth of B cells in a coordinated way, but they do so, apparently, through different signals.

In one aspect of the present invention, ligands that bind to Bp5O and increase the proliferation of activated B cells can be used to increase the immune response. These ligands that bind to Bp5O, for example, can be used with adjuvants ”to increase the immune response to a vaccine. Alternatively, these ligands can be used to increase the immune response to an individual with immune suppression.

In another aspect, the ligands of the present invention can be chemically modified in such a way that the cells to which the ligands are attached are eliminated. Since all B cells express the Bp5O antigen, this approach would suppress the immune response. For example, a cytotoxic drug connected with a ligand of the present invention, in vivo, can be used to generate immunosuppression in order to cross the barriers of histocompatibility in patients with transplants; alternatively, these modified ligands can be used to control immunological diseases.

In another aspect of the present invention, malignant conditions can be treated, such as tumor cells that express Bp5O, using a ligand of the present invention linked to a chemotherapeutic agent useful in the treatment of such neoplastic disease. These modified ligands can be used in vivo to direct the chemotherapeutic agent to any type of malignant cell that expresses the Bp5O antigen, including cells that are not B cells but that express Bp5O. When using the ligands of the present invention that increase the proliferation of B cells, a special advantage can be obtained when treating malignant situations of B cells if the therapeutic agent bound to the ligand is constituted by one that is more effective in killing the cell proliferation; in this circumstance, the potentiation of the drug's action may be obtained.

Alternatively, the ligands of the present invention can be used in vitro to identify or separate cells expressing the Bp5O antigen and / or to test organic fluids for the presence of

- 10 - / s <

Bp50 antigen that may or may not have spilled. In addition, the ligands of the present invention can be used in vivo to depict cells or tumors that express the Bp50 antigen.

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

3.1. DEFINITIONS

As used herein, the following abbreviations will have the following meanings:

AO = acridine orange

BCGF = B cell cell growth factor

BCGF (top) ~ 60 Kda human BCGF

BCGF (bottom) = 12 Kda human BCGF

Bp35 = specific B cell surface polypeptide defined by mAb G28-5

Fv = variable region or antigen combination site of an antibody molecule. This may consist of any fragment that contains the idiotype of the molecule including, but not limited to Fab, F (ab ') 2> Fab' e, similar.

IF = immunofluorescence

Ig = immunoglobulin

IL-1 = interleukin 1

IL-2 = interleukin 2 kDa = kilodalton mAb = monoclonal antibody

SDS-PAGE = electrophoresis on sodium cylsulfate-polyacrylamide gels

TPA = 12-0-tetra-decanoyl ~ phorbol-13-acetate

4. DESCRIPTION OF THE FIGURES

Fig. 1. Bp5O expression is limited to Bp35 + B cells. Bi-color cytometric analysis of 50,000 cells was performed, as described by (Clark, et al., 1985, Proc. Nat. Acad. Sei. USA 82 : 1766 - 1770). The data diagram was made as number of cells against the green fluorescence logarithm and the red fluorescence logarithm, where 4-5 points represent, approximately, a double fluorescence. Data are presented to show negative autofluorescent cells. The staining PE (red) - anti-Bp35 (1F5) against FICT (green) - anti-Bp50 (G28-5) indicates that all Bp5O + cells are also Bp35 +.

Fig, 2. Biochemical comparison of the Bp5O polypeptide with other cell surface antigens. Bp5O immunoprecipitation was performed from the cells

125 tonsils radiolabelled with I, as described. Isolated antigens were electrophoresed on SDS polyacrylamide gel plates, without reduction. Gels were visualized with radiographs and amplification screens. Panel A: lane 1, anti-Bp50 (G-28-5); lane 2, anti-Bp45 (BLAST-2); lane 3, anti-Bp39 (G28-1); lane 4, anti-Bp39 (41-H16); lane 5, only sepharose-rat anti-Ig developed in the goat. An exposure period of 2 days was chosen so that the bands in lanes 2 to 4 did not overexpress themselves and could be clearly distinguished from Bp5O. One of three experiences.

Fig. 3. Bicolor immunofluorescent analysis of Bp5O expression. Tonsillary or peripheral blood cells were isolated by centrifugation in a Ficoll apparatus and stained with G28-5 (anti-Bp50) conjugated to PE (red) in combination with reference antibodies, including 2G3 (anti-IgM), 1F5 (anti-Bp35), HBlOa (anti-DR) and

9.6 (anti-CD2, E receptor), conjugated to fluorescein (green). The cells were analyzed with a FACS IV adapted with logarithmic amplifiers of 4 decades in both red and green dimensions. Forward and right-angle light scattering was used to encleusitate the monocytes. The unstained cells were positioned at the rear of the grid; red fluorescence is on the right and green fluorescence on the left.

Fig. 4 «Dose response curves by increasing the proliferation of dense ER-B tonsillar cells by anti-Bp50 antibodies, as indicated; just half; anti-Bp50 only, Bp35 only (5 µg / ml); BCGF only; anti-Bp35 plus BCGF; anti-Bp35 plus graduated doses of anti-Bp5O. On the third day, mean proliferation + normalized error of quadruplicate samples was measured.

Fig. 5. The anti-Bp50 mAb is more effective and increases proliferation if added after a B cell activation signal. The dense Er-B tonsillar cells were incubated for 4 days with medium only, anti- Bp5O (0.5 µg / ml) at different times after incubation, anti-Bp35 (5 µg / ml) was added at different times after incubation; anti-Bp50 remained constant and anti-Bp35 was added thereafter at different time periods; anti-Bp-35 was kept constant and added to this anti-Bp50, for cultures, at different time intervals. Over the last 10 hours, 3H-thymidine was added and its incorporation was measured.

Fig. 6. Comparison of the anti-Bp35 and anti-Bp50 ability to induce the remaining B tonsillar cells to leave the GO stage of the cell cycle. Three days after treatment with only medium (____), only with anti-Bp35 (-----) and only with Ig (.....); A, without additional additives; B, anti-Bp50 (0.5 µg / ml) added to each

- 14 of the groups; C, 5% BCGF, added to each of the groups. Data were plotted against the number of cells against the AO red fluorescence logarithm (ARU).

Fig. 7. Kinetic effects of B cell proliferation after stimulation with anti-Bp50 versus BCGF. Dense tonsillar E-B cells were stimulated with only medium; only with 10% BCGF; with only anti-Bp35; only with anti-Bp50; with 10% anti-Bp35 + BCGF; with anti-Bp35 4-anti-Bp50; and with anti-Bp35 + anti-Bp50 +. 10% BCGF. Proliferation was measured on the days indicated by an 18-hour pulse of Ή-thymidine. Proliferation was measured in quadruplicate samples and normalized errors are indicated. One of three experiences.

Fig. 8. Periods of time after stimulation with anti-Bp35 when anti-Bp50 (A) or BCGF (B) optimally increase proliferation. Dense tonsillar cells, E-B, were stimulated as indicated and proliferation was measured on the third day by an 18-hour pulse of -Ή-thymidine. Medium; anti-Bp35 added only in the indicated periods; anti-Bp5O or BCGF only; anti-Bp35 added at the beginning of the culture followed by anti-Bp35. One of two experiences.

Proliferation was measured in quadruplicate samples and normalized errors are indicated. Doses used: anti-Bp35, 5 µg / ml; anti-Bp50, 0.2 µg / ml; BCGF (lower) than 5%. The concentrations used were as follows: anti-Bp35, 5 µg / ml; anti-Bp50, 0.2 µg / ml; BCGF, 5%.

Fig. 9. The anti-Bp50 and BCGF have additive effects on the proliferation of B cells. 'E-B dense tonsillar cells were stimulated with doses of BCGF (lower) together with only anti-Bp50; anti-Bp35 only; drops of anti-Ig only; anti-Bp-35 + anti-Bp50; or anti-Bp50 + + anti-Ig. Proliferation was measured 3 days after stimulation with an 18-hour pulse of 3 H-thymidine. Proliferation was measured in quadruplicate samples and standard errors are indicated. One of four experiences.

Doses used for 10 ^ cells: anti-Bp35, 5 µg / ml; anti-Bp50, 0.2 µg / ml; drops of anti-Ig, 50 µg / ml.

Fig. 10. Comparative effects of anti-Bp50 and BCGF on normal and healthy B cells. Peripheral blood E-B cells (A) or dense E-B tonsil cells (C) with or without TPA (75 ng / ml) were stimulated in the presence of 10% BCGF or 1 µg / ml anti-Bp50. Two B-cell lymphomas (panels B and D) were separately stimulated in the same way. Proliferation was measured on the third day by incorporating 3 H-thymidine over a 12-hour pulse. Proliferation was measured in quadruplicate samples and standard errors are indicated.

5. DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention relates to ligands that

a) bind to Bp50, a specific polypeptide on the surface of 50 Kda B cells, and b) increase the proliferation of activated B cells. The present invention also relates to the Bp50 antigen itself, which is defined by mAb G28-5 and '. * Plays a role in the proliferation of B cells. In addition, the present invention relates to ligands that bind to Bp5O but that do not demonstrate a biological effect or function such as increased proliferation of activated B cells.

The ligands of the present invention include antibody molecules, monoclonal antibody molecules and fragments of these antibody molecules that contain the antigen combining site for the Bp5O receptor, including chemically modified antibodies and fragments; such fragments include but are not limited to Fv, Fab, F (ab ') ₂ , Fab * and the like. In addition, the ligands of the present invention comprise lymphokines that bind to the Bp5O receptor. These may include, but are not limited to, BCGFs, as well as chemically modified and similar lymphokines. The ligands of the present invention can be used in their modified or unmodified forms to modulate and regulate immune responses and in the therapy of malignant diseases that express the Bp5O antigen. These uses are described in more detail in Section 5.4. forward.

When the ligand is a monoclonal antibody, or a fragment thereof, the monoclonal antibody against Bp5O can be prepared using any of the techniques that provide 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 recently become available, such as the B cell hybridoma technique (Kozbor et al. others, 1983, Immunology Today 4: 72) and the hybridoma technique for producing monoclonal antibodies (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) and similar techniques, are within the scope of the present invention.

Antibody fragments that contain the idiom of the molecule could be originated by known techniques. These fragments include, for example, but are not limited to: F (ab ') 2 fragment that can be originated by treating the antibody molecule with pepsin; Fab fragments which can be generated by reducing the disulfide bridges of the FíabOg fragment "Fragment FíabOg Q ^u can be generated by treating the antibody molecule with papain and a reducing agent to reduce the disulfide bridges.

When the ligand that binds to Bp5O is lymphokine, lymphokine can be obtained from natural sources or if its amino acid sequence is known or deduced, lymphokine can be synthesized by chemical synthetic methods.

Alternatively, if the lymphokine genetic sequence is known, recombinant DNA techniques can be used to clone the gene into an expression vector that provides for transcription and transfer of the genetic sequence into a suitable host cell.

Depending on the intended use, the appropriate ligand or fragments of the ligand can be chemically modified by binding any one of a variety of compounds to the ligand using techniques

- “Coupling cases known in the art. These techniques may include, but are not limited to, the use of carbo-diimide, cyanogen bromide, bifunctional reagents such as glutaraideide, N-succinimidyl-3 (2-pyridyl-di-thio) -propionate (SPDP), reactions Schiff's alkaline, bonds to sulfhydryl radicals, the use of sodium isothiocinate or enzymatic bond. When it is intended to bind a radioisotope to the ligand, this must be done by enzymatic means, oxidative substitution, chelation, etc. For verification of the enzymatic reagents that can be used for protein modifications, see Lundblad and Noyes, Chemical Reagents for Protein Modification, Volume II, CRG Press, Inc., Boca Raton, Florida, Ch. 5, pp. 123 - 139, 1984.

Chemical binding or coupling of a compound to the ligand can be directed to a site on the ligand that does not participate in Bp5O binding. It can be achieved by protecting the ligand binding site before making the coupling connection. For example, the ligand can first bind to Bp5O in order to protect the Bp5O binding site, then coupling can be carried out to bind the desired compound to the reactive sites available in the Iigando-Bp50 complex. Once the coupling reaction is complete, the complex can be disrupted, thereby creating a modified ligand to which the desired compound is attached in such a way that the Bp5O binding site of the molecule is minimally affected. When the ligand comprises a monoclonal antibody such as G28-5, in which the Fc domain of the molecule is not necessary for the ligand to exert its effects (see Section 5.3.3., Below)

- 19 it will be advantageous to direct the coupling of the desired compounds to the Fc domain of the molecule.

The following sub-sections describe the new 50 kDa B cell surface marker, Bp50, which apparently works on B cell proliferation, as well as ligands that bind to the new 50 Kda receptor and their uses. As an example of the ligands of the present invention, a monoclonal antibody that defines Bp50 and its F (ab ') ₂ fragments is also described, which, like BCGF, increases the proliferation of B cells, differently, the anti mAb -Bp35, which can induce the remaining B cells in GO to enter Gl, the anti-Bp50 mAb does not activate the remaining B cells. The anti-Bp35 mAb together with the anti-Bp50 mAb, without any additional exogenous signals, induce strong activation and proliferation of purified B cells.

The experiments described below also demonstrate that the activity of anti-Bp50 is similar to the activity of BCGF, but that of Bp50 is distinct from BCGF, since anti-Bp50 and low-molecular weight BCGF are clearly additive and act differently on different sub-groups of B cell or malignant diseases. The Bp50 can be a receptor for a different BCGF or for a transmembrane signal that modulates BCGF production or BCGF receptor expression.

5.1. METHODS USED TO CHARACTERIZE THE Bp50 RECEIVER

Cellular Preparations. Mononuclear cells were isolated from normal or leukemic and heparinized peripheral blood, using Ficoll-Hypaque density gradient (Pharmacia, Piscataway, NJ). Mononuclear cells were obtained from tonsillar tissues, as described (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766 - 1770). T cell depletion was performed with an assay for detecting mononuclear cells (rosetting) from goat erythrocytes treated with AET and separation in Ficoll-Hypaque density gradient. In some experiments, blood B cells were enriched by isolating the cells adhering to wool fabric. Monocytes were removed by incubation in plastic Petri dishes once or twice at 37 ° C for 45 minutes, unless otherwise stated. Floating or dense fractions of tonsillar B cells were isolated by Parcoll step gradients as described (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766 - 1770). Dense tonsillar B cell preparations consistently have more than 95% slg + Bp35 cells. The enriched blood B cell preparations have 60-85% slg + cells. B-cell lymphoma cells were isolated by gentle carding of the lymphoma cells in medium, followed by density gradient centrifugation in a Ficoll-Hipaque.

Monoclonal Antibodies. The G28-5 antibody to Bp50 was originated by immunizing BALB / c mice with human tonsillar E lymphocytes and splenic cell fusion.

only immunized with NS-1 myeloma (Kohler, et al., 1975, Nature 256: 495 - 497; Ledbetter, et al., 1979, Immunol. Rev. 47: 63 - 82). Hybrid cell cultures that secrete the antibody reactive with the B tonsillar cells and not with the T cells were identified using indirect immunofluorescence (IF) and analysis with a FACS IV cell selector; cultures with antibodies that provided histograms with similar configurations for the known mAb for all B cell markers (e.g. Bp35) were cloned and selected for further studies. The G28-5 clone produced an IgG mAb that only reacted with normal or malignant B cells or B cell lines. Another mAb used in this study was described in detail (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 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 and Histocompatibility, ASHI, New York, 6, pp. 325-340). Included are 1F5 (IgG _2a ) anti-Bp35, HBlOa (IgG _2a ), anti-HLA-DR, 2C3 (IgG ^) anti-u chain, G19-4 (IgG- ^) anti-CD3, FC-2 ( IgG _2a ) anti-Fc receptor, CD16, and 9.6 (IgG _2a ) anti-CD2 (receptor E) provided by Paul Martin (Martin, et al., 1983, J. Immunol. 131: 180). The IgGl mAbs were purified by precipitation using 45% or 50% saturated ammonium sulfate and Sephacryl ”DEAE column chromatography and the IgG2a mAbs were purified using protein A Sepharose columns. (ab *) 2 of G28-5, according to the method of Parham (Parham, et al., 1983, J. Immunol. 131: 2895) and purified in a column of 2 meters in length Sephacryl S2OO ”and, rehearsed purity is determined by SDS-PAGE (Ledbetter, et al., J. Immunol.

135: 1819). MAb 203 for u-chains was conjugated with drops of Sepharose 4B (Pharmacia Fine Chemicals, Uppsala,

Sweden) using cyanogen bromide coupling.

Conjugations with fluorescelna. and phycoerythrin. The purified mAbs were either directly conjugated to fluorescine using fluorescine isothiocyanate (FITO; Molecular Probes) (green) according to the method of Goding (Goding, et al., 1976, J. Immunol. Meth. 13: 215-226) or conjugated to R-phycoerythrin (PE) (red) using SPDP (Pharmacia) according to a method 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 micro-titration plates for 30 minutes with an appropriate dilution of green and / or red antibody, washed twice and then analyzed in a FACS IV cell sorter.

Bi-color immunofluorescence. Bi-color studies were carried out with a selector (FACS IV: Becton-Dickinson, Moutain View, CA) of fluorescence-activated cells using a 560-nm dichroic reflector to split the irradiation and a long-pass filter and pass-through filter (Distric Optics Hudson, MA) in front, respectively, of the red and green photomultiplication tubes. In addition, a two-color compensator (T. Nozaki, Stanford University) is used to correct the slightest loss of red and green signal. For each two-color stain, data was collected from 40,000 cells and stored on floppy disks. The data are presented as number of cells (vertical) against the green fluorescence logarithm against the red fluorescence logarithm in a 64x64 point grid. Approximately 4.5 points represent a doubling of fluorescence. The unstained cells were positioned in the rear corner of the grid; red fluorescence is on the right and green fluorescence on the left. The IF bicolor flow cytometry system with fluorescein and phycoerythrin is described in more detail (Ledbetter, et al., 1985, in Perspectives in Immunogenetics and Histocompatibility, ASHI, New York, 6, 119-129 and 325-340).

Cell Culture. Blood or lymphoid k lymphoid cells were cultured at 5-10 x 1 (K ml, in quadruplicate in 96-tube micro-titration plates, containing 200 µl of RPMI-1640 medium, enriched with 15% fetal bovine serum. %, antibiotics, glutamine and pyruvate (R15). After 1 to 7 days, cells were boosted with 0.5 µGi of 3H-thymidine per tube (Uev / England Nuclear,

6.7 Ci / mmol; 1 Ci = 37), for 18 hours. Then, cells were collected on glass fiber filters with a cell collection device and radioactivity was measured in a scintillation counter. In some experiments, antibodies or factors were added at various time points after starting cultures; In these experiments, proliferation was measured at 3 ² day.

Co-stimulation factors. Purified BCGF was purchased from Citokine Technology ”(Buffalo, New

York and contained no detectable IL-1, IL-2 or interferon activity. This BCGE was prepared according to the method of Maizel and his collaborators (Maizel, et al., 1982, Proc. Nat. Acad. Sei. USA 79: 5998), which demonstrated that the main BCGF activity in this material resides in a species 12-kDa, hereinafter referred to as BCG? (lower) (Mehta, et al., 1985, J. Immunol. 135: 3298). The purification steps included preparative scale DEAE affinity chromatography, followed by hydroxylapatite column chromatography. IL-1 purified to homogeneity was a generous gift from Dr. Steven Dower (Dower, et al., 1985, J. Exp. Ied. 162: 501). Recombinant IL-2 was kindly provided by Cetus Corporation. TPA (12-0-tetradecanoyl-phorbol-13-acetate) was purchased from Sigma.

Detection of cell activation. Changes in cell volume induced by mAbs and / or factors were measured using a cell selector and a light angled front diffuser. Cell cycle changes in cellular RNA and DNA were measured by staining the activated cells with acridine orange and measuring the cellular RNA (red) and ADK (green) content with a cell selector, according to the method of Darzynkiewics et al. (Darzynkievãcz, et al., 1980, Proc. Llat. Acad. Sci. USA 77: 6697-6702). Changes in the relative levels of cell surface antigens were monitored using mAbs directly conjugated to fluorescein and then quantified by direct IP fluorescence levels with an Epics V cell selector.

Biochemical Characterization of Bp5Q. Bp5O immunoprecipitation was performed on the surface of 1251-labeled tonsillar cells, as described (Ledbetter, et al., 1985, J. Immunol. 134: 4250-4254). Isolated antigens were electrophoresed on 10% SDS polyacrylamide gels, without reduction. Gels were visualized using auto-radiography at -70 ° C and Cronex lighting plus amplification screens (Dupont).

5.2. Bp50 RBCBPTOR CHARACTERIZATION

The following subsections describe the results of tests conducted using the methods previously described.

5.2.1. IDENTIFICATION OF A CELLULAR SURFACE MARKER

50 kDa SPECIFIC TO B-CELLS, Bp50

A mAb for Bp50 was obtained by immunizing BALB / c mice with human tonsillar lymphocytes and fusing splenic cells immunized with NS-1 myeloma. One clone, G28-5, produced an IgG1 mAb that did not contain the NS-1 light chain. After scrutiny by IF analysis, it was discovered that G28-5 only reacted with normal or malignant B cells or with B cell lines. An understandable screening of normal tissues, according to established methods (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770; Ledbetter, et al., 1986, Human Immunol. 15: 30-44; Ledbetter, 1985, in Perspectives in Immunogenetics and Histocompatibility, ASHI, New York, 6, pp. 325-340 ), revealed that the G28-5 antibody reacts with negative E cells to the

- 26 mononuclear (Er-) from blood or tonsils but not with T cells not adhering to the wool tissue, to PHA-induced T cell blasts, or with blood granulocytes, monocytes, red blood cells or platelets. It reacts strongly with all seven tested B lymphoblast cell lines and with three Burkit lymphoma lines (Raji, Daudi, Hamalwa), but not with four T cell lines (CEM, HSB-2, JURKAT, and HPB-ALL). All chronic lymphocytic leukemias, tested (3/3) and 90% (9/10) of the tested B lymphomas expressed the Bp50 marker, while only 28% (2/7) of acute non-T, non-B CALLA + lymphocytic leukemias expressed

Bp50.

The restricted distribution of Bp50 in normal tissues was subsequently confirmed by quantitative bicolor immunofluorescence analysis (bichromatic IF). It was found, using an antibody (red) conjugated with phycoerythrin (PE) to the Bp35 antigen of all B cells (Bl, CD29) and an anti-Bp50 antibody conjugated to fluorescein (green), that Bp50 is expressed only in cells B, Bp35 + (fig. 1) in the blood or tonsils. The cells consistently express Bp50 levels below those of tonsillar B cells; this resembles the expression of HLA-DR (Ledbetter, et al., 1936, Human Immunol. 15: 30-44), and the expression of gp54 (Wang, et al., 1979, J. Exp. Ied. 149: 1424 -1433) which are also lower in blood B cells. Bp50 was expressed at identical levels in the subpopulations of tonsillar B cells separated in Percoll gradients in floating and dense fractions. Using the

- 27 -I mAb of the present invention conjugated with PE for the T cell marker, CD3 (T3) θ, the marker associated with NK cells, CD16 (Fc receptor) (Ledbetter, et al., 1979, Immunol. Rev. 47: 63-82), Bp5O was found not to be expressed in T cells or UK cells. Using bichromatic IF, it was also found that PHA CD3 + blasts that express high levels of IL-2 do not express Bp5O.

G28-5 antibody reacted with a single polypeptide on tonsillar lymphocytes that migrated to approximately 50 Kd under non-reducing conditions (Fig. 2A). This molecule is larger than the previously mentioned B cell markers, in the same order of molecular weight 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 Ijeukocyte Typing II, eds. Reinherz, et al., Springer Verlag, Berlin, Chap. 12 Vol. 2, 155-167; Slovin, et al., 1982, Proc. Nat. Acad. Sci. USA 79: 2649-2653; Thorley-hawson, et al., 1985, J. Immunol. 134: 3007-3012, and Fig. 2B). The exposure time of this gel was selected in such a way that the molecular weights of the other B cell markers could be easily compared with Bp50. The marker Bp39, in a different way from Bp50, was expressed in granulocytes and, Bp45, in a different way from Bp50, was restricted to B cell blasts. Antibodies to Bp39 (41-H16) and to Bp45 (MNM6, Blasto-1, Blasto-2) became available through an international establishment (Clark, et al., 1986, in leukocyte Typing II, eds. Reinherz, et al., Springer Verlag, Berlin, Chap. 12 Vol. 2, 155 -167), did not block the binding of anti-Bp50 antibodies

treated with fluorescein to B cells. Thus, based on tissue distribution, biochemical analysis and blocking studies, monoclonal antibody G28-5 recognizes a 50 kDa structure distinct from that of other cell antigens

B known.

5.2.2. Bp50 EXPRESSION IS LIMITED TO B CELLS

Studies on the distribution of cell lines and hematopoietic tissues and the detailed bichromatic flow cytometric analysis revealed that Bp50 is expressed only in B lymphocytes. As shown in Fig. 3, Bp50 is expressed in a small subpopulation of blood lymphocytes and in a large population of tonsillar lymphocytes. All Bp50 + cells in the blood and tonsils express virtually Bp35 and HLA-DR, but do not express CD2 (Fig.l) or CD3, T cell molecules or Fc IgG receptors that have been discovered in UK cells. In addition, ConA-activated CD3 + T cell blasts express IL-2 receptors but do not express Bp50,

Bichromatic flow cytometric analysis allows quantitative measurement of the density affinity relationship between two surface antigens. The remaining dense B cells in the mantle area of the secondary follicles have previously been shown to express IgM and low levels of Bp35, whereas activated B cells floating in the germinal center were IgM negative and expressed high levels of Bp35 (Ledbetter, and others, Human Immunol. 15:30). Fig. 3 demonstrates that IgM positive and IgM negative B cell subpopulations express Bp50 in equal

amounts, which indicates that Bp5O is expressed in both the remaining B cells and B cells activated in vivo.

5.3. EXTENSION OF B-CELL PROLIFERATION WITH ANTI-Bp5O ANTIBODY

As explained above, B cells can be activated with low doses of specific anti-u-chain antibodies. It has recently been discovered that the B cell specific marker, Bp35 (Bi), a 35-kDa polypeptide, could also play a role in early B cell activation: mAb 1F5 to Bp35, such as low doses of anti -u, activate B cells to increase cell volume and RNA content and become capable of responding to BCGF (Clark, et al., 1985, Proc. Nat, Acad. Sci. USA 82: 1766-1770; Gollay, et al., 1985, J. Immunol. 135: 3795-3801). However, it is interesting to compare the effect of anti-Bp50 mAb on the proliferation of untreated B cells or activated B cells with either the anti-Bp35 antibody or the anti-u antibody (Table 1). Anti-Bp35 antibodies in solution or anti-u linked to Sepharose drops, only under suitable conditions, could stimulate some proliferation of B cells (Table 1, line 1); in contrast, only anti-Bp50 antibodies did not stimulate proliferation (Table 1, line 2). However, anti-Bp50 mAb considerably increased proliferation when cultured with anti-u or anti-Bp35 drops. In this case, anti-Bp50 resembles BCGF (Table 1, line 3). Thus, it was important to determine whether anti-Bp50 in conjunction with BCGF could induce B cell proliferation.

Table 1, line 4, anti-Bp50 together with BCGF did not induce proliferation, but increased the proliferation of cells activated by either anti-u or anti-Bp35, slightly more than any of the stimulants alone. BCGF above a logarithmic scale of three, has no effect on the proliferation of dense B cells, even when anti-Bp50 is used in doses between 0.1 and 10 µg / ml.

Table 1

Increase of Anti-Ig where Anti-Bp35 induced B cell proliferation with Anti-Bp50 antibodies

Average proliferation + SE of B cells cultured with:

Line Co-stimulant Middle anti-u drops Anti-Bp35 1 none 1,212 + 547 10,219 + 462 5.539 + 308 2 Anti-Bp5O 719 + 718 38,792 + 1,329 25,465 + 616 3 BCG? 456 + 217 14.217 + 445 9,443 + 343 4 Anti-Bp50 + BCGP 1.456 + 126 54.393 + 537 46,488 + 3,387 Measured > if proliferation of cells : B tonsillar : Er (95% of

surface IgM ⁺ cells) on the third day, as described. Briefly, they were cultured in quadruplicate 2 x 10 cells with RPMI 1640 medium, containing 15% bovine fetus serum plus additives, without antibodies with monoclonal antibody 2C3 for u chains coupled to Sepharose drops (”anti-u beads ”, 50 µg / ml) or with the free 1P5 anti-Bp35 antibody (5 µg / ml). Cultures containing medium were grown

anti-u or anti-Bp35 drops, only with BCGF (5% final concentration, Cytoxine, Technology, Buffalo, New York; they had no detectable IL-1 or IL-2 activity), with anti-Bp50 (dilution of 1: 1000 ascitic fluid) as a co-stimulant.

O

After 43 hours, cells were boosted with Ή-tinii. dine and its incorporation was measured after 18 hours.

5.3.1. ANTI-Bp50 mAb INCREASES PROLIFERATION ONLY AFTER B-CELLS ARE ACTIVATED BY ANTI-Bp35 OR ANTI-u ANTIBODIES

The results in Table 1 suggest that the anti-Bp50 mAb might not induce proliferation on its own. As shown in Fig. 4, doses of anti-Bp50 between 0.05 µg to 2.0 µg / ml have no effect on the release of 3 H-thymidine. However, in the presence of optimal levels of anti-Bp35 mAb, an amount as small as 0.1 to 0.5 µg / ml of anti-Bp50 antibodies substantially increased proliferation. 50,000 to 70,000 cpm were detected in the optimal proliferation period when highly purified B cells were cultured with only anti-Bp35 plus anti-Bp50. A consistent observation was that higher doses of anti-Bp50 (greater than 2-5 µg / ml) were less effective than doses in the 100-200 ng range.

These results suggest that anti-Bp50 can act only, after B cells are activated by other signals. The data presented in Fig. 5 suggest that this is actually the case. When B cells were first activated with anti-Bp35, anti-Bp50 could be added 24-48 hours later and still increase proliferation at 49 days. In contrast, when cells were treated with anti-Bp5O, anti-Bp35 was only effective when added a few hours after the start of cultures. Similar results were found when anti-u was used instead of anti-Bp35.

5.3.2. ANTI-Bp5O mAb DOES NOT ACTIVATE B-CELLS IN GO, BUT INDUCES ACTIVATED B-CELLS TO PROGRESS OVER THE CELL CYCLE

Anti-Bp35, such as low doses of anti-u antibodies, was previously found to induce the remaining tonsillar B cells in GO to increase (Clark, et al., 1986, Leukocyte Typing II, eds., Reinherz, et al. , Springer Verlag, Berlin, Vol. 2, 455-462) (Gollay, et al., 1985, J. Immunol. 135: 3795-3801). Thus, it was of interest to compare the ability of anti-Bp50 mAb with that of anti-Bp35 mAb in its effects on cell activation. As shown in Fig. ΒΑ, dense tonsillar cells, unstimulated even after 3 to 4 days of culture, had a uniform RNA profile characteristic of cells in GO (Darzynkiewicz, 1980, Proc. Nat. Acad. Sei. USA 77 : 6697-6702). However, about 15-30% of cells stimulated with anti-Bp35 or anti-u increased the RNA 0 content that indicates entry into Gl. In contrast, neither anti-Bp50 (Fig. 6B) nor BCGF (Fig. 6C) induced significant numbers of B cells to enter Gl. Nevertheless, 2 days after activation, 0 anti-Bp35 mAb and 0 anti-Ig mAb induced 13.5% and 20.9% of cells, respectively tonsils to enter was Gl, whereas the cells

-33- / treated with anti-Bp5O (2.7%) or BCGF (3.2%) only remained at the measured control levels (2.2%). However, when either anti-Bp50 or BCGF was added together with anti-Bp35 or anti-u antibodies, the proportion of cells that entered G1 increased dramatically. Similarly, anti-Bp50 and BCGF alone did not induce B cells to enter the S phase (Table 2), but together with both anti-Bp35 and anti-u the number of cells in the phase increased three-fold. S.

Table 2

Effect of Anti-Bp5O and BCGF on cell cycle progression in tonsillar lymphocytes

Competence sign Progression sign % % ^G cells 1 s / g ₂ / m middle none 89.9 7.1 2.5 anti-Bp35 Π 80.4 14.5 3.7 anti-Ig !! 65.6 27.6 5.7 middle anti-Bp50 83.6 12.0 3.3 anti-Bp35 n 54.1 35.5 9.7 anti-Ig II 43.6 36.2 16.2 middle BCGF 85.4 11.7 2.2 anti-Bp35 II 56.6 32.6 11.6 anti-Ig ii 48.4 36.1 14.1 Percentage of cells in or S and G ₂ , holds end

with the use of the acridine orange staining procedure (Darzynkiewicz, et al., 1930, Proc. lat. Acad. Sei.

- 34 6

USA 77: 6697-6702); 1x10 dense tonsillar lymphocytes with anti-Bp35 (5 µg / ml), drops of anti-u (50 µg / ml), anti-Bp50 (0.4 µg / ml), BCGF (5%) or combinations, as indicated.

5.3.3. OPTIMAL CONDITIONS FOR INCREASING B-CELL PROLIFERATION WITH ANTI-Bp50 ANTIBODIES

Antibodies to Bp50 on their own have little or no detectable effect on the remaining dense B cells (Table 3). However, in the presence of agents that can activate B cells, such as anti-Ig, anti-Bp35 and TPA, anti-Bp50 mAb unequivocally increases proliferation. The anti-Bp50 does not costimulate with several interleukins, including purified IL-1, recombinant IL-2 and BCGF (lower). A comparison of the effects of anti-Bp50 with those of BCGF (lower) demonstrated that the same agents that were co-stimulators with anti-Bp50 were also co-stimulators with BCGF (lower) (Table 3). Of particular interest is the discovery that BCGF and anti-Bp50, taken together, were not yet co-stimulators of the remaining cells.

Table 3

Increased proliferation of B cells with antibodies or B cell growth factors

Average proliferation + S.E. of B cells cultured with:

Anti-BpSO BUÕF

Go-stimulant ________ Medium __________ (200 ng / ml) ___________ (5%)

none 96 1 267 + 15 285 + 74 anti-Ig 5.833 + 391 41,634 + 2.103 25,094 + 61 anti-Bp35 (5 ug / ml) 457 + 45 8,143 4 * 280 1.733 + 32 TPA (2 ng / ml) 7.361 + 537 21,163 + 871 13,064 + 1,030 IL-1 (10 U / ml) 264 + 2 308 + 23 221 + 8 IL-2 (100 U / ml) 204 + 34 350 + 7 220 + 11 BGGF (5%) 220 + 7 851 + 28 270 + 18

Dense Er tonsillar B lines (greater than 95% of SIgM cells) cultured for 48 hours at 2 x 10 ³ cells / tube followed by a 24 hour pulse of ^ H-thymidine prior to counting.

An amount as small as 0.05 µg of anti-Bp50 antibodies increased proliferation. In subsequent studies, an optimum dose of 0.3 µg / ml was used. A consistent observation was that when the entire molecule was used, higher doses of anti-Bp50 (greater than 2-5 µg / ml) were less effective than doses in the range 0.1-0.5 g / ml.

Human B cells are strangely sensitive to the inhibitory effects mediated by the Fc ~ 36 receptors of antibodies that bind to surface Xg (Parker, 1980, Immunol. Rev. 52: 115; Bijsterbosch, et al., 1985, J. Exp. Med. 162: 1825). Thus, it was important to compare the effectiveness of the anti-Bp50 mAb in its entirety, with that of the anti-Bp50 F (ab ') 2 fragments. The F (ab ') 2 fragments in a dose range greater than 100 times were unequivocally as affective or more effective than the entire antibody in increasing proliferation (Table 4). Thus, the anti-Bp50 mAb Fc domain is not necessary for anti-Bp50 to exert its effects and may even be an inhibitor. In other words, anti-Bp50, such as BCGF can apparently act as a soluble mediator without the aid of the cellular accessory function mediated by the Fc receptor.

Table 4 Fc domain of Anti-BpJO Antibodies is not necessary for increased B cell proliferation

Anti-Bp50 Dose (ug / ml) Average cell proliferation B grown with: Means Anti-Bp35 none - 295 + 16 269 + 27 all the Ab 0.125 278 + 32 5.140 + 20 1.25 275 + 24 4,686 + 342 12.5 163 + 15 3.852 + 203 F (ab ’) g 0.125 594 + 21 10,635 + 449 1.25 531 + 3 10,893 + 575 12.5 279 1 8 9,411 + 870 The conditions of culture cell phone were described in Table 3

5.3.4. DIFFERENCES BETWEEN ANTI-Bp5O AND BCGF (LOWER) anti-Bp50 and BCGF (lower) ACTIVITY have similar effects on B cells and are co-stimulators with the same agents (Table 3). However, several lines of evidence indicate that anti-Bp50 and BCGF used in this study apparently operate through different signals. First, Bp5O molecules, unlike BCGF (lower) receptors (Bijsterbosch, et al., 1985, J. Exp. Med. 162: 1825), express in the remaining B cells

blood (Fig. 3). Secondly, although anti-Bp50 and BGGF (lower) work more effectively when added after anti-Bp35 or anti-Ig, anti-Bp5O is clearly effective, optimally when added 12 hours after the start of cultures (Pig 8A). In contrast, BGGF (lower) could be added 24 hours after the start of cultures and would still increase proliferation optimally (Fig. 8B). These kinetic assays that took shape after the Howard and Paul approach (1983, Ann. Rev. Immunol. 1: 307), suggest that a Bp50-dependent signal may exert its effects, normally, before BCGF.

Anti-Bp50 and BCGF (lower) increase the proliferation of B cells activated with anti-Bp35 or anti-Ig (Table 3). However, the effect of aati-Bp50 and BCGF (lower) were additive in many trials (Fig. 7). Figure 9 shows the BCGF titration (lower) in an assay in which anti-Bp50 was used in its optimum concentration (0.2 µg / ml). BGGF (lower) could subsequently increase the proliferation of the remaining B cells in the presence of anti-Bp50, after activation by anti-Ig or anti-Bp35. The optimal concentrations of BCGF (lower) were 5-10%, while 25 was inhibitory. Thus, when anti-Bp50 and BCGF (lower) are used at their optimum concentrations, they still have additive effects on B cell proliferation.

Finally, the subpopulations of normal and malignant B cells differed in their responses to anti-Bp50 and BGGF (lower). For example, some blood B cells responded to BCGF (lower) but did not respond to

anti-Bp50 (Table 5). An additional activation signal such as anti-Bp35 (Table 5) or TPA (Fig. 10) was consistently required to allow blood B cells to respond to anti-Bp50. While dense tonsillar B cells do not generally respond to BCGF or anti-Bp50, floating B cells (Table 5) respond. Malignant B cells also differ in their sensitivity to response to anti-Bp50 versus BCGF. For example, some B-cell lymphomas responded to TPA plus BCGF (lower) but did not respond to TPA plus G28-5 anti-Bp50 (Fig. 10B and D). In contrast, dense tonsillar B cells and peripheral blood B cells responded to TPA plus BCGF (lower) or more anti-Bp50 (Fig. 10A and C).

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5.4. USE OF Bp-5O ANTI-Bp5O B

The ligands of the present invention can be used in vivo or in vitro, in their modified or unmodified forms to modulate the immune response. The ligands themselves can be used, for example, as an adjuvant ”to increase the immune response to a vaccine or to increase the immune response in an individual with immune suppression. Alternatively, if coupled with cytotoxin ligands or anti-poliferative agents, these modified ligands can be used to decrease the immune response, for example in an autoimmune disease or in patients undergoing transplants to obviate the risk of rejection. These modified ligands can also be used to treat malignancies that comprise cells or tumors that express the Bp5O antigen whether or not the malignant disease originates from B cells.

The ligands of the present invention and / or Bp5O itself can be used in vitro. Such applications include in vitro assays, such as immunological assays for detecting cells that express the Bp5O antigen and / or for detecting the spread, if any, of the Bp5O antigen in organic fluids. In this situation, the ligand or Bp5O can be bridged with a radioactive marker, with fluorine, with an enzyme with an enzyme substrate, dyes, etc. in addition, ligands can be used to separate and / or identify cells expressing the Bp5O antigen, in which case the ligand can be coupled to an immobile support, or to a fluorine that can be used in a FACS fluorescence activated cells).

The applications of the ligands and Bp5O of the present invention are discussed in more detail below.

5.4.1. RECEPTOR Bp5O B USES OF SUCH LIGANDS AS ANTI-BUSTION TO INCREASE CELL PROLIFERATION.

Previous studies have suggested that the factors involved in the induction of GO B cells to the G1 phase of the cell cycle are distinct from the factors or needs for transit to the S phase. This form is mainly based on studies that demonstrate that agents such as as drops in anti-Ig, or anti-Bp35 B cell activation factors, alone have little or no effect on B cell proliferation. However, these same agents can drive B cells to a point of cell activation where they are sensitive to growth factors. In contrast, growth factors such as BCGF or IL-2 alone, have no effect on the remaining B cells but increase the growth of activated B cells.

Although the present invention is not limited by any theory or explanation, the results presented here provide additional support for a source of regulation other than cell activation and growth steps. It has been shown here that the activation and proliferation signals in human B cells can be transmitted through different cell surface structures. Although anti-Bp50 activates B cells to enter the G1 phase of the cell cycle, it alone induces little or no proliferation. The anti-Bp50 mAb has the opposite effect; cannot activate

- 43 B cells, but when added even 12-24 hours after activation it can induce cell growth.

The Bp5O molecule can, presumably, function as a receptor for a ligand such as a soluble growth factor or for a signal mediated by a cell-to-cell contact (i.e., a ligand discovered on the surface of another cell).

Previous studies have identified several

BCGRs derived from T cells, which, as anti-Bp50 increase the proliferation of B cells. Low and high molecular weight forms of B cell growth factors have been identified and different types have been shown to have additive effects (Kehrl, et al., 1984, Immunol. Rev. 18: 75-96; Kishimoto, 1985, Ann. Rev. Immunol. 3: 133-157; Sv / ain, 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). Thus, Bp50 can be the receptor for one of these factors.

With the exception of IL-2 receptors and the C3d receptor, receptors on B cells for growth signals have not yet been identified. The mAb reacts with a B cell marker that expresses only on activated B cells and blocks BCGF-dependent proliferation and therefore can recognize the BCG1 * receptor or a similar structure. Bp50 appears to be distinguishable from the AB-1 marker since mAb AB-1 does not block the binding of G28-5, anti-Bp50 antibody and, unlike mAb G28-5, only reacts with activated B cells ( Jung, et al., 1984,

J. Exp. Med. 160: 1919-1924). Bp50 is found in all B cells, which does not seem to be the case for BCGF receptors, based on absorption analyzes and direct binding assays. Current data indicate that Bp50 and the receptor for low molecular weight BCGF are distinct structures. Using a rabbit hetero-antiserum, Wang and his collaborators (Wang, 1979, J. Exp. Med. 149: 1424-1433), previously described a 54 kDa glycoprotein, gp54, which, like Bp50, is expressed in all B cells, but at lower levels in blood B cells and tonsillar B cells. It is possible, but unlikely, that rabbit hetero-antiserum and anti-Bp50 recognize the same or similar structures; differently from anti-Bp50 mAb, rabbit antiserum for gp54 alone, was sufficient to stimulate B cell proliferation.

anti-Bp35 alone, unlike anti-Bp50, can activate B cells from GO to G1 and therefore can be referred to as an activation signal. Whether or not Bp35 plays a role only in the early activation of B cells is not yet perfectly clear, since anti-Bp35 antibodies can stimulate the division of some B cells (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770). Similarly, Bp50 may not function strictly as just a sign of growth ”: anti-Bp50 antibodies, together with activation signals (anti-Bp35 or anti-u) not only increase proliferation but also increase the total number of B cells entering G1 (Chart 2). In other words, anti-Bp50 as

- 45 co-stimulator works by promoting the progression of the activation (GO to Gl) and growth (G to S) phases of the cell cycle. The BCGF used in these studies also has similar activity (Fig. 6C). Thus, anti-Bp35 θ anti-Bp5O (or BCGF) appear to be more analogous to competence and progression factors described in studies to regulate the growth of fibroblasts. The way in which B cells respond to anti-Bp35 or anti-BpSO may clearly depend on their differentiation or activation states.

It has been shown here that two mAbs, anti-Bp35 (a sign of competence) and anti-Bp50 (a sign of progression), can together induce a substantial proliferation of highly purified B cells in the absence of antigen or other factors known. The natural ligands for these structures are not yet known. However, since mAb for the appropriate epitopes can mimic soluble factors and signals mediated by cell-to-cell interactions, it may be possible to use appropriate combinations of mAb to command and regulate the proliferation or differentiation of human B cells. This, in turn, will assist in vivo invention strategies for the control of human diseases such as malignant B-cell diseases, immune deficiencies and certain autoimmune diseases.

The new monoclonal antibodies, G28-5, which react with a single chain polypeptide of approximately 50 Kd expressed on the surface of human B cells, and a particular aspect of the ligands of the present invention that

- 4b can increase the proliferation of activated B cells. Since the proliferation of human B cells can be increased in an identical manner by BCGFs derived from T cells including low and high molecular weight BCGF, the activity of anti-Bp50 was compared with that of a BCGF preparation containing predominantly low molecular weight BCGF. The anti-Bp50 and BCGF (lower) proved to be very similar due to the fact that they were co-stimulators with the same activating agents (anti-Ig, anti-Bp35 and TPA) but did not co-stimulate each other or with IL-1 or IL-2. In addition, the anti-Bp50, G28-5 activity was not dependent on its Fc domain since the G28-5 F (ab ') ₂ fragments were functionally active. This suggests that soluble anti-Bp50, like soluble BCGF, does not require auxiliary cells carrying the Fc receptor to exert an effect. In addition, anti-Bp50 and BCGF are only effective in the presence of an activation stimulus. In other words, anti-Bp50 and BCGF are not competence factors ”, but rather promote the“ progression ”of B cells through the cell cycle.

Although it is possible that Bp5O may function as a receptor for a ligand such as a growth factor for B cells, several results have suggested that Bp5O is not the receptor, at least, for BCGF (lower) used in this study: it is expressed in cells blood cells whereas BCGF (lower) receptors apparently do not. Candidate structures for the BCGF (lower) receptor, other than Bp5O, are also expressed only in activated B cells. In addition, normal or malignant B cell populations differ in their sensitivity to response to anti-Bp5O versus BCGF (lower) (Table 5 and Figure 10). However, some lymphomas proliferated in response to BCGF (lower), but not in response to anti-Bp50.

Finally, in several trials, the optimal concentrations of anti-Bp50 and BCGF, together, induced more proliferation than either one alone. The anti-Bp50 simulates the activity of another BCGF, such as BCGF (superior) which is co-stimulating with anti-IgM (Ambrus, et al., 1985, J. Exp. Med. 162: 1319; Ambrus, et al., 1985 , J. Clin Invest 75: 732). This suggests that Bp50 could function as the receptor for BCGF (higher).

Although Bp50 may be a receptor for a soluble ligand, Bp50 may function as a receptor for a cell-to-cell mediated signal that regulates BCGF receptor levels and / or autocratic production. The precedence for differentiation antigens that serve as autocratic receptor threshold amplifiers comes from studies of T cells. 0 mAb for the common leukocytic antigen Lp220, increases proliferation by raising the expression of the IL-2 receptor on activated T cells (Ledbetter, et al., 1985, J. Immunol. 135: 1819). An analogous mechanism can work with anti-Bp50 and with the expression of certain BCGF receptors. Bp50 and BCGF (lower) are apparently under some coordination controls since, like IL-1 and IL-2 receptors, BCGF increases the expression of Bp50 in certain leukemic cells. The Bp50 molecule also shares similarities with the Tp44 molecule that influences the pro

- 48 production of IL-2. Antibody 9.3, anti-Tp44, has been shown to increase the proliferation of anti-CD3 or TPA-activated T cells (Ledbetter, et al., 1985, J. Exp. Med. 161: 1513). Similarly, anti-Bp50 increases the proliferation of B cells activated by anti-Bp35 or TPA. The Tp44 signal performs its function by stimulating the production of IL-2, preferably the stimulation of the growth of T cells. The Bp5O signal, presumably, could function in an analogous manner by stimulating the production of autocratic B cells (G- ordon, et al., 1984, Nature, Lond. 310: 145).

5.4.2. MODIFIED LEAGUES USED FOR IMMUNE SUPPRESSION

LOGIC OR TREATMENT OF DISEASES

According to this aspect, the ligand of the present invention can be modified by binding an anti-proliferative agent, such that the resulting molecule can be used to destroy cells that express the Bp50 antigen. Such modified ligands can be used in the treatment of autoimmune diseases in order to suppress B cell proliferation, thereby suppressing the immune response. These modified ligands can also be used as immunological suppressants in patients who have undergone transplants to prevent graft rejection. According to the previously described, cytotoxic agents that are used to suppress immune responses can be linked to the ligands of the present invention. When ligands are used that increase the proliferation of B cells, an increased effect may result as the drug will be directed to the proliferating B cells.

In another aspect, the ligands of the present invention that have been modified by binding of an anti-proliferative agent can be used to treat diseases in which tumors or cells express the Bp5O antigen. The binding of these chemotherapeutic agents to the ligands of the present invention would result in an increased increase in drug specificity for malignant cells. In addition, a particular advantage could be obtained when ss treated a malignant B cell disease with a ligand coupled to a cytotoxin, which is more effective in destroying proliferating cells, than in cells that are not proliferating; treatment with such a ligand could result in potentiation of the action of the cytotoxin.

Accordingly, chiropractic agents that can bind to the ligands of the present invention include but are not limited to the agents listed in Table 6, below, which is derived from Goodman and Giman (The Pharmacological Basis of Therapeutics, Sixth Edition, MacMillan Publishing Co ., Inc., Hew York, pp. 1249-1313, 1980) which is indicated here by reference.

⁵ ”· ζ <

Table 6

Chemotherapeutic agents that can couple with Anti-Bp50 ligands

Alkylating agent class

Type

Mustard

Nitogen

Antimetabolites

Products

Natural

Derivatives of

Ethylenimine

Alkyl sulphonates

Nitrosoureas

Triazenres

Folic acid analogs

Pyridine analogues

Purine analogues

Alkaloids of

Vinca

Antibiotics

Enzymes

Agent

Mechloroethamine

C iclofo sfamida

Melfalano

Uracil mustard

Chlorambucil

Tiotepa

Busulfan

Carmustine

Lomustine

Semustina

Bstreptozocin

Decarbazine

Methotrexate

Fluoro-uracil

Cytarabine

Azarabine

Mercaptopurine

Thioguanine

Vinblastine

Vincristine

Dactinomycin

Daunorubicin

Doxo-rubicin

Bleomycin

Mithramycin

Mitomycin

L-asparaginase

Agents

Mixed

Complexes

Plastic Coordinates

Cisplatin

Single Subs- Hydroxy-urea replaced Matil-Hydrazine derivatives Procarbazine Adrenocortical suppressors Mitotane Hormones and Adrenocorticos- Prednisone Antagonists tenoids Progestenones Hydroxyprogesterone Caproate Medroprogestenone Acetate Magestrol Acetate Estrogens Di-ethyl-stylbestrol Ethinyl estradiol Anti-estrogens Tamoxifen Androgens Testosterone propionate Fluoxymesterone Isotopes Phosphorous Sodium phosphate Radioac ti te ³² P Iodine Sodium iodide 131 _z

Any method known in the art can be used to couple the ligand to the chemotherapeutic or anti-proliferative agent. Examples of such methods have previously been listed (see Section 5, above).

-ZÍ

5.4.3. OTHER OTILIZAgCBS FROM MGAKDOS B PS Bp5O

In addition to the therapeutic applications, the ligands and Bp5O have other applications in diagnostic tests, separation schemes, etc., in vivo and in vitro.

The Bp5O receptor can be used to manufacture and / or design the ligands of the present invention. Bp5O can also be used with the ligands of the present invention in in vitro assays which require a standard to quantify the amount of Bp5O detected in a sample. Finally, Bp5O itself can be useful as a soluble factor that mediates immunity, for example, a lymphokine.

The ligands of the present invention can be used, in addition to therapeutic treatment and diagnostic assays, to identify or separate cells expressing the Bp5O antigen. In addition, if a radiolabeling compound or a radio-opaque compound binds to the ligand, the ligand can be used to visualize tumors that express the Bp5O antigen in vivo.

From the foregoing description, other possible uses will become apparent to the specialist.

6. DEPOSIT OF CELLULAR LINES

The following hybridoma was deposited at the American Type Culture Collection, Rockville, MD ”, which took on the following deposit number:

Hybridoma ATCC Deposit Number

G-28-5

HB911 The scope of the present invention is in no way limited to the deposited hybridoma, since the deposit aspect should be understood as a simple illustration of an aspect of the present invention and any equivalent cell line is covered by the Within the scope of the present invention, it is clear that several modifications of the present invention, in addition to those demonstrated and described here, will become evident to the expert, from the previous description accompanying the graphs. Such modifications should be considered within the scope of the appended claims.

Claims (38)

1, - Process for the preparation of an anti-Bp50 monoclonal antibody, characterized by the fact that: a) to propagate a hybrid continuous cell line as deposited in A.T.C.C. and to which the acceptance number HB 9110, or a mutant, recombinant or genetically constructed derivative, has been assigned; and b) collecting the antibody produced by the continuous cell line and (a) binding to a 50-qulodalton B-cell surface antigen called Bp50 and defined by the monoclonal antibody G28-5; and (b) after binding to an activated B cell, stimulating the activated B cell in such a way that it travels its cell cycle to increase the proliferation of B cells. 2. Process according to claim 1, characterized in that the antibody is cleaved to form a fraction of the antibody molecule Fv, Fab, F (ab ') ₂ or Fab'. . 3. A process according to claim 1, characterized in that the antibody molecule consists of G28-5 or any of its fractions Fv, Fab, F (ab ') ^ ^or Fab'. 4. - Process for the preparation of monoclonal antibodies anti-Bp50 antigen characterized by the fact: a) to propagate a hybridoma formed by Ci fusion) a cell capable of producing antibodies (a) that bind to a 50-guLlodalton B cell surface antigen, called Bp50 and defined by the monoclonal antibody G28-5 and ( b) that, after binding to an activated B cell, stimulate the activated B cell in such a way that it runs through its cell cycle and increases the proliferation of B cells; (ii) with a myeloma cell; and b) to collect the antibodies produced by the hybridoma. 5. A process according to claim 4, characterized in that the antibody is cleaved to form an Fv, Fab, F (ab *) or Fab 'fraction of the antibody molecule. 6. A process according to claim 4, characterized in that the antibody molecule consists of G28-5 or any of its fractions Fv, Fab, F (ab ') or Fab'. 7 Process to increase B-cell proliferation, characterized by the fact that activated B cells are treated with an effective dose of a ligand that binds to Bp50, a 50-kilodalton B cell surface antigen and defined as an antibody monoclonal G28-5, so that B cells cycle and increase their proliferation. 8. A process according to claim 7, characterized in that B cells are activated by treatment with an effective dose of a second ligand that binds to Bp35, a 35 kilodalton B cell surface antigen, in such a way that cell B progresses from stage G to stage G of the cycle 0 1 cell. 9. Process according to claim 7 or 8, characterized in that it is carried out in vivo. 10. A process according to claim 7 or 8, characterized in that it is carried out in vitro. 11. - Method according to claim 7 or 8, ^char £ ized in that the ligand contain an antibody molecule that binds to Bp50, or an Fv, Fab, F (ab ') ^ ^or Fab' molecule of the antibody that also binds to Bp50. • * B 12. A process according to claim 7 or 8, characterized in that the linker contains a monoclonal antibody molecule that binds to Bp50 or an Fv, Fab, F (ab ') or 2' fragment. Fab 'of a monoclonal antibody molecule that also binds to Bp50. 13. The method of claim 12 wherein the monoclonal antibody molecule contains G28-5, or a fragment thereof Fv, Fab, F (ab ') ₂ i ^or Fab'. 14. The method of claim 12 wherein the monoclonal antibody is obtained from a hybridoma cell line, designated by the ATCC acceptance number HB9110 or a mutant, recombinant or ge- derivative. netically constructed. 15. A process according to claim 7 or 8, characterized in that the ligand contains lymphokine, 16. The method of claim 15 wherein the lymphokine contains a B cell development factor. 17. The method of claim 15 wherein the lymphokine is located on the surface of a cell. 18. The method of claim 8 wherein the second linker contains an antibody molecule that binds to Bp35. 19. The method of claim 18 wherein the antibody that binds to Bp35 further contains a monoclonal antibody. 20. Process according to Claim 8, characterized. due to the fact that activated B cells are treated with the ligand that binds to Bp50 in a period of about 12 hours, after activation by the second ligand that binds to Bp35. 21. - Process to suppress the proliferation of cells that express Bp50, a 50 kilodalton B cell surface antigen defined by monoclonal antibody G28-5, characterized by the fact that the referred cells are treated with an effective dose of a ligand that binds to Bp50 and is coupled to an antiproliferative agent. 22. The method of claim 21 wherein the cells expressing Bp50 include B cells. 23. The method of claim 21, wherein the cells expressing Bp50 include malignant cells. 24. The method of claim 21, characterized in that it is carried out in vivo. 25. The method of claim 21, characterized in that it is carried out in vitro. 26. The method of claim 21, wherein the ligand contains an antibody molecule or an Fv, Fab, FCab ') ^ ^or Fab' fragment thereof. 27. The method of claim 26, wherein the antibody molecule contains a monoclonal antibody molecule or an Fv, Fab, F (ab ') or Fab' fragment thereof. 28. The method of claim 27 wherein the monoclonal antibody contains G28-5 or a fragment thereof Fv, Fab, F (ab ') 29. The method of claim 27 wherein the monoclonal antibody is produced from a hybridoma cell line, such as that designated by the ATCC / -60Cr acceptance number HB9110, or a mutant thereof , derives from the recombinant or genetically constructed. 30. The method of claim 21, wherein the ligand contains a lymphokine. 31. The method of claim 30 wherein the lymphokine contains a B-cell development factor. 32. The method of claim 21, 26 or 30, characterized in that the antiproliferative agent contains an alkylating agent. 33. The method of claim 21, 26 or 30, characterized in that the antiproliferative agent contains an antimetabolite. 34. The method of claim 21, 26 or 30, characterized in that the antiproliferative agent contains an antibiotic. 35. The method of claim 21, 26 or 30, characterized in that the antiproliferative agent contains a vinca alkaloid. 36. The method of claim 21, 26 or 30, characterized in that the antiproliferative agent contains an enzyme. 37. The method of claim 21, 26 or 30, characterized in that the proliferative agent contains a coordinated platinum complex. 38. The method of claim 21, 26 or 30, characterized in that the antiproliferative agent contains a radioisotope, Lisbon, April 14, 1988 The agent of brcpnedade riclusfria « J RESUME Process to increase B cell proliferation using ligands The present invention describes a process for increasing the proliferation of B cells, which consists of treating activated B cells with an effective dose of a ligand such as lymphokines, antibody molecules or their Fv fragments, which bind to Bp50 , a new B cell receptor also described in the present invention as a 50-kilodalton polypeptide, such that B cells cycle and increase their proliferation. These ligands that increase the proliferation of activated B cells can be used to regulate the proliferation and differentiation of these cells. Figure 1. ΑΝΤΙ -βρ 35 \ JtRVAE.LMO V5. ANT ^ - b _? 50 ν · Ε.κτ> ί. SÁNCrUE PEWVÉWCO V •> í «‘ '' 'i · · λζ & ς &; Χ ·,. > ·, ._ »‘ Wtj »* Ι **» * Ή J «1 ^ · l <« »·. . ’* ·· ύΤί ·.> ·· ίχ £ .Ϊ · Λ’Χ. ·% · Z TONS'LA. .;>. > -¾ <:. >? ·· «» '. '* »·'» 10 ··· '/. “« «ΛλΪίΑ * _t ';% &;' * 'V ·' · '• í.v. · ·.» * · »£' * ¹ i, · '• vw. / U.Tli · < .'-. Λ «Vju. - · »· ν · <· * η ··· *« Μ ··> · ♦ · «-. * Λ ’^ Η * Λ -Λ *’ * ’V ·’ · ί ’ffií · Figurd; 2. 30- ^ V Figure. 3. t. sanwe pe wíç «* c.o AKU-Bf 50 ONLY RED ANTl-Bp 50 VERHEVHO V5 AOT-KVl VERVE VOViSV UA ANTl-Bf 50 RED vs Green anti-CDZ. (receiverE)