NL8701371A - LIGANDS AND METHODS FOR STRENGTHENING B-CELL EXPANSION. - Google Patents

Description

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Ligands and Methods for Enhancing B Cell Expansion.

Table of contents.

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1 Introduction ............................................... .. 2 2. Background of the invention. —......................... 2 3. Summary of the invention ................. ........... 4 3.1. Definitions .................................. 8 5 4. Figure description .......... ........................ 8 5. Detailed description of the invention ............. 11 5.1. Methods used for characterization of the

Bp5Q receptor ................... 14 5.2. Characterization of the Bp50 receptor ............... 17 10 5.2.1. Identification of a B cell

Specific 50 kDa cell surface marker

Bp50 ...................................... 17 5.2.2. Bp50 expression is limited to B cells. 19 5.3. Amplification of B cell extension with 15 Anti-Bp50 antibodies ............................. 20 5.3.1. Anti-Bp50 mAh enhances expansion only after B cells are activated by anti-Bp35 or anti-u antibodies .......... 21 5.3.2. Anti-Bp50 mAb do not activate 20 B cells from GQ but do induce activated B cells to continue the cell cycle .................. 22 5.3.3. Optimal conditions for enhancement of B cell extension with anti-Bp50 antibodies 23

25 5.3.4. Differences between anti-Bp50 and BCGF

(low) activity ........................ 26 5.4. Application of anti-Bp50 ligands and Bp5Q -------.... 29 5.4.1. Bp50 receptor and use of ligands such as anti-3p50 to enhance B cell expansion .......................... 29 5.4.2. Modified ligands used for

Immunosuppression or treatment of malignancies .....................'........ 34 5.4.3. Other applications of ligands and Bp50 .. 37 35 6. Deposits of cell lines ................................... ... 37

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>. :? V »ƒ» -2- 1. Introduction

The present invention is directed to ligands such as antibody molecules or fragments of antibody molecules or other ligands such as lymphokines that bind to a 50kBa B cell surface marker, further referred to as Bp50, which plays a role in B cell extension but not in early B- cell activation. The present invention is also directed to the Bp50 B cell antigen itself. In a particular embodiment of the present invention, a G28-5 monoclonal antibody is defined which defines Bp50 and appears to play a role in the expansion of activated B cells but has no detectable effect on resting B cells. .

The ligands such as antibodies, lymphokines and fragments thereof of the present invention can be used to direct and regulate human B cell extension and / or differentiation.

In addition, the ligands of the present invention can be modified by attachment thereto of other compounds that can be used in the treatment and / or detection of malignant cells expressing the Bp50 antigen.

2. Background of the Invention The activation of resting B cells from to G1 phase of the cell cycle and the subsequent induction of activated B cells to extension are distinct steps that require distinct control mechanisms. Some agents, including mouse B cell stimulating factor-pl (BSF-pl) (Rabin, et al., 1985, Proc. Nat. Acad. Sci. 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, 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. This means that they induce B cells to grow larger, synthesize more RNA, and pass into G ^, but only do not induce DNA synthesis in B cells. Other "growth" factors such as hymane B cell growth factor (BCGF) and interleukin-2 (IL-2) cause activated B cells to go through the cell cycle and enter S phase but do not induce 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.

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A number of factors that promote B cell growth have now been described by researchers in both mouse and human systems.

These include B cell growth factors (BCGF), which are derived from several diverse sources including T cell lines or hybridomas, B cell 5 lines, or dendrite cells. Although both interleukin-1 (11-1} and inter-leukin-2 (IL-2) have been shown to enhance B cell growth, they are apparently different from certain BCGFs, for example, monoclonal antibodies (mAb) against a mouse-BCGF (O'Hara, et al., 1985,

Nature (Lond. 315: 333) or human BCGF (Ambrus, et al., 1985, J. Exp.

10 Med. 162: 1319) blocking of the BCGF activity but not of the 11-1 or 11-2 activity. Although distinguished from 11-1 or 11-2, the BCGFs themselves appear to be heterogeneous, based on biochemical data and. activity differences on different B cell subsets or costimulation assays. For example, 60-kilodaltons (kDa) high molecular weight human 15 BCGF, BCGF (high) has been identified which is different from a 12-kDa low molecular weight form, BCGF (low) (Ambrus, et al., 1985, J. Clin. Invest. 75: 732 ). Recently, the cDNA encoding a 20-kDa mouse BCGF, which has been tentatively referred to as B cell stimulation factor pl (BSF-pl), has been cloned and sequenced (Noma et al., 1986, Nature 319: 640).

The recombinant lymphokine not only has BCGF activity, but can also activate resting B cells and induce the differentiation of IgG producing cells; therefore the difference of human BCGF (high) and BCGF (low) in both its molecular weight and in its activity range.

These activation and growth signals presumably regulate cells by interacting with specific B cell surface structures. In addition to the surface Ig antigen-specific signal, several other candidate B cell surface polypeptides have been identified that may play some role in the activation or growth of B cells. For example, the cell surface receptors for 11-130 (Dower, et al., 1985, J. Exp. Med. 160: 501) and IL-2 (Robb et al., 1984, J. Exp. Med. 160: 1126 ), and recently functional 11-2 receptors on B cells have been identified (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 have yet to be fully characterized.

-4- *

Several candidate B cell surface polypeptides have been identified that may play some role 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 5 (mAb) against mouse B cell antigen produce Lyb2 activation, and Recently evidence has emerged that Lyb2 may be the receptor for BSF-pl (Yakura, 1985, Fed. Proc. 44: 1532). Likewise, we have found that suitable mAb (1F5) against a 35 kDa polypeptide, Bp35, activates human B cells from GQ to G (Clark, et al., 1985, 10 ': Proc. Nat. Acad. Sci. USA 82 : 1766-1770; Gollay, et al., J. Immunol.

135: 3795-3801). Aggregated C3d or antibodies to the 140 kDa C3d receptor, Bpl40, cause extension of T cell-dependent B 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 15: 73-76). Although BCGFs have been identified in both mouse and human, the receptors for these factors have not yet been isolated. A polyclonal antiserum which identified a 54 kDa polypeptide (gp54) on human B cells and has been shown to be produced by Wang et al. (Wang, et al., 1979, J. Exp. Med. 149: 1424-1433). the rabbit antiserum against 20 gp54 tonsillary B cells spurred division. Recently, an mAb (AB-1) against a 55-kDa, restricted B-activated antigen containing BCGF was isolated by Jung and Fu (Jung, et al., 1984, J. Exp. Med. 160: 1919-1924). -dependent extension blocks. However, whether or not anti-gp54 or AB-1 recognize a BCGF receptor is not yet known.

3. Summary of the invention

The present invention is directed to ligands that (a) bind to Bp50, a 50kDa B cell specific surface polypeptide described herein, and (b) enhance the extension of activated B cells. The invention is also directed to the Bp50 antigen itself, which is defined by monoclonal antibody G28-5 and plays a role in the extension of activated B cells. In addition, the invention is directed to ligands that bind to Bp50, but show no biological effect or function such as enhancement of the extension of activated B cells.

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The ligands of the present invention include antibody molecules, monoclonal antibody molecules, and fragments of these antibody molecules containing the antigen-combining site / or chemically modified antibodies and fragments; such 5 fragments include, but are not limited to, Fv, Fab, F (ab ') 2,

Fab1 and the like. In addition, the ligands of the present invention include 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, for example, by binding or coupling a compound to the ligand. Such compounds include, but are not limited to, cytotoxic agents, therapeutic agents, chernotherapeutic agents, labels such as radiolabels, dyes, enzymes, radicopaque compounds, and the like. The ligands of the present invention can be used in their modified or unmodified form for targeting, regulating and modifying human 3 cell extension and / or differentiation.

The present invention is based on the discovery that two human B cell differentiation antigens, Bp35 and the B cell-20 antigen, Bp50 described herein, have apparently distinct functions as signal receptors in B cell activation. Monoclonal antibodies (mAb) to Bp35 and Pb50 both give positive signals to B cells that stimulate their cycle cycle. Monoclonal antibodies to 3p35, as well as anti-Ig antibodies, basically function by activating resting B cells to become competent to enter the cell cycle phase. In contrast, a monoclonal antibody or its F (ab ') 2 fragment described herein functions against Bp5Q, a 50 kDa polypeptide expressed on all B cells, by stimulating activated B cells to progress through the cell cycle and enhancing the -30 extension of activated B cells. Monoclonal antibodies against

Bp35, as well as anti-Ig antibodies, activate tonsillar B cells and elicit low levels of B cell expansion. In contrast, anti-Bp50 monoclonal antibody alone does not lead to B cell activation or to induce B cell expansion, but together with anti-Bp35 or anti-Ig antibodies, enhances B cell extension. In this regard, the action of anti-Bp50 antibody resembles the activity of f '*: * * -6- B cell growth factors (BCGF). Only a small amount of 0.05 µg / ml of anti-Bp50 is needed to enhance extension, and like BCGF, anti-Bp50 is effective even 12 to 12 hours after B cells are activated with anti-Ig or anti-Bp35 is added. Without additional exogenous signals, anti-Bp35 and anti-Bp50 antibodies together elicit a strong expansion of purified resting B cells. These results indicate that the Bp35 and Bp50 surface molecules play a role in the regulation of B cell activation and cell cycle progression. Because of the significance of anti-Bp35 and like molecules 10 'on the effect and action of the ligands of the present invention, the publication of Clark et al., 1985, Proc.

Natl. Acad. Sci. (USA) 82: 1766-1770 incorporated herein by reference.

Although the activity of anti-Bp50 is similar to that of BCGF (low) because both anti-Bp50 and BCGF (low) do not have a costimulating effect with the same agents but both anti-Bp50 and BCGF (low) only affect activated B cells and act in a soluble form, the activity of anti-Bp50 can be distinguished from the activity of BCGF (low), in that the extension of B cells containing optimal amounts of anti-Bp50 and anti- Bp35 (or anti-Ig) is stimulated, can be further enhanced with BCGF (low) and both blood B cells and certain B cell lymphomas respond differently to anti-Bp50 versus BCGF. For optimal activity, anti-Bp50 should be added within 12 hours of B cell activation, while BCGF (low) 25 a. maintains optimal activity even when added 24 hours after activation. In addition, Bp50 is expressed on all B cells, while receptors or BCGF (low) are limited to activated B cells. Therefore, anti-Bp50 and BCGF (layer) can coordinate B cell growth in a coordinated manner, but apparently they do so via distinct signals.

In one embodiment of the present invention, the ligands that bind to Bp50 and enhance the extension of activated B cells can be used to enhance an immune response. For example, these ligands that bind Bp50 can be used as an "adjuvant" to enhance an immune response to a vaccine. On the other hand, these ligands can be used to enhance the immune response of an immunosuppressed individual.

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In another embodiment, the ligands of the invention can be chemically modified in such a way that the cells to which the ligands bind are killed. Because all B cells express the Bp50 antigen, this approach would result in a suppression of the immune response. For example, a cytotoxic drug bound to a ligand of the present invention can be used in vivo to cause immunosuppression in order to exceed limits of hist compatibility in transplant patients? on the other hand, these modified ligands can be used to control 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 coupled to a chemotherapeutic agent useful for the treatment of such neoplastic diseases. These modified ligands can be used in vivo to direct the chemotherapeutic agent to any type of malignant cell expressing the Bp50 antigen, including cells that are not B cells but do express Bp50. When using the ligands of the invention that enhance B cell proliferation, a particular advantage would be achieved when treating B cell malignancies where the chemotherapeutic agent bound to the ligand comprises a substance more effective in killing expanding cells ; in this case an enhancement of the drug action would be obtained.

On the other hand, the ligands of the invention can be used in vitro to identify or separate cells expressing the Bp50 antigen and / or to test body fluids for the presence of the Bp50 antigen which may or may not have been shed. In addition, the ligands of the invention can be used in vivo to image imaging cells or tumors expressing the Bp50 antigen.

The purified Bp50 antigen of the present invention can be used to prepare antibodies and to prepare or design other ligands of the invention. In addition, the Bp50 antigen could be used in assays such as diagnostic * c -8 immunoassays. In addition, Bp50 itself can be used as an agent for promoting cell immunity in vivo or in vitro.

3.1 «Definitions

As used herein, the following abbreviations will have the indicated meanings: AO = acridine orange BCGF = B cell growth factor

BCGF (high) = a 60 kDa human BCGF

BCGF (low) = a 12 kDa human BCGF

Bp35 = a 35 kDa B cell specific surface polypeptide (CD20) defined by mAb 1F5 Bp50 = a 50 kDa B cell specific surface polypeptide defined by mAb G28-5 Fv = the variable region or the antigen-combining 15 site of an antibody molecule. This can be any fragment that contains the idiotype of the molecule, including (without limitation)

Fab, F (ab '), Fab', and the like IF = immunofluorescence t Ig = immunoglobulin IL-1 - interleukin 1 IL-2 = interleukin 2 kDa = kilodaltons mAb = monoclonal antibody 25-SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis TPA = 12-0-tetradecanoylforbol-13-acetate 4. Figure description

Fig. 1. Expression of Bp50 is limited to Bp35 ± B cells. A two-color flow cytometric analysis of 50,000 cells was performed in the manner described by Clark, et al., 1985, Proc. Wet. Acad. Sci. USA 82: 1766-1770. The data are plotted as cell number versus the green fluorescence logarithm and the red fluorescence logarithm where 4-5 dots represent approximately a doubling of -9- * fluorescence. The data is provided to show auto-fluorescent negative cells. PE (red) -anti-Bp35 (1F5) versus FITC (green) -anti-Bp5Q (G28-5) staining shows that all Bp50 + cells are also Bp35 +.

FIG. 2. Biochemical comparison of Bp50 polypeptide with other 3-cell surface antigens. Immunoprecipitation of Bp50 from the 125 surface with I-labeled tonsillary cells was performed as described. Isolated antigens were treated electrophoretically on 10% SDS polyacrylamide slab gels without reduction. The gels were visualized with auto-radiography and reinforcement screens.

Panel Ailane 1, anti-Bp50 (G28-5); lane 2, anti-Bp95 (G28-8); lane 3, sepharose-goat anti-mouse Ig alone.

Exposure time: 4 days. Panel B: lane 1, anti-Bp50 (G28-5); lane 2, anti-Bp45 (3LAST-2); lane 3, anti-Bp39 (G28-1); lane 4, anti-Bp39 (41-H16); lane 5, sepharose-goat anti-mouse Ig alone.

An exposure time of 2 days was chosen so that the bands in lanes 2-4 were not overexposed and could be clearly distinguished with respect to Bp50. One of three experiments.

Fig. 3. Two-color immunofluorescence analysis of Bp50 express-20 sion. Peripheral blood or tonsillar mononuclear cells were isolated by centrifugation on Ficoll and stained with PE (red) conjugated G28-5 (anti-3p50) in combination with fluorescein (green) conjugated reference antibodies, including 2C3 (anti-IgM); 1F5 (anti-Bp35); HB10a (anti-DR); and 9.6 (anti-CD2), E receptor). Cells were analyzed with a FACS IV equipped with four decade log amplifiers in both red and green dimensions. Forward and right angle light scattering was used to exclude monocytes. Unstained cells are located on the back of the grid; red fluorescence on the right side and green fluorescence on the left.

FIG. 4. Dose response curves for enhancing expansion of dense tonsillary ErB cells by anti-Bp50 antibodies as indicated: Media only; anti-Bp50 only; anti-Bp35 (5 µg / ml) only; BCGF only; anti-Bp35 plus BCGF; anti-Bp35 plus staged doses of anti-Bp50. Mean extension ± standard error of quadruplicate samples was measured on day 3.

-10- *

Fig. 5. Anti-Bp50 mAh are very effective in enhancement enhancement when added after a B cell activation signal. Dense tonsillar Er-B cells were incubated for 4 days with media alone, anti-Bp50 (0.5 µg / ml) added at different times after incubation, anti-Bp35 (5 µg / ml) added at different times after incubation ; anti-Bp50 kept constant to which anti-Bp35 was added later at different times; anti-Bp35 kept constant to which anti-Bp50 was added to cultures at different times. During the last 10 hours, H-thymidine was added and 10. its uptake measured.

Fig. 6. Comparison of the ability of anti-Bp35 and anti-Bp50 to induce resting tonsillary B cells to exit the Gq stage of the cell cycle. Day 3 after treatment media only (-), anti-Bp35 only (-----); and Ig alone (.....), A, no additional additives; B, anti-Bp50 (0.5 µg / ml) added to each group; C, 5% BCGF added to each group. The data are plotted as a relative cell number versus the logarithm of AO red fluorescence (RNA).

Fig. 7. Kinetics of B cell extension after stimulation with anti-Bp50 versus BCGF. Dense tonsillar E-B cells were stimulated with media alone; 10% BCGF alone? anti-Bp35 only? anti-Bp50 only? anti-Bp35 + 10% BCGF? anti-Bp35 + anti-Bp50? and anti-Bp35 + anti-Bp50 + 10% BCGF. Extension was measured on the indicated days by a 3 shot H-thymidine for 18 hours. Extension was measured in quadruplicate and standard errors are indicated. One of three experiments.

FIG. 8. Times after stimulation with anti-Bp35 at which anti-Bp50 (A) or BCGF (B) optimally enhance the extension. Dense tonsillary E-B cells were stimulated in the manner shown and the extension was measured by an 18-hour burst of H-thymidine on day 3.

Media? anti-Bp35 added only at the indicated times; anti-30 Bp50 or BCGF alone; anti-Bp35 added at the start of the culture followed by addition of anti-Bp50 or BCGF at the indicated times? anti-Bp50 or BCGF added at the start of the culture followed by anti-Bp35. One of two experiments. The extension was measured in quadruplicate and standard errors are indicated. The doses used: 35 anti-Bp35, 5 µg / ml; anti-Bp50, 0.2 µg / ml? BCGF (low) 5%. The concentrations used were as follows: anti-Bp35.5 µg / ml; anti-Bp50, 0.2 µg / ml; BCGF, 5%.

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Fig. 9. Anti-Bp50 and BCGF have additive effects on B cell extension. Dense tonsillar E-B cells were stimulated with staged doses of BCGF (low) together with anti-Bp50 alone; anti-Bp35 only? anti-Ig beads only; anti-3p35 + anti-Bp50; or anti-Bp50 + 5 anti-Ig. The extension was measured on day 3 after stimulation with a 3-hour pulse of H-thymidine for 18 hours. The extension was measured in quadruplicate and standard errors are indicated. One of four experiments. The doses used for 10 cells: anti-Bp35, 5 µg / ml? anti-3p50, 0.2 µg / ml; anti-Ig beads, 50 µg / ml.

FIG. 10. Comparative effects of anti-Bp50 and BCGF on normal and malignant B cells. Peripheral blood E-B cells (A) or dense tonsillary E-B cells (C) were stimulated with or without TPA (75 ng / ml) in the presence of 10% BCGF or 1.ug/ml anti-Bp50. Two separate 3-cell lymphomas (panels B and D) were stimulated in the same manner.

The extension was measured on day 3 by incorporation of H-thymidine over a 12 hour burst. The extension was measured in quadruplicate and standard errors are indicated.

B

5. Detailed Description of the Invention The present invention is directed to ligands that (a) bind to Bp50, a 50 kDA B cell-specific surface polypeptide, and (b) enhance the extension of activated B cells. The invention is also directed to the Bp50 ahtigen itself, which is defined by mAb G28-5 and plays a role in B cell extension. In addition, the invention is directed to ligands that bind to Bp50 but show no biological effect or function such as enhancement of extension of activated B cells.

The ligands of the present invention include antibody molecules, monoclonal antibody molecules, and fragments of these antibody molecules containing the antigen-combining site that bind to the Bp5Q receptor including chemically modified antibodies and fragments; such fragments include, but are not limited to, 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 can include, but are not limited to, 35 BCGFs as well as chemically modified lymphokines and the like.

-12-

The ligands of the invention can be used in their modified or unmodified forms to modulate and regulate immune responses and. in the therapy of malignancies expressing the Bp50 antigen. These applications are discussed in more detail in chapter 5.4.

When the ligand is a monoclonal antibody or a fragment thereof, the anti-Bp50 monoclonal antibody may be prepared by any technique that leads to the production of antibody molecules through a culture of continuous cell lines. For example, the 10 hybrid ida technique originally developed by Kohier and

Milstein (1975, Nature 256: 495-497) as well as other techniques that have become more recently available such as the human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72) and the EBV hybridoma technique for preparing human monoclonal antibodies (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) and the like to the scope of the present invention.

Antibody fragments containing the idiotype of the molecule can be generated according to known techniques. For example, such fragments include, but are not limited to: the 20 F (ah ') 2 fragment that can be generated by treating the antibody molecule with pepsin; the Fab 'fragments that can be generated by reducing the disulfide bridges of the F (ab') 2 fragment; the F (ab ') 2 fragment that can be generated by treating the antibody molecule with papain; and the 2Fab or Fab fragments that can be generated by treating the antibody molecule with papain and a reducing agent to reduce the disulfide bridges.

When the ligand that binds Bp50 is a lymphokine, the lymphokine can be obtained from natural sources or, when its amino acid sequence is known or derived, the lymphokine can be prepared by chemical synthetic methods. On the other hand, when the gene sequence of the lymphokine is known, recombinant DNA techniques can be used to clone the gene into an expression vector that transcribes and translates the gene sequence into a suitable host cell.

Depending on the intended use, the ligand or suitable fragments of the ligand can be chemically modified by attachment to the ligand of one of several compounds by itself. i ♦ Λ fj -13- • r * known coupling techniques are used. Such techniques may include, but are not limited to, the use of carbodiimide, cyanogen bromide, bifunctional reagents such as glutaraldehyde, N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), Schiff base reactions, attachment to sulfhydryl groups, use of sodium isothiocyanate, or enzymatic attachment, to name just a few possibilities. When a radioisotope is to be bound to the ligand, it can also be performed via 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, CSC press, Inc., Boca Raton, Florida, Chapter 5, p. 123-139, 1984.

The chemical binding or coupling of a compound to the ligand could be directed to a site on the ligand that does not participate in the binding to Bp50. This could be reduced by protecting the binding site of the ligand before performing the coupling reaction. For example, the ligand can first be bound to Bp50 to protect the 3p50 binding site, after which the coupling reaction can be performed to attach the desired compound to available reactive sites on the Igand-Bp50 complex. Once the coupling reaction is complete, the complex can be broken to form a modified ligand to which the desired compound is bound so that the Bp50 binding site of the molecule is minimized. When the ligand comprises a monoclonal antibody such as G28-5, in which the Fc domain of the molecule is not required for the ligand to exert its effect (see chapter 5.3.3.), It may be advantageous to couple desired compounds. send to the Fc domain of the molecule.

30 The subsections below describe the new, 50-kDa B cell

surface marker, Bp50, which apparently plays a role in B cell extension, as well as ligands that bind to the new 50-kDa receptor, and their gene. fits. As an example of the ligands of the present invention, a monoclonal antibody that defines Bp5Q as well as its F (ab1) 2 fragments which, as well as BCGF B cell extension, are described. Other than anti-Bp35 mAb, which is resting B cells in GQ

Ji * -14- can induce transition to G ^ does not give anti-Bp50 mAb activation of resting B cells. Anti-Bp35 and anti-Bp50 mAb together, without any additional exogenous signals, induce strong activation and expansion of purified B cells.

5 The experiments described below also show that anti-

Bp50 activity is similar to BCGF activity but that anti-Bp50 differs from one BCGF because anti-Bp50 and low molecular weight BCGF are clearly additive and act differently on various B cell subsets or malignancies. BpSO can be a receptor for a distinct BCGF 1D 'or for a transmembrane signal that modulates BCGF production or BCGF receptor expression.

5.1. Methods Used for Characterization of the Bp50 Receptor Cell Preparations. Mononuclear cells were isolated from normal or leukemic heparanized peripheral blood by Ficoll-Hypaque gradients (Pharmacia, Piscataway, NJ). Mononuclear cells were obtained from tonsillary tissues as described by (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770). Depletion of T cells was accomplished with AET-treated sheep erythrocyte rosetting and Ficoll-Hypaque gradient separation. In some experiments, enrichment of blood B cells was accomplished by isolating cells attached to nylon wool. Monocytes were removed by incubation on plastic Petri dishes once or twice for 45 minutes at 37 ° C unless otherwise indicated. Floating or dense tonsillar B cell fractions were isolated by Percoll step gradients as described by 25 (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770). Dense tonsillar B cell preparations consistently contain more than 95% slg +

Bp35 + cells. Preparations enriched for blood B cells contained 60-85% slg + cells. B cell lymphoma cells were isolated by gently pesting lymphoma cells in the medium followed by Ficoll-30 Hypaque gradient centrifugation.

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

35 Rev. 47: 63-82). Hybrid cell cultures that secrete antibody that reac- = ·. * ij - · - Λ -15- is active towards tonsillar B cells and not towards T cells were identified by indirect immunofluorescence (I?) and analysis with a FACS IV cell sorter? cultures with antibody showing similar histogram patterns as known mAb against 5 pan 3 cell markers (e.g. Bp35) were cloned and selected for further study. The G28-5 clone produced an IgG ^ mAb that reacted only with normal or malignant B cells or B cell lines. Other mAb used in this study have been described in detail (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770? Clark, et al., 19S6, Human Immunol. 16: 100? Ledbetter, et al., 1986, Human Immunol. 15: 30-44; Ledbetter, et al., 1985, in Perspectives in Immunogenetics and Histocompatibility, ASHI, New York, 6, 325-340). These include 1F5 (IgG) anti-Bp35, HB10a (IgG), anti-HLA-DR, 2C3 (IgG.) Anti-u chain,

Zd Zd X.

G19-4 (IgG) anti-CD3, FC-2 (IgG.) Anti-Fc receptor CD16, and 9.6 (IgG) X £ ci anti-CD2 (E receptor) supplied by Dr. Paul Martin (Martin, et al., 1983, J. Immunol. 131: 180). The IgG ^ mAbs were purified by precipitation using 45% or 50% saturated ammonium sulfate and DEAE Sephacryl column chromatography, and the IgG mAbs were purified with 4 »3.

help from protein A Sepharose columns. The F (ab *) ^ fragments of G28-520 were prepared by the method of Parham (Parham, et al., 1983, J. Immunol. 131: 2895), purified on a 2-meter Sephacryl S200 column, and tested for purity by SDS-PAGE (Ledbetter, et al., 1985, J. Immunol. 135: 1819). The 2C3 mAb against u chains was conjugated to Sepharose 4B beads (Pharmacia Fine Chemicals, Uppsala, Sweden) using cyanogen-bromide coupling.

Fluorescelne and Phycoerythrin Conjugations. Purified mAb were either conjugated directly with fluorescence using fluorescence isothiocyanate (FITC; Molecular Probes) (green) following method from Goding (Goding, et al., 1976, J. Immunol. Meth. 13: 215-226), or conjugated to R-phycoerythrine (PE) (red) using SPDP

(Pharmacia) by a method detailed by us 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 on a FACS IV cell sorter.

X * -16-

Two-color immunofluorescence. Two-color studies were performed with a fluorescence-activated cell sorter (FACS IV: Becton-Dickinson, Mountain View, CA) using a 560-nm dichroic mirror to split the beam and a 580 long-5 pass filter and 540 short-pass filter (Ditric Options, Hudson, MA) arranged for the red and green photomultiplier tubes, respectively. In addition, a two-color compensator (T. Nozaki, Stanford University) was used to correct for minor overflow of green and red signals. For each two-color staining, data from. 40.00 cells collected and stored on floppy disks. Data are presented as cell number (vertical) versus the green fluorescence logarithm versus the red fluorescence logarithm on a 64 x 64 dot grid. About 4.5 dots represent doubling of the fluorescence. Unstained cells are located at the rear corner of the grid; red fluorescence is on the right side and green fluorescence on the left. Our flow cytometry system for two-color IF with fluorescence and phycoerythrine is described in more detail by (Ledbetter, et al., 1985, in Perspectives in Immunogenetics and Histocompatibility, ASHI, New York, 6, 119-129 and 20 325-340 ).

Cell culture. Blood or tonsillar lymphoid cells were grown at 5-10 x 105 ml in quadruplicate in 96-well microtiter plates containing 200 µl KPMI-1640 medium supplemented with 15% fetal bovine serum, antibiotics, glutamine, and pyruvate (R15). After 1-7 days, the cells were administered a burst of H-thymidine of 0.5 µCi / well (New England Nuclear, 6.7 Ci / mmol; 1 Ci = 37) for 18 hours. The cells were then harvested on glass fiber flashes with a cell 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 the culture; extension in these experiments was measured on day 3.

Cost-stimulating factors. Purified BCGF was purchased from Cytokine Technology (Buffalo, New York) and contained no detectable IL-1, 11-2 or interferon activity. This BCGF was prepared by the method according to Maizel et al. (Maizel, et al., 1982, Proc. Nat. Acad.

Sci. USA 79: 5998), which have demonstrated that the major BCGF active

τ · * X

In this material, there is a 12-kDa species further referred to as nBCGF (layer) "(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 5. homogeneity was a generous gift from Dr. Steven Dower (Dower, et al., 1985, J. Exp. Med. 162: 501). The Cetus Corporation was kind enough to provide recombinant IL-2. TPA (12-0-tetradeconoyl phorbol 13 acetate) was purchased from Sigma.

Detection of cell activation. Changes in cell volume, cpge-10 calls by mAb and / or factors, were measured with a cell sorter and a forward angular light scatterer. Cell cycle changes in cellular RNA and DNA levels were measured by staining activated cells with acridine orange and measuring the relative cellular RNA (red) and DNA (green) content with a cell sorter according to the method of Darzynkiewicz et al. (Darzynkiewicz, et al ., 1980, Proc Nat Acad.

Sci. USA 77: 6697-6702). Changes in relative levels of cell surface antigens were monitored using mAb, conjugated directly with fluorescein, and then quantitated by direct IP fluorescence levels with an Epics V cell sorter.

20 Biochemical characterization of Bp50. Immunoprecipitation of 125

Bp50 of surface I-labeled tonsillar cells was performed as described (Ledbetter, et al., 1985, J. Immunol. 134: 4250-4254). Isolated antigens were electrophoresed on 10% SDS polyacrylamide bibs without reduction. The gels 25 were visualized by autoradiography at -70 ° C and Cronex illumination plus enhancement screens (Dupont).

5.2. Characterization of the Bp50 receptor The subsections below describe the results of the experiments performed with the methods described above.

30 5.2.1. Identification of a B cell specific _50 kDa cell surface marker, Bp50_

An mAb against Bp50 was generated by immunizing BALB / c mice with human tonsillar lymphocytes and fusing immune spleen cells with the NS-1 myeloma. One clone, G28-5, produced an IgG ^ mAb that did not contain the NS-1 light chain. After examination with IF analysis, G28-5 was found

# * - * ·· 'V

'l'>; _ * * · -18- to react only with normal or malignant B cells or B cell lines. A comprehensive study of normal tissues by known 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 5 in Immunogenetics and Histocompatibility, ASHI, New York, 6, pp. 325-340) revealed that the G28-5 antibody reacts with E rosette negative (Er) cells from blood or tonsils but not with no nylon wool adhering T cells, PHA-induced T cell blasts, or with blood granulocytes, monocytes, red cells, or platelets. It responded strongly with 10. all. seven B lymphoblastoid cell lines tested and three Burkitt's lymphoma lines (Raji, Daudi, Namalwa), but not four T cell lines (CEM, HSB-2, JURKAT, and HPB-ALL). All tested chronic lymphocytic leukemias (3/3) and 90% (9/10) of tested B lymphomas expressed the Bp50 marker while only 28% (2/7) of non T, non B CALLA + 15 acute lymphocytic leukemias Bp50 expressed.

The limited distribution of Bp50 on normal tissues was further confirmed by quantitative two-color immunofluorescence (two-color IF) analyzes.

Using an R-phycoerythrine (PE) -conjugated anti-20 body (red) against the pan B cell antigen Bp35 (B1, CD20) and fluorescein-conjugated anti-Bp50 antibody (green), we found that Bp50 only was expressed on Bp35 + B cells (Fig. 1) in blood or tonsils. Blood B cells consistently expressed slightly lower levels of Bp50 than tonsillar B cells; this is consistent with HLA-DR expression, (Ledbetter, et al., 1986, Human Immunol. 15-30-44) and with gp54 expression (Wang, et al., 1979, J. Exp. Med. 149 : 1424-1433) which are also lower in blood B cells. Bp50 was expressed at corresponding levels on tonsillar B cell subpopulations separated on Percoll gradients into buoyant and dense fractions. Using our PE-conjugated mAb against the T cell marker, CD3 (T3), and NK cell bound marker, CD16 (Fc receptor) (Ledbetter, et al., 1979, Immunol. Rev. 47: 63-82), we found that Bp50 was not expressed on T cells or NK cells. Using two-color IF, we also found that CD3 + PHA blasts expressing high levels of IL-2 receptors did not express Bp50.

jW1 -Λϊ .i # ^ i1 f ‘'· 1 -19-

The G28-5 antibody reacted with a single polypeptide on tonsillary lymph ocytes, which migrated at approximately 50 Kd under non-reducing conditions (Fig. 2A). This molecule is larger than previously described B cell markers in the same molecular weight range as Bp39 or Bp45 (Zipf, et al., 1983, J. Immunol. 131: 3064-3072; Kitner, et al., 1981, Nature 294, 458- 460; Clark, et al., 1986, in Leukocyte Typing II, eds. Reinherz, et al. Springer Verlag, Berlin, Chapter 12 Vol. 2, 155-167; Slovin, et al., 1982, Proc. Nat Acad Sci US 79: 2649-2653; Thorley-Lawson, et al., 1985, J. Immunol. 134: 3007-3012 and Fig. 2B).

The exposure time for this gel was chosen so that the molecular weights of the other B cell markers could easily be compared to Bp50.

The Bp39 marker, unlike Bp5Q, is expressed on granulocytes and Bp45, unlike Bp50, is limited to B cell blasts. Antibodies to Bp39 (41-H16) and Bp45 (MNM6, Blast-1, Blast-2) 15 made available through an international workshop (Clark, et al., 1986, in Leukocyte Typing II, eds. Reinherz, et al., Springer Verlag, Berlin, Chapter 12 Vol. 2, 155-167) did not block the binding of fluorescent anti-3p50 antibodies to B cells. Therefore, based on tissue distribution, biochemical analysis, and blocking studies, the G28-5 monoclonal antibody recognizes a 50-Kd structure different from other known B cell anti-genes.

5.2.2. Bp50 expression is limited to B cells. Both hematopoietic tissue and cell line distribution studies and detailed two-color flow cytometric analyzes revealed that Bp50 is expressed only on B lymphocytes. As shown in Figure 3, Bp50 is expressed on a small subset of blood lymphocytes and on a large population of tonsillar lymphocytes. Virtually all Bp5Q + cells in both blood and tonsils also expressed 3p35 and HLA-DR, but did not express the CD2 (Figure 1) or CD3, T cell molecules or the IgG Fc receptors found on NK cells. . Furthermore, ConA-activated CD3 + T cell blasts expressed IL-2 receptors, but did not express Bp50.

35 Two-color flow cytometric analyzes allow a quantitative measurement of the density relationship between two surface 1 ·. > «1 **" v ft * ~ -20 antigens. We have previously been shown that the dense resting B cells in the mantle zone of secondary follicles express IgM and low levels of Bp35, while the floating, activated B cells in the germinal center are IgM negative and elevated levels of 5 Expressing Bp35 (Ledbetter, et al., Human Immunol. 15:30).

Fig. 3 shows that both IgM positive and IgM negative B cell subsets expressed Bp50 in equal amounts, indicating that Bp50 expresses both resting B cells and B cells activated in vivo.

10 5.3, Enhancement of B cell extension _with anti-Bp50 antibody_

As previously explained, B cells can be activated with low doses of anti-u chain-specific antibodies. We recently found that the B cell-specific marker Bp35 (Bl), a 35-kDa polypeptide, may also play a role in early B cell activation: it activates 1F5 mb against Bp35 as well as low doses of anti-u antibody, B cells "to increase cell volume and RNA content and to responsiveness to BCGF (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770; Gollay, et al., 1985, J. Immunol. 135: 3795-3801) It was therefore interesting to note the effect of anti-Bp50 mAb in the extension of untreated B cells or B cells activated with either anti-Bp35 or Compare anti-u antibodies (Table A) Anti-Bp35 in solution or Sepharose beads bound anti-u antibodies could only stimulate some B cell extension under appropriate conditions (Table A 25, line 1); Bp50 antibodies alone do not extend (Table A, line 2) However, anti-Bp50 mAb significantly enhanced the extension when cultured with anti-u beads or with anti-Bp35.

In this respect it seemed. anti-Bp50 on BCGF (Table A, line 3). It was therefore important to determine whether anti-Bp50 and BCGF together could elicit B cell extension. As illustrated in Table A, line 4, anti-Bp50 and BCGF together did not elicit extension, but extension of either anti-u or anti-Bp35 activated cells was slightly more enhanced than by any stimulant alone. BCGF over a three log range, when used with anti-Bp50 without other signals, had no effect on the expansion of dense B cells, even when anti-Bp50 was used at doses ranging from 0.1 to 10 µg / ml .

.. i. j --vs -4 "" ""> * f • W 5 * Δ * »-21-

TABLE A

Amplification of anti-Ig or anti-Bp35 induced B cell extension with anti-Bp50 antibodies

Mean extension ± S.E. of B cells, cultured with: 5 line Co-stimulant_media_anti-u-granules_anti-Bp35 1 none 1,2121547 10,219 ± 462 5,539 + 308 2 anti-Bp50 719 ± 718 38,792 + 1,329 25,465 + 616 3 BCGF 456 ± 217 14,217 ± 445 9,443 +343 4 1.456Ü26 54.393 + 2.537 46.488 + 3.387

10 + BCGF

• 4-

Expansion of dense Ertonsillar B cells (95% surface IgM cells) was measured on day 3 as described. Briefly, 2 x IQ5 cells / 200 µl well were grown in quadruplicate for 48 hours with RPMI 1640 medium containing 15% fetal bovine serum plus additives without antibody or with either 2C3 monoclonal anti-u chain antibody coupled to sepharose beads ("anti- u beads, 50 µg / ml) either • free 1F5 anti-Bp35 antibody (5 µg / ml). Cultures containing media, "anti-u beads", or anti-Bp35, were alone or measured BCGF (5% final Concentration, Cytokine Technology, Buffalo, New York; without detectable IL-1 or IL-2 activity), cultured with anti-BpSO (1: 1000 dilution of ascites) as costimulants. After 40 hours, the 3 cells were boosted with H-thymidine, and recorded numbers were measured after 18 hours.

5.3.1. Anti-Bp5Q mAb enhances extension only after B cells have been activated by anti-Bp35 or anti-u antibodies

The results in Table A indicate that anti-Bp50 mAb alone would not be able to induce extension. As shown in Figure 4, doses of anti-3p50 ranging from 0.05 µg to 2.0 µg / ml 3 had no effect on the uptake of H-thymidine. However, in the presence of optimal levels of anti-Bp35 mAb, small amounts of anti-Bp50 antibodies of only 0.1 to 0.5 µg / ml significantly enhanced the extension. Large amounts as high as 50,000 to 70,000 cpm were detectable at the optimal time of expansion when highly purified B cells were grown only with anti-Bp35 plus anti-Bp50.

v · * -22-

Consistently, higher doses of anti-Bp50 (greater than 2-5 µg / ml) were observed to be less effective than doses in the range of 100-200 ng.

These results indicated that anti-Bp50 can only function after B cells have been activated by other signals. The data shown in Fig. 5 indicate that this is indeed the case. When B cells were first activated with anti-Bp35, anti-Bp50 could be added as late as 24-48 hours later and still enhance extension on day 4. In contrast, in the case that the cells were first treated with anti-Bp50, anti-Bp35 was effective only when within a 10; couple of hours. was added after the start of the culture. Similar results were found when anti-u was used instead of anti-Bp35.

5.3.2. Anti-Bp50 mAb do not release B cell activation but do induce 15 activated B cells to continue through the cell cycle.

It was previously found by us that anti-Bp35, as well as small doses of anti-u antibodies, elicit quiescent tonsillary B cells in GQ (Clark, et al., 1986, Leukocyte Typing II, eds., 20 Reinherz, et al Springer Verlag, Berlin, Vol. 2, 455-462} and transition to the G phase of the cell cycle (Gollay, et al., 1985, J. Immunol. 135: 3795-3801). therefore, interesting to compare the ability of anti-Bp50 mAb to that of anti-Bp35 mAb in their effects on B cell activation As shown in Figure 6A, unstimulated dense tonsillary B cells even after 3- 4 days in culture a uniform RNA profile characteristic of cells in G ^ (Darzynkiewicz, 1980, Proc. Nat. Acad. Sci. USA 77: 6697-6702). About 15-30% of with anti-Bp35 or anti- u stimulated cells, however, had an increased RNA content indicating transition to G ^. In contrast, neither anti-Bp50 (Fig. 6B) nor BCGF (Fig. 60) induced only a transition of G ^. n significant numbers of B cells to G ^. For example, 2 days after activation, anti-Bp35 and anti-Ig mAb induced 13.5% and 20.9% of tonsillar cells to transition to G ^, respectively, while cells containing only anti-Bp50 (2.7%) or BCGF (3.2%) were treated 35, remained at media control levels (2.2%). However, when either anti-Bp50 or BCGF together with anti-Bp35 or anti-u antibodies, C-7 n -r, v / V j J ƒ 1 1 *.

-23- added, the amount of cells that switched to G ^ increased dramatically. Similarly, anti-Bp50 and BCGF alone did not induce B cells to transition to the S phase (Table B), but together with either anti-B35 or anti-u they led to a two to three fold increase in the number of cells in the S phase.

TABLE B

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

Competency- Progression- GQ% Cells S / G ^ / M

IQ signal signal G ^ media none 89.9 7.1 2.5 anti-Bp35 * 80.4 14.5 3.7 anti-Ig "65.6 27.6 5.7 media anti-Bp50 83.6. 12.0 3.3 15 anti-Bp35 ”54.1 35.5 9.7 anti-Ig" 43.6 36.2 16.2 media BCGF 85.4 11.7 2.2 anti-Bp35 n 56, 6 32.6 11.6 anti-Ig "48.4 36.1 14.1 20 Percentage of cells in G ^, G ^, or S and G ^ determined using the acridine orange staining procedure (Darzynkiewicz, et al ., 1980, Proc.

g

Wet. Acad. Sci. USA 77: 6697-6702); 1 x 10 dense tonsillary lymphocytes with anti-Bp35 (5 µg / ml), anti-u on beads (50 µg / ml), anti-Bp50 (0.4 µg / ml), BCGF (5%) or combinations as shown.

5.3.3. Optimal conditions for enhancement of B cell extension with anti-Bp50 antibodies

Antibodies to Bp50 alone have little or no detectable effect on dense resting B cells (Table C). However, in the present age of agents that can activate B cells such as anti-Ig, anti-Bp35 and TPA, anti-Bp50 mAb markedly enhanced extension.

-24-

Anti-Bp50 showed no costimulation with various interleukins, including purified IL-1, recombinant IL-2 and BCGF (low). A comparison of the effects of anti-Bp50 with those of BCGF (low) showed that the same agents that co-stimulated with anti-Bp50 also co-stimulated with BCGF (low) (Table C). Of particular interest was the finding that BCGF and anti-Bp50 together did not yet yield resting cell costimulation.

TABLE · C

Enhancement of B cell extension with anti-Bp50 10 'antibodies or B cell growth factor

Mean extension ± S.E. of B cells, characterized by:

Co-stimulant media anti-Bp50 BCGF

(200 ng / ml) (5%) 15 none 96 ± 1 267 ± 15 285 ± 74 'anti-Ig 5,833 ± 391 41,634 ± 2,103 25,094 ± 61 anti-Bp35 457 + 45 8,143 ± 280 1,733 ± 32 (5 µg / ml) TPA (2 ng / ml) 7,361 ± 537 21,163 ± 871 13,064 ± 1,030 20 IL-1 (10 U / ml) 264 ± 2 308 ± 23 221 ± 8 11-2 (100 U / ml) 204 ± '34 350 ± 7 220 ± 11 BCGF (5%) 220 ± 7 851 ± 28 270 ± 18

Dense Ertonsillar B cells (more than 95% slgM cells) cultured for 48 hours at 2 x 105 cells / well, followed by a 24 hour burst of H-thymidine before counting.

The kinetics of the anti-Bp50 amplified extension are shown in Figure 7. The extension peak occurred on day 4 and then declined regardless of whether the cells were activated with anti-Bp35 or other activators such as anti-Ig or TPA. The kinetics of the enhancement enhanced by BCGF or anti-30 Bp50 were the same.

A small amount of anti-Bp50 antibodies as low as 0.05 µg enhanced the extension. An optimal dose of 0.3 µg / ml was used in subsequent studies. A consistent observation was - ·. * '<Ί' -.v * - ·? * -25- that when whole antibody molecules were used, higher doses of anti-PB50 (greater than 2-5 µg / ml) were less effective than doses in the range 0.1-0.5 µg / ml.

Human B cells are highly sensitive to inhibitory effects exerted via the Fc receptors of surface Ig binding antibodies (Parker, 1980, Immunol. Rev. 52: 115; Bijsterbosch, et al., 1985, J. Exp. Med 162: 1825). It was therefore important to compare the efficacy of complete anti-Bp50 mAb with that of anti-3p50 F (ab '> 2 fragments. Over a 100-fold dose range, 10 F (ab1) 2 fragments were clearly as effective as or more effective than complete antibody in terms of B cell extension enhancement (Table D). Therefore, the Fc domain of anti-Bp50 mAb is not necessary for anti-Bp50 to exert its effect and may act at most inhibitory. -Bp50 as well as BCGF apparently act as a soluble mediator without assistance of Fc receptor mediated accessory cell function.

TABLE D

The Fc domain of anti-Bp50 antibodies is not required to enhance B cell extension. 20 Average B cell extension grown with: ___

Dose

Anti-Bp50 (µg / ml) media anti-Bp35 none - 295 ± 16 269 ± 27 25 complete Ab 0.125 278 ± 32 5.140 ± 20 1.25 275 ± 24 4.686 ± 342 12.5 163 ± 15 3.852 ± 203 F (ab2) 2 0.125 594 ± 21 10.635 ± 449 1.25 531 ± 3 10.893 ± 575 30 12.5 279 ± 8 9.411 ± 870

The cell culture conditions were as described in Table C.

*** 2 y Λ -26- 5.3.4. Differences between anti-Bp50 and BCGF (low) activity

Anti-Bp50 and BCGF (low) had a similar effect on B cells and were costimulatory with the same agents (Table C). However, different types of evidence indicate that anti-Bp50 and the BCGF used in this study apparently act via different signals. Firstly, Bp50 becomes molecules other than BCGF

(low) receptors (Bijsterbosch, et al., 1985, J. Exp. Med. 162: 1825) expressed on resting blood B cells (Fig. 3). Second, although both anti-Bp50 and BCGF (low) function most effectively when added after anti-Bp35 or anti-Ig, anti-Bp50 was clearly most effective when applied 12 hours after the start of ksreek was added '(fig. 8A). In contrast, BCGF (low) could be added even 24 hours after the start of the cultures and still give an optimal enhancement of the extension (Fig. 8B). These kinetics experiments, using the approach of Howard and Paul (1983, Ann. Rev. Immunol. 1: 307) as a model, indicate that a Bp50 dependent signal can normally exert its effect on BCGF.

Both anti-Bp50 and BCGF (low) enhanced extension of B-20 cells activated with anti-Bp35 or anti-Ig (Table C). However, the effect of anti-Bp50 and BCGF (low) was additive in many experiments (Fig. 7). Fig. 9 shows a titration of BCGF (low) in an experiment using anti-Bp50 at its optimal concentration (0.2 µg / ml). BCGF (low) could further enhance the extension of resting B cells in the presence of anti-Bp50 upon activation with anti-Ig or with anti-Bp35. Optimal concentrations of BCGF (low) were 5-10%, while 25% were inhibitor. Thus, when anti-Bp50 and BCGF (layer) were both used at their optimal concentrations, they still showed additive effects on B cell extension.

Finally, both normal and malignant B cell subsets differed in their responses to anti-Bp50 and to BCGF (low). For example, some blood B cells responded to BCGF (low) but not anti-Bp50 (Table E).

An additional activation signal such as anti-Bp35 (Table E) or TPA (Fig. 10) was consistently needed to allow blood B cells to respond to anti-Bp50, while dense tonsillar B cells generally did not respond to BCGF or anti-Bp50, floating B cells did respond - ct

ƒ 'J i O / I

-27- y (Table E). B cell malignancies also differed in their response to anti-Bp50 versus BCGF. For example, some B cell lymphomas responded to TPA plus BCGF (low), but not to TPA plus G28-5 anti-BpSO (Fig. 10B and D). In contrast, dense tonsillar B cells and peripheral blood 5 B cells responded to TPA plus either BCGF (low) or anti-Pb50 (Figures 10A and C).

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?; I '</ 1 + f -vi j ~ -J & (· + t Λ -29-) 5.4 Uses of anti-Bp50 ligands and Bp50 The ligands of the present invention can be used in vivo or in vitro in their unmodified or modified form. used to modulate the immune response For example, the ligands themselves can be used as an "adjuvant" to enhance an immune response to a vaccine or to enhance the immune response of an individual in an immunosuppressive state.

Also, when cytotoxins or anti-extenders are coupled to the ligands, these modified ligands can be used to lower an immune response, for example, in autoimmune diseases or in transplant patients to avoid rejection of the grafted material. These modified ligands could also be used to treat malignancies comprising cells or tumors expressing the 3p50 antigen, regardless of whether the malignant text is originally a B cell.

Both the ligands of the present invention and / or Bp5Q itself can be used in vitro. Such uses include in vitro assays, such as immunoassays for the determination of cells expressing the Bp50 antigen and / or for the detection of any shed Bp5Q in body fluids. In this case, the ligand or Bp50 could be labeled with a radiolabel, fluorine, enzyme, enzyme substrate, dye, etc. In addition, the ligands can be used to separate and / or identify cells expressing the 3p50 antigen, in which case the ligand may be linked to an immobile support, or to a fluorine which can be used in a FACS (fluorescence activated cell sorter).

The various uses and uses of the ligands and Bp50 of the present invention are discussed in greater detail below.

30 5.4.1. Bp50 receptor and applications of ligands such as anti-Bp50 for enhancement of B cell extension

Previous studies indicated that the factors involved in the induction of B cells from GQ to the cell cycle phase differ from the factors or requirements for transition in the S phase.

. , i -30-

This model is essentially based on studies demonstrating that agents such as low doses of anti-Ig B cell activation factors or anti-Bp35 alone have little or no effect on B cell proliferation.

However, these same agents can bring B cells to a point in cell activation where they are sensitive to growth factors. In contrast, growth factors such as BCGF or IL-2 alone do not affect resting B cells but enhance the growth of activated B cells.

Although the present invention is not limited to any theory or explanation, the results presented herein provide further support for a model of distinct regulation of B cell activation and growth steps. It has been shown herein that activation and extension signals in human B cells can be passed through distinct cell surface structures. Although anti-Bp35 mAb activated B cells to transition to the cell cycle phase, it alone induced little or no extension. Anti-Bp50 mAb had the opposite effect. It could not activate B cells, but if added even 12-24 hours after activation, it could induce B cell growth.

The Bp50 molecule would likely function normally as either a receptor for a ligand such as a soluble growth factor 20 or for a cell-to-cell-mediated signal (i.e.

a ligand found on the surface of another cell). Previous studies have identified several T cell-derived BCGFs which, like anti-BP50, enhance B cell expansion. Both high and low molecular weight forms of B cell growth factors have been identified and have been shown to have additive effects for different types (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).

Bp50 could therefore be a receptor for one of these factors.

With the exception of IL-2 receptors and the C3d receptor, the receptors on B cells for growth signals have not yet been identified. The AB-1 mAb reacts with a B cell marker that is only expressed on activated B cells and blocks BCGF-dependent extension, 35. ' and therefore could recognize the BCGF receptor or a related structure. Bp50 appears to differ from the AB-1 marker because the AB-1 mAb ^ igi 3 / f '* * -31- does not block the binding of the G28-5 anti-Bp50 antibody, and unlike the G28-5 mSb alone reacts with activated B cells (Jung, et al, 1984, J. Exp. Med. 160: 1919-1924). Bp50 is present on all B cells, which does not appear to be the case for BCGF receptors based on absorption analysis and direct binding assays. Our current data indicate that Bp50 and the low molecular weight BCGF receptor are distinct structures. Using a rabbit heteroanti serum, Wang and co-workers (Wang, 1979, J. Exp. Med. 149: 1424-1433) previously described a 54-kDa glycoprotein, gp54, which, like Bp50, is expressed on all B- cells but at lower levels on blood B cells than on tonsillar B cells. It is possible, but unlikely, that the rabbit heteroantiserum and anti-Bp50 recognize the same or related structures: other than anti-BpSO mAb, the rabbit antiserum to gp54 alone was sufficient to stimulate B cell expansion.

Anti-Bp35 alone can activate B cells other than anti-Bp50

Gq to G1 and can therefore be referred to as an "activation" signal. Whether or not Bp35 functions alone in early B cell activation is not yet clear because anti-Bp35 antibodies can stimulate some B cells to divide (Clark, et al., 1985, Proc. Nat. Acad. Sci.

20 USA 82: 1766-1770). Likewise, it is possible that Bp50 does not function solely as a "growth" signal: anti-Bp50 antibodies together with activation signals (anti-Bp35 or anti-u) not only enhance extension but also increase total B cells that transition to G ^ (Table B). In other words, anti-3p50 as a costimulant promotes the progression of both the activation (Gq to G ^) and the growth (G to S) phases of the cell cycle. The BCGF used in these studies also had a similar activity (Fig. 6C). Thus, anti-Bp35 and anti-Pb50 (or BCGF) appear to be very analogous to the "competence" and "progression" factors described in studies of fibroblast growth regulation. How B cells respond to anti-Bp35 or anti-Bp50 can clearly depend on their state of differentiation or activation.

Herein, we demonstrated that two mAb, anti-3p35 (a "competence" signal) and anti-Bp50 (a "progression" signal), together can significantly expand highly purified B cells in the absence of antigen or other known factors. induce. The natural 2 -32 ligands for these structures are not yet known. However, since mAb against suitable epitopes can mimic both soluble factors and cell-cell interaction-mediated signals, appropriate combinations of mAb may be used to direct and regulate human B cell expansion or differentiation. This, in turn, will contribute to designing strategies in vivo for the control of human diseases such as B cell malignancies, immunodeficiencies and certain autoimmune diseases.

The novel G28-5 monoclonal antibody, which reacts with a 10) approximately 50 Kd single chain polypeptide expressed on the surface of human B cells, is only one particular embodiment of the ligands of the present invention that extend activated B cells can strengthen. Because human B cell extension can also be enhanced by T cell-derived BCGFs including low and high molecular weight BCGF, we compared the activity of anti-Bp50 G28-5 with that of a BCGF preparation containing predominantly low molecular weight BCGF. Anti-Bp50 G28-5 and BCGF (low) showed great similarity in that they gave costimulation with the same activators (anti-Ig, anti-Bp35 and TPA), but did not cost stimulation with each other or with IL-1 or IL-2. Furthermore, the activity of anti-Bp50 G28-5 did not depend on its Fc domain because F (ab ') 2 fragments of G28-5 were functionally active. This indicates that soluble anti-Bp50 as well as soluble BCGF does not require an Fc receptor-carrying accessory cells to exert an effect. Furthermore, both anti-Bp50 are 25. and. BCGF effective only in the presence of an activation stimulus. With. 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 Bp50 functions as a receptor for a ligand such as a B cell growth factor, several results indicate that Bp50 is at least not the receptor for the BCGF (low) used in this study: it is expressed on blood B cells while BCGF (low) receptors apparently are not expressed thereon. Candidate structures for the PCGF (low) receptor other than Bp50 are expressed only on activated B cells. Furthermore, both normal and malignant B cell populations differ in their response to anti-Bp50 versus BCGF (low) (Table E and Figure 10). For example, some B lymphomas show extension in response to BCGF (low), but not in response to anti-Bp50. Finally, in a number of experiments, optimal concentrations of anti- Bp50 and BCGF taken together are more extensive than either, anti-Bp50 mimics the activity of other BCGF, such as 5 BCGF (high), which costimulate with anti-IgM (Ambrus, et al., 1985, J. Exp. Med 162: 1319 Ambrus, et al. # 1985, J. Clin Invest 75: 732).

This indicates that Bp50 could function as the receptor for BCGF (high).

On the other hand, although Bp50 may be a receptor for a soluble ligand, Bp50 may function as a receptor for a cell-cell-mediated signal that regulates BCGF receptor levels and / or autocrine production. Evidence for the service of differentiation antigens such as enhancers of an autocrine receptor pathway comes from T cell studies. Monoclonal antibody to the Lp220 general leukocyte-15 antigen enhances extension by increasing IL-2 receptor expression on activated T cells (Ledbetter, et al., 1985, J. Immunol. 135: 1819). An analogous mechanism may work with anti-Bp50 and expression of certain BCGF receptors. Bp50 and BCGF (low) are apparently under some coordinated control because BCGF, like IL-1 and IL-2 receptors, enhances expression of Bp50 on certain leukemic cells. The Bp50 molecule also has similarities to the Tp44 molecule that influences IL-2 production. It has been shown by us and others that the 9.3 anti-Tp44 antibody enhances the extension of T cells activated by anti-CD3 or TPA (Ledbetter et al., 1985, J. Immunol. 135: 2331? Hara, et al., 1985, J Exp Med 161: 1513).

Likewise, anti-Bp50 enhances the expansion of B cells activated by anti-Bp35 or TPA. The Tp44 signal functions by stimulating IL-2 production rather than by stimulating T cell growth. The Bp50 signal could function in an analogous fashion by stimulating B-cell autocrine production (Gordon, et al., 1984, Nature, Lond. 310: 145).

Λ -: ** ·! . > t "C" -34- 5.4.2. Modified ligands, 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 anti-extension agent so that the resulting molecule can be used to kill cells expressing the Bp50 antigen. Such modified ligands can be used in the treatment of autoimmune diseases to suppress B cell proliferation and thereby suppress the autoimmune response. These modified ligands can also be used for immunosuppression of a transplant patient to prevent rejection of a grafted material. Accordingly, cytotoxic agents used to suppress the immune response can be bound to the ligands of the invention. When using ligands that enhance B cell expansion, an enhanced effect should result because the drug will be directed to B cell expansion.

In another embodiment, the ligands of the present invention that have been modified by the attachment of an anti-proliferative agent can be used to treat malignancies in which tumors or cells express the Bp50 antigen. Attachment of these chemotherapeutic agents to the ligands of the invention should lead to greater drug specificity for the malignant cells. In addition, a particular advantage would be obtained if a B cell malignancy is treated with a ligand linked to a cytotoxin that is more effective in killing expanding cells than non-expanding cells; treatment with such a ligand should enhance the action of the cytotoxin.

Accordingly, the chemotherapeutic or anti-extension agents which can be coupled to, but are not limited to, the ligands of the present invention include those listed in Table F, which table is derived from Goodman and Gilman, The Pharmacological Basis of Therapeutics, sixth edition, 35 MacMillan Publishing Co., Inc. New York, p. 1249-1313, 1980 to be incorporated by reference herein.

** "-Λ Vf Mf · /> * i 1.) J .1 ï -35-

TABLE F

Chemotherapeutic Agents That Can Be Linked to Anti-3p5Q Ligands

Class Type Medium 5 alkylating agent nitrogen mustard Mechlorethamine

Cyclophosphamide Melphalan Uracil Mustard Chlorambucil 10 ethyleneimine Thiotepa derivatives alkyl sulfonates Busulfan nitrosoureas Carmustine

Lomustine 15 Semustine

Streptozocin Tririazenes Dacarbazine Antimetabolites Folinic Acid Methotrexate Analogs 20 Pyrimidine Fluorouracil Analogs.

Cytarabme

Azaribine purine Mercaptopurine analogues.

Thioguamne 25 natural Vinca Vinblastine products alkaloids. . .

c Vxncristxne I S -36 antibiotics Dactinomycin

Daunorubicin

Doxorubicin

Bleomycin

Mithramycin

Mitomycin enzymes L-Asparaginase various agents platinum Cisplatin coordination complexes substituted Hydroxyurea urea methylhydrazine- Procarbazine derivative adrenocortical Mitotane suppressant hormones and adrenocorti- Prednisone antagonists costeroids progestins Hydroxyprogesterone caproate

Medroprogesterone Acetate

Megestrol acetate estrogens Diethylstilbestrol

Ethinyl estradiol antiestrogen. Tamoxifen Androgens Testosterone Propionate

Fluoxymesterone -ΛΚ - «·« », · '' V '" »' 5- -37- radioactive phosphorus Sodium phosphate, 32

. "- P

isotope iodine Sodium iodide, 131 ^

Any method known per se can be used to couple the ligand to the chemotherapeutic or anti-extension agent. Examples of such methods are listed above (see chapter 5.).

5.4.3. Other applications of ligands and 3p50

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

The Bp50 receptor can be used to prepare and / or design the ligands of the invention. Bp50 can also be used with the ligands of the invention in in vitro assays that require a standard for quantifying the amount of Bp50 detected in a sample, ultimately Bp50 itself can be useful as a soluble factor that promotes 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 expressing the Bp50 antigen. In addition, when an appropriate radiolabel or radiopaque compound is bound to the ligand, the ligand could be used for in vivo imaging of tumors expressing the Bp5Q antigen. Other applications will become apparent to those skilled in the art from the foregoing description.

6. Deposits of cell lines

The following hybridoma has been deposited with the American Type Culture Collection, Hockville, MD, and has been assigned the indicated registration number: 30 Hybridoma ATCC registration number G28-5 HB9110 ****. 4 * 'tf 4 ^ -38-

The present invention is not limited in scope by the deposited hybridoma because the deposited embodiment is intended as a single illustration of one aspect of the invention and all cell lines that are functionally equivalent to the scope of the invention 5. Indeed, various modifications of the invention in addition to the variants shown and described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. It is intended that such modifications fall within the scope of the accompanying claims.

10. v '·· «? ft} ^ v / 'i

Claims (67)

1. Essentially pure ligand which (a) binds to Bp50, a 50 kilo-dalton B cell surface antigen defined by monoclonal antibody G28-5 and (b) upon binding to an activated B cell stimulates the activated B cell to go through the cell cycle so that expansion of the B cell is enhanced. The ligand of claim 1, comprising an antibody molecule or an Fv, Fab, F (ab ') 2 or Fab' portion of the antibody molecule. Ligand according to claim 2, wherein the antibody molecule comprises a monoclonal antibody molecule or an Fv, Fab, Fiab1) or Fab 'portion 10 of the monoclonal antibody molecule. The monoclonal antibody molecule of claim 3, comprising G28-5, or an Fv, Fab, F (ab ') 2 or Fabr portion thereof. Monoclonal antibody molecule according to claim 3, produced by a hybridoma cell line as deposited with the ATCC with accession number HB9110, or a mutant, recombinant or genetically engineered derivative thereof. Ligand according to claim 1, comprising a lymphokine. Lymphokine according to claim 6, comprising a B cell growth factor. 8. Ligand according to claim 6, wherein the lymphokine is on the surface of a cell. The ligand according to claim 1, 2 or 6, further comprising a compound linked to the ligand. The ligand according to claim 9, wherein the compound coupled to the ligand comprises an anti-propagating agent. The ligand according to claim 9, wherein the compound coupled to the ligand comprises an alkylating agent. The ligand according to claim 9, wherein the compound coupled to the ligand comprises an anti-metabolite. Ligand according to claim 9, wherein the compound coupled to the ligand comprises an antibiotic. The ligand according to claim 9, wherein the compound coupled to the ligand comprises a vinca alkaloid. Ligand according to claim 9, wherein the compound coupled to the ligand comprises an enzyme. . - '5> -i j *> t -40- The ligand of claim 9, wherein the compound coupled to the ligand comprises a platinum coordination complex. The ligand of claim 9, wherein the compound coupled to the ligand comprises a radioisotope. The ligand of claim 9, wherein the compound coupled to the ligand comprises a fluorescent compound. 19. Essentially pure ligand that binds to Bp50, a 50 kilodalton B cell surface antigen defined by monoclonal antibody G28-5. Ligand according to claim 19, comprising an antibody molecule or an Fv, Fab, Fiab ') or Fab' portion of the antibody molecule. A ligand according to claim 20, wherein the antibody molecule comprises a monoclonal antibody molecule or an Fv, Fab, F (ab ') 2 or Fab' portion of the monoclonal antibody molecule. The ligand of claim 19, which comprises a lymphokine. Lymphokine according to claim 22, comprising a B cell growth factor. The ligand according to claim 19, 20 or 22, further comprising a compound linked to the ligand. 25 Fab; portion of the monoclonal antibody molecule. The ligand according to claim 24, wherein the compound coupled to the ligand comprises an anti-propagating agent. The ligand of claim 24, wherein the compound coupled to the ligand comprises an alkylating agent. The ligand of claim 24, wherein the ligand-linked compound comprises an anti-metabolite. The ligand of claim 24, wherein the compound coupled to the ligand comprises an antibiotic. The ligand of claim 24, wherein the compound coupled to the ligand comprises a vinca alkaloid. ATCC with accession number Hb9110, or a mutant, recombinant or genetically engineered derivative thereof. 30. The ligand of claim 24, wherein the compound coupled to the ligand comprises an enzyme. The ligand of claim 24, wherein the ligand linked compound comprises a platinum coordination complex. The ligand of claim 24, wherein the compound coupled to the ligand comprises a radioisotope. The ligand of claim 24, wherein the compound coupled to the ligand comprises a fluorescent compound. "·· ·" - 7 * 4,. ·· "J · ï * ί" *. -41- 34. Essentially pure 50 kilodalton B cell surface antigen defined by monoclonal antibody G28-5. Essentially pure 50 kilodaltone antigen according to claim 34 comprising a polypeptide. A method for enhancing B cell expansion, comprising treating activated B cells with an effective dose of a ligand that binds to Bp50, a 50 kilodalton B cell surface antigen defined by monoclonal antibody G28-5, so that the activated B cells go through the cell cycle and extension is enhanced. The method of claim 36, wherein the B cells are activated by treatment with an effective dose of a second ligand, which binds to 3p35, a 35 kilodaltons of B cell surface antigen, such that the B cell moves from the GQ to the stage of the cell cycle rings. The method of claim 36 or 37, which is performed in vivo. The method according to claim 36 or 37, which is performed in vitro. The method of claim 36 or 37, wherein the ligand comprises an antibody molecule that binds to Bp50 or an Fv, Fab, F (ab ') ^ or Fab' 20 portion of the antibody molecule that binds to Bp50. The method of claim 36 or 37, wherein the ligand comprises a monoclonal antibody molecule that binds to Bp50 or an Fv, Fab, F (ah 'or Fab * portion of a monoclonal antibody molecule that binds to 3p50. The method of claim 41, wherein the monoclonal antibody comprises molecule G28-5, or an Fv, Fab, F (ab ') 2 or Fab1 portion thereof. 43. The method of claim 41, wherein the monoclonal antibody is produced by a hybridoma cell line as deposited with the ATCC with accession number HB9110, or a mutant, recombinant or genetically engineered derivative thereof. The method of claim 36 or 37, wherein the ligand comprises a lymphokine. The method of claim 44, wherein the lymphokine comprises a B cell growth factor. The method of claim 44, wherein the lymphokine is on the surface of a cell. .- '* -42- The method of claim 37, wherein the second ligand comprises an antibody molecule that binds to Bp35. The method of claim 47, wherein the antibody that binds to Bp35 further comprises a monoclonal antibody. The method of claim 37, wherein the activated B cells are treated with the ligand that binds to Bp50 within about 12 hours of activation by the second ligand that binds to Bp35. 50. A method of suppressing the extension of cells expressing Bp5Q, a 50 kilodalton B cell surface 1.0, antigen defined by monoclonal antibody G28-5 comprising treating the cells expressing Bp50 with an effective dose of a ligand that binds to 3p50, which ligand is linked to an anti-extension agent. 51. The method of claim 50, wherein the cells expressing Bp50 comprise B cells. The method of claim 50, wherein the cells expressing Bp50 comprise malignant cells. a The method of claim 50, which is performed in vivo. The method of claim 50, which is performed in vitro. The method of claim 50, wherein the ligand comprises an antibody molecule or an Fv, Fab, F (ab ') 2 or Fab' portion of the antibody molecule. The method of claim 55, wherein the antibody molecule comprises a monoclonal antibody molecule or an Fv, Fab, FCab 'or The method of claim 56, wherein the monoclonal antibody comprises G28-5, or an Fv, Fab, Fiab '^ or Fab' portion thereof. The method of claim 56, wherein the monoclonal antibody is produced by a hybridoma cell line as deposited in the The method of claim 50, wherein the ligand is a lymphokine. includes. The method of claim 59, wherein the lymphokine comprises a B cell 35 growth factor. * - *. -43- A method according to claim 50, 55 or 59, wherein the anti-extension agent comprises an alkylating agent. The method of claim 50, 55 or 59, wherein the anti-spreading agent comprises an anti-metabolite. A method according to claim 50, 55 or 59, wherein the anti-spreading agent comprises an antibiotic. The method of claim 50, 55 or 59, wherein the anti-propagating agent comprises a vinca alkaloid. The method of claim 50, 55 or 59, wherein the anti-101 extension agent comprises an enzyme. A method according to claim 50, 55 or 59, wherein the anti-extension agent comprises a platinum coordination complex. The method of claim 50, 55 or 59, wherein the anti-propagating agent comprises a radicototope. 15 t