Note: Descriptions are shown in the official language in which they were submitted.
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1. Il~TRODUCTION
The present invention is directed to ligands, such as
antibody molecules or fragments of antibody molecules or
other ligands such as lymphokines which bind to a 50kDa B-
cell surface marker, herein referred to as Bp50, thatfunctions in B-cell proliferation 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 monoclonal antibody, G28-5, is
described that defines BpS0 and appears to play a role in
the proliferation of activated B-cells but has no detectable
effect on the proliferation of resting B-cells.
The ligands, such as antibodies, lymphokines and
fragments thereof of the present invention can be used to
direct and regulate human B-cell proliferation and/or
differentiation. In addition, the ligands of the present
invention may be modified by the attachment of other
compounds which can be used in the treatment and/or
detection of malignant cells that express the Bp50 antigen.
2. BACKGROUND OF THE INVENTION
The activation of resting B-cells from Go to Gl phase
of the cell cycle and the subsequent induction of activated
B-cells to proliferate are distinct steps requiring distinct
regulatory mechanisms. Some agents, including murine B-cell
stimulating factor-pl (BSF-pl) (Rabin, et al., 1985, Proc.
N_ 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 nactivation~ or ~competencen factors.
That is, they induce B-cells to enlarge, synthesize more
-~'
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RNA, and enter Gl, but alone they do not induce DNA
synthesis in B-cells. Other ~growth~ factors, such as human
B-cell growth factor (BCGF) and interleukin-2 (IL-2) cause
activated B-cells to traverse the cell cycle and enter S
phase but do not trigger resting B-cells (Kehrl, et al.,
1984, Immunol. Rev. 18:75-96; Muraguchi, et al., 1984, J.
Immunol. 132:176-180; Zubler, et al. 1984, J. Exp. Med.
160:1170-1183; Jung, et al., 1984, J. Exp. Med. 160:1597-
1604).
A number of factors that promote the growth of B-cells
have now been described by investigators of both murine and
human systems. These include B-cell growth factors (BCGF)
derived from several different sources including T-cell
lines or hybridomas, B-cell lines, or dendritic cells.
Although both interleukin-l (IL-l) and interleukin-2 (IL-2)
have been shown to augment B-cell growth, they apparently
are distinct from certain BCGFs. For instance, monoclonal
antibodies (mAb~ to a murine BCGF (O'Hara, et al., 198S,
~ature (Lond.) 315:333) or human BCGF tAmbrus, et al., 1985,
J. Exp. Med. 162:1319) block BCGF activity but not IL-l or
IL-2 activity. Although distinct from IL-l or IL-2, the
BCGFs themselves appear to be heterogeneous based on
biochemical data and differential activity on different B-
cell subsets or costimulation assays. For instance, 60-
kilodalton (kDa) high-molecular-weight human BCGF, BCGF
(high), has been identified that is distinct from a 12-kDa
low-molecular-weight form, DCGF (low) (Ambrus, et al., 1985,
J. Clin. Invest. 75:732). The cDNA encoding a 20-kDa murine
BCGF, tentatively designated B-cell stimulating factor pl
(BSF-pl), has recently been cloned and se~uenced (Noma et
al., 1986, ~ature 319:640). The recombinant lymphokine not
only has BCGF activity but can also activate resting B-cells
~ 5 ~ 1 33 8 781
and induce the differentiation of IgGl producing cells; thus
it differs from human BCGF (high) and BCGF (low) both in its
molecular weight and in its range of activity.
These activation and growth signals presumably regulate
cells by interacting with specific B-cell surface
structures. In addition to the antigen-specific signal
through surface Ig, several other candidate B-cell surface
polypeptides have been identified that may in some way
function in the activation or growth of B-cells. For
instance, the cell surface receptors for IL-l (Dower, et
al., 1985, J. Exp. Med. 160:501) and IL-2 (Robb et al.,
1984, J. Exp. Med. 160:1126) have been characterized, and
recently functional IL-2 receptors have been identified on
B-cells (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. Several candidate B-cell surface
polypeptides have been identified that may in some way
function in the activation or growth of B-cells. For
example, Subbarao and Mosier (Subbarao, et al., 1983,
Immunol. Rev. 69:81-97) found that monoclonal antibodies
(mAb) to the murine B-cell antigen Lyb2 activate B-cells,
and recently evidence has been presented suggesting Lyb2 may
be the receptor for BSF-pl (Yakura, 1985, Fed. Proc.
44:1532). Similarly, we have found that appropriate mAb
(lF5) to a 35 kDa polypeptide, Bp35, activates human B-cells
from Go into Gl (Clark, et al., 1985, Proc. Nat. Acad. Sci.
USA 82:1766-1770; Gollay, et al., 1985, J. Immunol.
135:3795-3801). Aggregated C3d or antibodies to the 140 ~Da
C3d receptor, Bpl40, cause proliferation of B-cells that are
T-cell dependent tMelchers, et al., 1985, Nature 317:264-
267; Nemerow, et al., 1985, J. Immunol. 135:3068-3073; Frade
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et al., 1985, Eur. J. Immunol. 15:73-76). Although BCGFs
have been identified in both mouse and man, the receptors
for these factors have not yet been isolated. Wang and
coworkers tWang, et al., 1979, J. Exp. Med. 149:1424-1433)
made a polyclonal antisera that identified a 54-kDa
polypeptide (gp54) on human B-cells and showed that the
rabbit antisera to gp54 induced tonsillar B-cells to divide.
Recently, Jung and Fu (Jung, et al., 1984, J. Exp. Med.
160:1919-1924) isolated a mAb (AB-l) to a 55-kDa antigen
restricted to activated 8-cells that blocks BCGF-dependent
proliferation. However, whether or not either anti-gp54 or
AB-l recogni2e $~a BCGF receptor is not yet known.
3. SUMMARY OF THE INVENTION
The present invention is directed to ligands which (a)
bind to Bp50, a 50kDa B-cell specific surface polypeptide
described herein, and (b) augment the proliferation of
activated B-cells. The invention is also directed to the
Bp50 antigen itself, which is defined by monoclonal antibody
G28-5 and functions in proliferation of activated B-cells.
In addition the invention is directed to ligands which bind
to Bp50, but do not demonstrate a biological effect or
function such as augmentation of the proliferation of
activated B-cells.
The ligands of the present invention include antibody
molecules, monoclonal antibody molecules and fragments of
these antibody molecules which contain the antigen combining
site or chemically modified antibodies and fragments; such
fragments include but are not limited to the Fv, Fab,
F(ab')2, Fab' and the like. In addition, the ligands of
the present invention comprise lymphokines, which can
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 linking or coupling a compound to the ligand.
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Such compounds include but are not limited to cytotoxic
agents, therapeutic agents, chemotherapeutic agents, labels
such as radiolabels, dyes, enzymes, radioopaque compounds,
and the like. The ligands of the present invention can in
their modified or unmodified for~, be used to direct,
regulate and modify human B-cell proliferation and/or
differentiation.
The present invention is based upon the discovery that
two human B-cell differentiation antigens, Bp35 and the B-
cell antigen described herein, Bp50, apparently playdistinctive roles as signal receptors in B-cell activation.
Monoclonal antibodies (mAb) to Bp35 and Bp50 both deliver
positive signals to B-cells that stimulate their transition
through the cell cycle. MAb to Bp35, like anti-Ig
antibodies, functions principally to activate resting B-
cells to become competent to enter the Gl phase of the cell
cycle. In contrast, a monoclonal antibody described herein
or its F(ab')2 fragment to Bp50, a 50-kDa polypeptide
expressed on all B-cells, functions to stimulate activated
8-cells to traverse the cell cycle and augments 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,
anti-Bp50 monoclonal antibody alone neither activates 8-
cells nor induces B-cells to proliferate, but together with
anti-Bp35 or anti-Ig antibodies, augments B-cell
proliferation. In this respect the action of anti-Bp50
antibody resembles the activity of B-cell growth factors
(BCGF). As little as 0.05 ug/ml of anti-Bp50 is needed to
augment proliferation and, like BCGF, anti-Bp50 is effective
even when added 12 to 24 hours after B-cells are activated
with anti-Ig or anti-Bp35. Without additional exogenous
signals, anti-Bp35 and anti-Bp50 antibodies together induce
strong proliferation of purified resting B-cells. These
results suggest that the Bp35 and Bp50 surface molecules
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function in the regulatory control of B-cell activation and
progression through the cell cycle. Because of the
significance anti-9p35 and like molecules have on the effect
and action of the ligands of the present invention, Clark et
al., 1985, Proc. Natl. Acad. Sci. (USA) 82:1766-1770.
Although the activity of anti-Bp50 resembles that of
BCGF ~low) since both anti-Bp50 and BCGF (low) are
costimulatory with the same agents but not with each other
and both anti-Bp50 and BCGF (low) affect only activated B-
cells and work in a soluble form, the activity of anti-Bp50
can be distinguished from the activity of BCGF (low), since
the proliferation of B-cells stimulated with optimal amounts
of anti-Bp50 and anti-Bp35 (or anti-Ig) can be augmented
further with BCGF (low) and both blood B-cells and certain
B-cell lymphomas respond differently to anti-BpS0 versus
BCGF. For optimal activity, anti-Bp50 should be added
within 12 hours of B-cell activation, whereas BCGF (low)
retains optimal activity even when added 24 hours after
activation. In addition, BpS0 is expressed on all B-cells
while receptors or BCGF (low) are restricted to activated
B-cells. Thus anti-Bp50 and BCGF (low) may coordinately
regulate B-cell growth, but apparently do so through
distinct signals.
In one embodiment of the present invention, the ligands
which bind to Bp50 and augment the profliferation of
activated B-cells can be used to increase an immune
response. For example, th~se ligands which bind BpS0 can be
used as an "adjuvant~ to increase an immune response to a
vaccine. Alternatively, these liqands can be used to
increase the immune response of an immunosuppressed
individual.
In another embodiment, the ligands of the invention can
be chemically modified so that the cells to which the
ligands bind are killed. Since all B-cells express the Bp50
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antigen, this approach would result in suppression of the
immune response. For example, a cytotoxic drug linked to a
~igand of the present invention can be used ln vivo to cause
immunosuppresion in order to cross histocompatibility
barriers in transplant patients; alternatively, these
modified ligands may be used to control autoimmune diseases.
In another embodiment of the present invention,
malignancies such as tumor cells that express Bp50 can be
treated using a ligand of the invention linked to a
chemotherapeutic agent useful in treating such neoplastic
disease. These modified ligands can be used in vivo to
direct the chemotherapeutic agent to any type of malignant
cell which expresses the Bp50 antigen including cells which
are not B-cells but which do express Bp50. When using the
ligands of the invention which augment B-cell proliferation,
a particular advantage should be realized when treating B-
cell malignancies where the chemotherapeutic agent linked to
the ligand comprises one that is more effective in killing
proliferating cells; in this instance a potentiation of the
drug action should be obtained.
Alternatively, the ligands of the invention can be used
in vitro to identify or separate cells which express the
Bp50 antigen and/or to assay body fluids for the presence of
the Bp50 antigen which may or may not be shed. In addition,
the ligands of the invention can be used in vivo in order to
image cells or tumors which express the Bp50 antigen.
The purified Bp50 antigen of the present invention can
be used to make antibodies and to make or design other
ligands of the invention. In addition the Bp50 antigen
could be used in assays such as diagnostic immunoassays.
Moreover, Bp50 itself may be used as a mediator of cell
immunity in vivo or in vitro.
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3.1. DEFINITIONS
As used herein, the following abbreviations will
have the meanings indicated:
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 lF5
Bp50 = a 50 kDa B-cell specific surface
polypeptide defined by mAb G28-5
Fv = the variable region or antigen-combining
site of an antibody molecule. This may be
any fragment which contains the idiotype of
the molecule including but not limited to
the Fab, F(ab')2, Fab', and the like.
IF = immunofluorescence
Ig = immunoglobulin
IL-l = interleukin 1
IL-2 = interleukin 2
kDa = kilodalton
mAb = monoclonal antibody
SDS-PAGE = sodium dodecyl sulphate-polyacrylamide
gel electrophoresis
TPA = 12-0-tetradecanoylphorbol-13 acetate
4. DESCRIPTION OF THE FIGURES
Fig. 1. Expression of Bp50 is restricted to Bp35+ B-
cells. Two-color flow cytometric analysis of 50,000 cells
was performed as described (Clark, et al., 1985, Proc. Nat.
Acad. Sci. USA 82:1766-1770). The data are plotted as cell
number versus log of green fluorescence and log of red
fluorescence where 4-5 dots represent approximately a
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doubling of fluorescence. The data are presented to show
autofluorescent negative cells. PE ~red) -anti-Bp35 (lF5)
versus FITC (green) -anti-Bp50 (G28-5) staining shows that
all Bp50+ cells are also Bp35+.
Fig. 2. Biochemical comparison of BpS0 polypeptide
with other B-cell surface antigens. Immunoprecipitation of
BpS0 from surface 125I-labeled tonsillar cells was performed
as described. Isolated antigens were electrophoresed on 10%
SDS polyacrylamide slab gels without reduction. Gels were
visualized with autoradiography and intensifying screens.
Panel A: lane 1, anti-BpS0 (G28-5); lane 2, anti-Bp95
~G28-8); lane 3, sepharose-goat anti-mouse Ig only.
Exposure time: 4 days. Panel B: lane 1, anti-Bp50 (G28-
5); lane 2, anti-Bp45 (BLAST-2); lane 3, anti-Bp39 (G28-1);
lane 4, anti-Bp39 (41-H16); lane 5, sepharose-goat anti-
mouse Ig only. An exposure time of 2 days was selected so
that the bands in lanes 2 to 4 were not overexposed and
could be clearly distinguished relative to Bp50. One of
three experiments.
Fig. 3. Two-color immunofluorescence analysis of Bp50
expression. Peripheral blood or tonsillar mononuclear cells
were isolated by centrifugation on Ficoll and stained with
PE (red)-conjugated G28-5 (anti-Bp50) in combination with
fluorescein (green)-conjugated reference antibodies,
including 2C3 (anti-IgM); lF5 (anti-Bp35); HBlOa (anti-DR);
and 9.6 (anti-CD2, E receptor). Cells were analyzed with a
FACS IV fitted with four decade log amplifiers in both red
and green dimensions. Forward and right angle light scatter
was used to gate out monocytes. Unstained cells are
positioned at the back of the grid; red fluorescence is to
the right and green fluorescence is to the left.
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Fig. 4. Dose response curves for augmentation of
proliferation of dense tonsillar Er- B-cells by anti-Bp50
antibodies as indicated: Media only; anti-BpS0 only anti-
Bp35 (5 ug/ml) only; BCGF only; anti-Bp3S plus BCGF; anti-
Bp3S plus graded doses of anti-BpS0. Mean proliferation +
standard error of quadruplicate samples was measured on day
3.
Fig. 5. Anti-BpS0 mAb are most effective at augmenting
proliferation if added after a B-cell activation signal.
Dense tonsillar Er- B-cells were incubated for 4 days with
media only, anti-Bp50 (0.5 ug/ml) added at different times
after incubation, anti-8p35 (5 ug/ml) added at different
times after incubation; anti-BpS0 kept constant to which
anti-Bp3S was added later at different times; anti-Bp35
kept constant to which anti-BpS0 was added to cultures at
different times. During the last lO hr 3H-thymidine was
added and its incorporation was measured.
Fig. 6. Comparison of the ability of anti-Bp3S and
anti-Bp50 to induce resting tonsillar B-cells to leave the
Go stage of the cell cycle. Day 3 post treatment media
only ( ), anti-Bp35 only (-----); and Ig only (........ ),
A, no additional additives; B, anti-Bp50 (0.5 ug/ml) added
to each group; C, 5% BCGF added to each group. Data is
plotted as relative cell number versus log of A0 red
fluorescence (RNA)-
Fig. 7. Kinetics of B-cell proliferation after
stimulation with anti-Bp50 versus BCGF. Dense tonsillar E-
B-cells were stimulated with media alone; 10% BCGF only;
anti-Bp35 only; anti-Bp50 only; anti-Bp35 + 10% BCGF; anti-
Bp35 + anti-Bp50; and anti-Bp35 + anti-Bp50 + 10% BCGF.
Proliferation was measured on the days indicated by an 18-
/3 133 87 81
hour pulse of 3H thymidine. Proliferation was measured inquadruplicate and standard errors are shown. One of three
experiments.
Fig. 8. Times after anti-Bp35 stimulation when anti-
S Bp50 (A) or BCGF (B) optimally augment proliferation. Densetonsillar E- B-cells were ~timulated as shown and
proliferation was measured by an 18 hour pulse of 3H
thymidine on day 3. Media; anti-Bp35 only added at times
indicated; anti-Bp50 or BCGF only; anti-Bp35 added at start
of culture followed by addition of anti-8pS0 or BCGF at
times indicated; anti-Bp50 or BCGF added at start of culture
followed by anti-Bp35. One of two experiments.
Proliferation was measured in quadruplicate and standard
errors are shown. Doses used: anti-Bp35, 5 ug~ml; anti-
lS Bp50, 0.2 ug/nl; BCGF (low) 5%. Concentrations used were asfollows: anti-Bp35, S ug/ml; anti-Bp50, 0.2 ug/ml; BCGF, 5%.
Fig. 9. Anti-Bp50 and BCGF have additive effects on B-
cell proliferation. Dense tonsillar E- B-cells were
stimulated with graded doses of BCGF (low) together with
anti-Bp50 only; anti-Bp35 only; anti-Ig-beads only ; anti-
Bp35 + anti-Bp50; or anti-BpS0 + anti-Ig. Proliferation was
measured on day 3 after stimulation with an 18-hour pulse of
3H thymidine. Proliferation was measured in quadruplicate
and standard errors are shown. One of four experiments.
Doses used 10 cells: anti-Bp35, S ug/ml; anti-BpS0, 0.2
ug/ml; anti-Ig-beads, 50 ug/ml.
Fig. 10. Comparative effects of anti-BpS0 and BCGF on
normal and malignant B-cells. Peripheral blood E- B-cells
(A) or dense tonsillar E- B-cells (C) were stimulated with
or without TPA (75 ng/ml) in the presence of 10% BCGF or 1
ug/ml anti-Bp50. Two separate B-cell lymphomas (panels B
and D) were stimulated in the same way. Proliferation was
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measured on day 3 by incorporation of 3H thymidine during a
12-hour pulse. Proliferation was measured in quadruplicate
and standard errors are shown.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to liqands which (a)
bind to Bp50, a 50kDa 8-cell specific surface polypeptide,
and (b) augment the proliferation of activated B-cells.
The invention is also directed to the Bp50 antigen itself,
which is defined by mAb G28-5 and functions in B-cell
proliferation. In addition, the invention is directed to
ligands which bind to Bp50 but do not demonstrate a
biological effect or function such as augmentation of
proliferation of activated B-cells.
The ligands of the present invention include antibody
molecules, monoclonal antibody molecules and fragments of
these antibody molecules which contain the antigen combining
site that binds to the Bp50 receptor including chemically
modified antibodies and fragments; such fragments include
but are not limited to the Fv, Fab, F(ab')2, Fab' and the
like. In addition, the ligands of the present invention
comprise lymphokines, which bind to the Bp50 receptor.
These may include but are not limited to BCGFs as well as
chemically modified lymphokines and the like. The ligands
of the invention can be used in their modified or unmodified
forms to modulate and regulate immune responses and in the
therapy of malignancies which express the BpS0 antigen.
These uses are discussed in more detail in Section 5.4
below.
Where the ligand is a monoclonal antibody, or a
fragment thereof, the monoclonal antibody can be prepared
against 8p50 using any technique which provides for 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)
_ l5 ~ 3~7~1
as well as other techniques which have more recently become
available, such as the human B-cell hybridoma technique
(Rozbor et al., 1983, Immunology Today 4:72) and the EBV-
hybridoma technique to produce human monoclonal antibodies
(Cole et al., 1985, Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96) and the like are
within the scope of the present invention.
Antibody fragments which contain the idiotype of the
molecule could be generated by known techniques. For
example, such fragments include but are not limited to: the
Ftab')2 fragment which can be generated by treating the
antibody molecule with pepsin; the Fab' fragments which can
be generated by reducing the disulfide bridges of the
F(ab')2 fragment; the F(ab')2 fragment which can be
generated by treating the antibody molecule with papain; and
the 2Fab or Fab fragments which can be generated by treating
the antibody molecule with papain and a reducing agent to
reduce the disulfide bridges.
Where the ligand that binds Bp50 is a lymphokine, the
lymphokine may be obtained from natural sources or if its
amino acid sequence is known or deduced the lymphokine can
be synthesized via chemical synthetic methods.
Alternatively, if the gene sequence of the lymphokine is
known, recombinant DNA techniques may be utilized to clone
the gene in an expression vector which provides for
transcription and translation of the gene sequence in an
appropriate host cell.
Depending upon its itended use, the ligand or
appropriate fragments of the ligand may be chemically
modified by the attachment of any of a variety of compounds
to the ligand using coupling techniques known in the art.
Such techniques 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
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sulfhydryl moieties, the use of sodium isothiocyanate, or
enzymatic linkage, to name but a few. Where a radioisotope
is to be attached to the ligand this may also be
accomplished via enzymatic means, oxidative substitution,
chelation etc. For a review of the chemical reagents which
can be used for protein modification see, Lundblad and
Noyes, Chemical Reagents for Protein Modification, Volume
II, CRC Press, Inc., Boca Raton, Florida, Ch. 5, pp.123-139,
1984.
The chemical linkage or coupling of a compound to the
ligand could be directed to a site on the ligand that does
not participate in binding to Bp50. This could be
accomplished by protecting the binding site of the ligand
prior to performing the coupling reaction. For example, the
ligand can first be bound to BpS0 in order to protect the
Bp50 binding site, then the coupling reaction can be
accomplished to link the desired compound to available
reactive sites on the ligand-Bp50 complex. Once the
coupling raction is complete, the complex can be disrupted
thereby generating a modified ligand to which the desired
compound is attached so that the Bp50 binding site of the
molecule is minimally affected. Where the ligand comprises
a monoclonal antibody such as G28-5, in which the Fc domain
of the molecule is not required for the ligand to exert its
effect (see Section 5.3.3. infra) it may be advantageous to
direct the coupling of desired compounds to the Fc domain of
the molecule.
The subsections below describe the new, 50-kDa B-cell
surface marker, Bp50, which apparently functions in B-cell
proliferation as well as ligands which bind to the new 50kDa
receptor, and their uses. As an example of the ligands of
the present invention a monoclonal antibody which defines
Bp50 and its ~(ab')2 fragments are also described which,
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like BCGF, augments B-cell proliferation. Unlike anti-Bp35
mAb, which can induce resting B-cells in Go to enter Gl,
anti-BpS0 mAb does not activate resting B-cells. Anti-Bp35
and anti-Bp50 mAb together, without any additional exogenous
S signals, induce strong activation and proliferation of
purified B-cells.
The experiments described below also demonstrate that
anti-Bp50 activity resembles BCGF activity but that anti-
Bp50 is distinct from one BCGF since anti-Bp50 and low
molecular weight BCGF are clearly additive and act
differently on various B-cell subsets or ~alignancies. Bp50
may be a receptor for a distinct BCGF or for a transmembrane
~ignal that modulates BCGF production or BCGF receptor
expression.
5.1. METHODS USED TO CHARACTERIZE 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 tonsillar tissues as
described (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA
82:1766-1770j. T cells were depleted with AET-treated sheep
erythrocyte rosetting and Ficoll-Hypaque gradient
separation. In some experiments blood B-cells were enriched
by isolating nylon wool adherent cells. Monocytes were
removed by incubation on plastic petri dishes one or two
times at 37 C for 45 minutes unless otherwise stated.
Buoyant or dense tonsillar B-cell fractions were isolated by
Percoll step gradients as described (Clark, et al., 1985,
Proc. Nat. Acad. Sci. USA 82:1766-1770). Dense tonsillar
B-cell preparations consistently had greater than 95% sIg+
Bp35+ cells. Blood B-cell-enriched preparations had 60-85%
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sIg+ cells. B-cell lymphoma cells were isolated by gently
teasing lymphoma cells into medium followed by Ficoll-
Hypaque gradient centrifugation.
Monoclonal antibodies. The G28-5 antibody to BpS0 was
generated by immunizing BALB/c mice with human E- tonsillar
lymphocytes and fusing immune spleen cells with the NS-l
myeloma (Xohler, et al., 1975, Nature 256:495-4g7;
Ledbetter, et al., 1979, Immunol. Rev. 47:63-82). Hybrid
cell cultures secreting antibody reactive with tonsillar B-
cells and not with T cells were identified by the use ofindirect immunofluorescence (IF) and analysis with a FACS IV
cell sorter; cultures with antibody giving histogram
patterns similar to known mAb to pan B-cell markers ~e.g.,
Bp35) were cloned and selected for further study. The G28-5
clone produced an IgGl 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 (Clar~, 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 Perspectives
_ Immunogenetics and Histocompatibility, ASHI, New York, 6,
pp. 325-340). These include lF5 (IgG2a) anti-Bp35, HBlOa
(IgG2a), anti-HL~-DR, 2C3 (IgGl) anti-u chain, G19-4 (IgGl)
anti-CD3, FC-2 (IgG2a) anti-Fc receptor CD16, and 9.6
(IgG2a) anti-CD2 (E receptor) provided by Dr. 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 DEAE Sephacryl column chromatography,
and the IgG2a mAbs were purified by the ùse of protein A
Sepharose columns. The Ftab')2 fragments of G28-5 were
prepared by the method of Parham (Parham, et al., 1983, J.
Immunol. 131:2895) purified on a 2-meter long sephacryl S200
column, and assayed for purity by SDS-PAGE (Ledbetter, et
Trade Mark
s :~
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al., 1985, J. Immunol. 135:1819). The 2C3 mAb to u-chains
was conjugated to Sepharose 4B beads (Pharmacia Fine
Chemicals, Uppsala, Sweden) using cyanogen bromide coupling.
Fluorescein and phycoerythrin conjugations. Purified
~Ab were either directly conjugated with fluorescein using
fluorescein-isothiocyanate (FITC; Molecular Probes) (green)
by the method of Goding (Goding, et al., 1976, J. Immunol.
Meth. 13:215-226), or conjugated to R-phycoerythrin (PE)
~red) by using SPDP tPharmacia) with a method we have
detailed in Ledbetter, et al., 1985, in Perspectives in
Immunogenetics and Histocompatibiity, ASHI, New York, 6,
119-129. ~ymphoid 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.
Two-color immunofluorescence. Two-color studies were
done with a fluorescence-activated cell sorter (FACS IV:
Becton-Dickinson, Mountain View, CA) by using a 560-nm
dichroic mirror to split the beam and a 580 long-pass filter
and a 540 short-pass filter (Ditric Optics, Hudson, MA) in
front of the red and green photomultiplier tubes,
respectively. In addition, a two-color compensator (T.
Nozaki, Stanford University) was used to correct for minor
spillover of green and red signals. For each two-color
stain, data from 40,000 cells were collected and stored on
floppy disks. Data are presented as cell number (vertical)
versus log green fluorescence versus log red fluorescence on
a 64 x 64 dot grid. Approximately 4.5 dots represents a
doubling of fluorescence. Unstained cells are positioned at
the back corner of the grid; red fluorescence is to the
right and green fluorescence is to the left. Our flow
cytometry system for two-color IF with fluorescein and
-~- 1338781
phycoerythrin is described in more detail ~Ledbetter, et
al., 1985, in Perspectives in Immunogenetics and
Histocompatibiity, ASHI, New York, 6, 119-129 and 325-340).
Cell culture. Blood or tonsillar lymphoid cells were
cultured at 5-10 x 105 ml in quadruplicate in 96-well
microtiter plates containing 200 ul RPMl-1640 medium
supplemented with 15% fetal bovine serum, antibiotics,
glutamine, and pyruvate (R15). After 1 to 7 days, cells
were pulsed with 0.5 uCi of H thymidine per well (New
England Nuclear, 6.7 Ci/mmol; 1 Ci=37) for 18 hours. Cells
were then harvested onto glass-fiber filters with a cell
harvester, and radioactivity was measured in a scintillation
counter. In some experiments, antibodies or factors were
added at various times after the start of cultures;
proliferation in these experiments was measured on day 3.
Costimulatory factors. Purified BCGF was purchased from
Cytokine Technology (Buffalo, ~ew York) and contained no
detectable IL-l, IL-2, or interferon activity. This BCGF
was prepared by the method of Maizel and coworkers (Maizel,
et al., 1982, Proc. Nat. Acad. Sci. USA 79:5998), who have
shown that the major BCGF activity in this material resides
in a 12-kDa species, hereinafter referred to as ~BCGF (low) n
(Mehta, et al., 1985, J. Immunol. 135:3298). The
purification steps included preparative scale DEAE affinity
chromatography followed by hydroxylapatite column
chromatography. IL-l purified to homogeneity was the
generous gift of Dr. Steven Dower (Dower, et al., 1985, J.
Exp. Med. 162:501). Recombinant IL-2 was kindly provided by
Cetus Corporation. TPA (12-0-tetradeconoyl phorbol 13-
acetate) was purchased from Sigma.
Detection of cell activation. Changes in cell volumeinduced by mAb and/or factors were measured using a cell
sorter and forward angle light-scatter. Cell cycle changes
j 21
1338781
in cellular RNA and DNA levels were measured by staining
activated cells with acridine orange and measuring relative
cellular RNA (red) and DNA (green) content with a cell
sorter by the method of Darzynkiewicz et al. (Darzynkiewicz,
et al., 1980, Proc. Nat. Acad. sci. USA 77:6697-6702).
Changes in relative levels of cell surface antigens were
monitored by use of mAb directly conjugated with fluorescein
and then quantitated by direct IF fluorescence levels with
an Epics V cell sorter.
Biochemical characterization of Bp50. Immunoprecipi-
tation of Bp50 from surface 12 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 slab gels without reduction. Gels
were visualized using autoradiography at -70C and Cronex
lightening plus intensifying screens (Dupont).
5.2. CHARACTERIZATION OF THE Bp50 RECEPTOR
The subsections below describe the results of the
experiments conducted using the methods described above.
5.2.1. IDENTIFICATION OF A B-CELL SPECIFIC
50 kDA CELL SURFACE MARKER, Bp50.
A mhb to Bp50 was raised by immunizing BALB/c mice
with human tonsillar lymphocytes and fusing immune spleen
cells with the ~S-1 myeloma. One clone, G28-5, produced an
IgGl mAb that did not contain the NS-l light chain. Upon
scrutiny by IF analysis, G28-5 was found to react only with
normal or malignant 8-cells or B-cell lines. A
comprehensive screening of normal tissues by 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
*
Trade Mark
1338781
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 nylon wool
nonadherent T cells, PHA-induced T-cell blasts, or with
blood granulocytes, monocytes, red cells, or platelets. It
reacted strongly with all seven B lymphoblastoid cell lines
tested and with three Burkitt's lymphoma lines (Raji, Daudi,
Namalwa), but not with four T cell lines (CEM, HSB-2,
J~RKAT, and HPB-ALL). All chronic lymphocytic leukemias
tested (3/3) and 90% (9/10) of B lymphomas tested expressed
the Bp50 marker while only 28% (2/7) of non T, non B CALLA
acute lymphocytic leukemias expressed Bp50.
The restricted distribution of Bp50 on normal tissues
was further confirmed by quantitative two-color
immunofluorescense (two color IF) analyses. Using an R-
phycoerythrin (PE)-conjugated antibody (red) to the pan B-
cell antigen Bp35 (Bl, CD20) and fluorescein-conjugated
anti-BpS0 antibody (green), we found that Bp50 was expressed
only on Bp35+ B-cells (Fig. 1) in blood or tonsils. Blood
B-cells consistently expressed somewhat lower levels of Bp50
than tonsillar B-cells; this is similar to HLA-DR
expression, (Ledbetter et al., 1986, Human Immunol.15:30-44)
and to qp54 expression (Wang, et al., 1979, J. Exp. Med.
149:1424-1433) which are also lower on blood B-cells. Bp50
was expressed at similar levels on tonsillar B-cell
subpopulations separated on Percoll gradients into buoyant
and dense fractions. Using our PE-conjugated mAb to the T
cell marker, CD3(T3), and NK cell-associated marker, CD16(Fc
receptor) (Ledbetter, et al., 1979, Immunol. Rev. 47:63-82),
we found that Bp50 is not expressed on T cells or NK cells.
Using two-color IF, we also found that CD3+ PHA blasts that
expressed high levels of IL-2 receptors did not express
Bp50.
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The G28-S antibody reacted with a single polypeptide on
tonsillar lymphocytes that migrated at approximately 50 Kd
under non-reducing conditions (Fig. 2A). This molecule is
larger than previously reported B-cell markers in the same
molecular weight range such as Bp39 or Bp45 (Zipf, et al.,
1983, J. Immunol. 131:3064-3072; Kitner, et al., 1981,
Nature 294, 458-460; Clark, et al., 1986, in Leukocyte
Typing II, eds. Reinherz, et al., Springer Verlag, Berlin,
Chap. 12 Vol. 2, 155-167; Slovin, et al., 1982, Proc. Nat.
Acad. Sci. USA 79:2649-2653; Thorley-Lawson, et al., 1985,
J. Immunol. 134:3007-3012, and Fig. 2B). The exposure time
for this gel was selected so that the molecular weights of
the other B-cell markers could be readily compared with
Bp50. The Bp39 marker, unlike Bp50, is expressed on
granulocytes and Bp45, unlike Bp50, is restricted to B-cell
blasts. Antibodies to Bp39 (41-H16) and Bp45 (MNM6, Blast-
1, Blast-2) made available through an international workshop
tClark, 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 fluoresceinated anti-Bp50
antibodies to B-cells. Thus, based on tissue distribution,
biochemical analysis, and blocking studies, the G28-5
monoclonal antibody recognizes a 50-Kd structure distinct
from other known B-cell antigens.
5.2.2. EXPRESSION OF Bp50 IS RESTRICTED TO B-CELLS
Both hematopoietic tissue and cell-line distribution
studies and detailed two-color flow cytometric analyses
revealed that Bp50 is expressed only on B lymphocytes. As
illustrated in Fig. 3, Bp50 is expressed on a small subset
of blood lymphocytes and on a large population of tonsillar
lymphocytes. Virtually all Bp50+ cells in both blood and
tonsils also expressed Bp35 and HLA-DR, but did not express
the CD2 (Fig. 1) or CD3, T-cell molecules or the IgG Fc
receptors that are found on NK cells. Furthermore, ConA-
~J~~ 1338781
activated CD3+ T-cell blasts expressed IL-2 receptors but
did not express Bp50.
Two-color flow cytometric analyses allow the
quantitative measurement of the density relationship between
two surface antigens. We previously showed that the dense,
resting B-cells in the mantle zone of secondary follicles
express IgM and low levels of Bp35, whereas the buoyant,
activated B-cells in the germinal center are IgM-negative
and express elevated levels of Bp35 (Ledbetter, et al.,
~0 Human Immunol. 15:30). Figure 3 shows that both IgM-
positive and IgM-negative B-cell subsets expressed Bp50 in
equal amounts, indicating that Bp50 is expressed on both
resting B-cells and B-cells activated in vivo.
5.3. AUGMENTATION OF B-CELL PROLIFERATION
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 function in early B-cell activation:
the lF5 mAb to Bp35, like low doses of anti-u antibody,
activates B-cells to increase in cell volume and RNA content
and to become responsive to BCGF (Clark, et al., 1985, Proc.
Nat. Acad. sci. USA 82:1766-1770; Gollay, et al., 1985, J.
Immunol. 135:3795-3801). Therefore, it was of interest to
compare the effect of anti-Bp50 mAb in the proliferation of
untreated B-cells or B-cells activated with either anti-Bp35
or anti-u antibodies (Table 1). Anti-Bp35 in solution or
anti-u antibodies attached to Sepharose beads, under
appropriate conditions alone, could stimulate some B-cell
proliferation (Table 1, line 1); in contrast, anti-Bp50
antibodies alone did not stimulate proliferation (Table 1,
line 2). However, anti-Bp50 mAb augmented proliferation
considerably when cultured with anti-u beads or with anti-
35 Bp35. In this respect anti-Bp50 resembled BCGF (Table 1,
line 3). Thus, it was important to determine whether
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anti-Bp50 and BCGF together could induce B-cell
proliferation. As illustrated in Table 1, line 4, anti-Bp50
and BCGF together induced no proliferation, but did augment
proliferation of either anti-u or anti-Bp35 activated cells
somewhat more than either stimulant alone. BCGF over a
three-log range, when used with anti-BpS0 without other
signals, had no effect on proliferation of dense B-cells
even when anti-Bp50 was used at doses ranging from 0.1 to 10
ug/ml.
Table 1
Augmentation of Anti-Ig or Anti-Bp35
Induced B Cell Proliferation With Anti-Bp50 Antibodies
Mean Proliferation + S.E. of B Cells Cultured With:
Line Co-stimulant Media Anti-u-beads Anti-Bp35
1 None 1,212+547 10,219+462 5,539+308
2 Anti-Bp50719+718 38,792+1,329 25,465+616
3 BCGF456+217 14,217+4459,443+343
4 Anti-Bp50
+ BCGF1,456+126 54,393+2,537 46,488+3,387
Pro~iferation of dense Er- tonsillar B-cells ( 95% surface
IgM c~ells) was measured on day 3 as described. Briefly,
2 x 10 cells/200 ul well were cultured in quadruplicate for
48 hrs with RPMI 1640 medium containing 15% fetal bovine
serum plus additives without antibody or with either 2C3
monoclonal antibody to u chains coupled to sepharose beads
(nanti-u beads,~ 50 ug/ml) or free lF5 anti-Bp35 antibody (5
ug/ml). Cultures containing media, nanti-u beads,~ or
anti-Bp35 were cultured alone or with BCGF (5% final
concentration, Cytokine Technology, Buffalo, New York; has
no detectable IL-1 or IL-2 activity), with anti-Bp50 (1:1000
dilution of ascit~s) as co-stimulants. After 40 hrs cells
were pulsed with H-thymidine, and counts incorporated were
measured after 18 hrs.
-~c- 1~38781
5.3.1. ANTI-Bp50 mAb AUGMENTS PROLIFERATION ONLY
AFTER B-CELLS ARE ACTIVIATED BY ANTI-Bp35
OR ANTI-u-ANTIBODIES
The results in Table 1 suggest that anti-Bp50 mAb could
not induce proliferation by itself. As shown in Fig. 4,
doses of anti-Bp50 ranging from 0.05 ug to 2.0 ug/ml
had no effect on H-thymidine uptake. However, in the
presence of optimal levels of anti-Bp35 mAb, as little as
0.1 to 0.5 ug/ml of anti-Bp50 antibodies augmented
proliferation substantially. As much as 50,000 to 70,000
cpm were detectable at the optimal time of proliferation
when highly purified B-cells were cultured only with anti-
Bp35 plus anti-Bp50. A consistent observation was that
higher doses of anti-Bp50 (greater than 2-5 ug/ml) were less
effective than doses in the 100-200 ng range.
These results suggested that anti-Bp50 may function
only after B-cells are activated by other signals. Data
shown in Fig. 5 suggest that this is indeed t~e case. If B-
cells were first activated with anti-Bp35, anti-Bp50 could
be added as late as 24-48 hours later and still augment
proliferation at day 4. In contrast, when cells were first
treated with anti-Bp50, anti-Bp35 was effective only if
added within a few hours after the start of cultures.
Similar results were found when anti-u rather than anti-Bp35
was used.
5.3.2. ANTI-Bp50 mAb DO NOT ACTIVATE B-CELLS OUT OF
G BUT DO INDUCE ACTIVATED B-CELLS TO PROGRESS
T~RO~GH THE CELL CYCLE
Previously, we have found that anti-Bp35, like low
doses of anti-u antibodies, induce resting tonsillar B-cells
in Go to enlarge (Clark, et al., 1986, Leukocyte Typing II,
eds., Reinherz, et al., Springer Verlag, Berlin, Vol. 2,
1338781
455-462) and to enter the G1 phase of the cell cycle
(Gollay, et al., 198S, J. Immunol. 135:3795-3801). Thus, it
was of interest to compare the ability of anti-Bp50 mAb to
anti-Bp35 mAb for their effects on B-cell activation. As
shown in Fig. 6A, unstimulated dense tonsillar B-cells even
after 3 to 4 days in culture had a uniform RNA profile
characteristic of cells in Go (Darzynkiewicz, 1980, Proc.
Nat. Acad. Sci. USA 77:6697-6702). However, about 15-30% of
cells stimulated with anti-Bp3S or anti-u had increased RNA
content indicative of entry into G~. In contrast, neither
anti-Bp50 (Fig. 6B) nor BCGF (Fig. 6C) alone induced
significant numbers of B-cells to enter Gl. For instance, 2
days after activation, anti-Bp35 and anti-Ig mAb induced
respectively 13.5% and 20.9% of tonsillar cells to enter Gl,
lS whereas cells treated with only anti-Bp50 (2.7%) or BCGF
(3.2%) remained at media control levels (2.2%). However,
when either anti-Bp50 or BCGF were added together with
anti-Bp35 or anti-u antibodies, the proportion of cells
entering Gl increased dramatically. Similarly, anti-BpS0 and
BCGF alone did not induce B-cells to enter S phase (Table
2), but together with either anti-Bp35 or anti-u did
increase the number of S phase cells two- to threefold.
1338781
Table 2
Effect of Anti-BpS0 and BCGF on
Cell Cycle Progression in Tonsillar Lymphocytes
CompetenceProgression ~ Cells
Signal Signal Go Gl / 2/
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-BpS0 83.6 12.0 3.3
anti-Bp35 ' 54.1 35.5 9.7
t5 anti-Ig ~ 43.6 36.2 16.2
media BCGF 85.4 11.7 2.2
anti-Bp35 ~ 56.6 32.6 11.6
anti-Ig ~ 48.4 36.1 14.1
Percentage of cells in G , G , or S and G determined with
the use of the acridine 8ran~e-staining p~ocedure
(Darzynkiewicz, et al , 1980 Proc. Nat. Acad. Sci. USA
77:6697-6702); 1 x 106 dense tonsillar lymphocytes with
anti-Bp35 (5 ug/ml), anti-u on beads (S0 ug/ml), anti-BpS0
(0.4 ug/ml), BCGF (S%) or combinations as shown.
5.3.3. OPTIMAL CONDITIONS FOR AUGMENTING B-CELL
PROLIFERATION WITH ANTI-Bp50 ANTIBODIES
Antibodies to Bp50 by themselves have little or no
detectable effect on dense resting 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
clearly augmented proliferation. Anti-Bp50 did not
costimulate with several interleukins, including purified
- ~S 1338781
IL-l, 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 were costimulatory with anti-Bp50 were
also costimulatory with BCGF (low) (Table 3). Of particular
interest was the finding that together BCGF and anti-Bp50
~till were not costimulatory for resting cells.
Table 3
Augmentation of B-Cell Proliferation with
Anti-Bp50 Antibodies or B-Cell Growth Factor
Mean Proliferation + S.E. of 8-Cells
Cultured with:
Anti-Bp50 BCGF
15 Co-stimulant Media (200 ng/ml)(5%)
none 96 + 1 267 + 15285 + 74
anti-Ig 5,833 + 391 41,634 + 2,10325,094 + 61
anti-Bp35
(5 ug/ml)457 + 45 8,143 + 2801,733 + 32
TPA (2 ng/ml)7,361 + 537 21,163 + 87113,064 + 1,030
IL-l (10 U/ml)264 + 2 308 + 23 221 + 8
IL-2 (100 U/ml)204 + 34 350 + 7 220 + 11
BCGF (5%) 220 + 7 851 + 28270 + 18
Dense Er- tonsillar B-cells ~greater than 95% sIgM cells)
cultured fo~ 48 hr at 2 x 10 cells/well followed by 24 hr
pulse with H-thymidine before counting.
The kinetics of proliferation augmented by anti-Bp50 is
shown in Fig. 7. The peak of proliferation occurred at day 4
and then waned whether or not cells were activated with
anti-Bp35 or other activators such as anti-Ig or TPA. The
kinetics of proliferation augmented by BCGF or by anti-Bp50
were slmilar.
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As little as 0.05 ug of anti-Bp50 antibodies augmented
proliferation. An optimal dose of 0.3 ug~ml was used in
subsequent studies. A consistent observation was that when
using whole antibody molecules, higher doses of anti-Bp50
~greater than 2-5 ug~ml) were less effective than doses in
the 0.1-0.5 ug/ml range.
Human B-cells are exquisitely sensitive to inhibitory
effects mediated by the Fc receptors of antibodies binding to
surface Ig lParker, 1980, Immunol. Rev. 52:115; Bijsterbosch,
et al., 1985, J. Exp. Med. 162:1825). Thus, it was important
to compare the efficacy of whole anti-Bp50 ~Ab with that of
anti-Bp50 F(ab')2 fragments. Over a 100-fold dose range
F(ab')2 fragments were clearly as effective as, or more
effective than, whole antibody at augmenting B-cell
proliferation (Table 4). Thus, the Fc domain of anti-Bp50 mAb
is not required for anti-Bp50 to exert its effect and, if
anything, may be inhibitory. In other words, anti-Bp50, like
BCGF, apparently can act as a soluble mediator without the
aid of Fc receptor-mediated accessory cell function.
- 31 -
I 338 781
Table 4
The Fc Domain of Anti-Bp50 Antibodies
Sis Not Required for Augmenting B-Cell Proliferation
Mean Proliferation of B Cells
Cultured with:
Dose
10 Anti-Bp50 (ug/ml) MediaAnti-Bp35
none -- 295 + 16269 + 27
whole Ab 0.125 278 + 325,140 + 20
1.25 275 + 244,686 + 342
12.5 163 + 153,852 + 203
F(ab')2 0.125 594 + 2110,635 + 449
1.25 531 + 310,893 + 575
12.5 279 + 89,411 + 870
Cell culture conditions were as described in Table 3.
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 3).
However, several lines of evidence indicate that anti-Bp50
and the BCGF used in this study apparently operate through
different signals. First, Bp50 molecules, unlike BCGF (low)
receptors (Bijsterbosch, et al., 1985, J. Exp. Med. 162:
1825), are expressed on resting blood B-cells (Fig. 3).
Second, although both anti-BpS0 and BCGF (low) function most
effectively when added after anti-Bp35 or anti-Ig, anti-Bp50
clearly was optimally effective when added 12 hours after
cultures began (Fig. 8A). In contrast, BCGF (low) could be
;
1338781
added as long as 24 hours after start of cultures and still
optimally augment proliferation (Fig. 8B). These kinetic
experiments, which are modeled after the approach of Howard
and Paul (1983, Ann. Rev. Immunol. 1:307), suggest that a
Bp50-dependent signal may normally exert its effect before
BCGF.
Both anti-Bp50 and BCGF (low) augmented proliferation
of B-cells activated with anti-Bp35 or anti-Ig (Table 3).
However, the effect of anti-Bp50 and BCGF (low) were
additive in many experiments (Figure 7). Figure 9 shows a
titration of BCGF (low) in an experiment where anti-Bp50 was
used at its optimal concentration (0.2 ug/ml). BCGF (low)
could further augment proliferation of restinq B-cells in
the presence of anti-Bp50 after activation by either anti-Ig
or by anti-Bp3S. Optimal concentrations of BCGF (low) were
5-10%, while 25% was inhibitory. Thus, when anti-Bp50 and
BCGF (low) were both used at their optimal concentrations,
they still showed additive effects on B-cell proliferation.
Finally, both normal and malignant B-cell subsets
differed in their responses to anti-Bp50 and to BCGF (low).
For example, some blood B-cells responded to BCGF (low) but
did not respond to anti-Bp50 (Table S). An additional
activation signal such as anti-Bp35 (Table 5) or TPA (Fig.
10) was consistently necessary to allow blood B-cells to
respond to anti-Bp50. While dense tonsillar B-cells
generally did not respond to either BCGF or anti-Bp50,
buoyant B-cells did respond (Table 5). B-cell malignancies
also differed in their responsiveness to anti-Bp50 versus
BCGF. For example, some B-cell lymphomas responded to TPA
plus BCGF (low) but not to TPA plus GZ8-5 anti-BpS0 (Fig. 10B
and D). In contrast, dense tonsillar B-cells and peripheral
blood B-cells responded to TPA plus either BCGF (low) or
anti-Bp50 (Fig. 10A and C).
- 33- 1338781
~ +l +l +l +l +l +l
O oo
m ~ ~
V'
0 5 ~ ~ o ,< O~
O . ~ ~0
~ +l +l +l +l +l +l
O C
~ ~d
~ a
.
.~ ~
t_ - ~ O
Q~ +l +l +l +l
-
mY
E E m C~
O ~ O ~ O O
~ V m m m m,
cO ~ c
Cell culture conditions as described in Table 1. Blood Nylon wood adherent lymphycytes (B cells plus monocytes) were depleted
of monocytes by incubation on plastic dishes overnight prior to stim~ tion (Exp. 1 and Exp. 2). In Exp. 3, blood E-lymphocytes
were depleted of monocytes by incubation on plastic dishes for 1 hr. prior to stim~ tion. Tonsillar Er- lymphocytes were
fractionated using Percoll gradients into dense (pellet) or buoyant (fraction 1) subsets (32).
1338781
5.4. USES OF ANTI-Bp50 LIGANDS AND Bp50
The ligands of the present invention may be used in
vivo or _ vitro, in their unmodified or modified forms to
modulate immune responses. For example, the ligands
themselves may be used as an ~adjuvantn to increase an
immune response to a vaccine or to increase the immune
response of an immunosuppressed individual. Alternatively,
~f cytotoxins or anti-proliferative agents are coupled to
the ligands, these modified ligands may be used to decrease
an immune response, for example, in autoimmune disease or in
transplant patients to obviate graft rejection. These
modified ligands could also be used to treat malignancies
that comprise cells or tumors which express the Bp50 antigen
whether or not the malignancy is B-cell in origin.
Both the ligands of the present invention and/or BpS0
itself can be used in vitro. Such applications include in
vitro assays, such as immunoassays for the detection of
cells which express the Bp50 antigen and/or for the
detection of shed Bp50 antigen, if any, in body fluids. In
this instance the ligand or Bp50 could be labeled with a
radiolabel, fluor, enzyme, enzyme substrate, dye, etc. In
addition, the ligands may be used to separate and/or
identify cells which express the Bp50 antigen, in which case
the ligand may be coupled to an immobile support, or to a
fluor which can be used in a FACS (fluorescence activated
cell sorter).
The various applications and uses of the ligands and
Bp50 of the present invention are discussed in more detail
below.
0 5.4.l. Bp50 RECEPTOR AND USES OF LIGANDS SUCH AS
3 ANTI-BpS0 TO AUGMENT B-CELL PROLIFERATION
Previous studies have suggested that the factors
involved in the induction of B-cells from Go into the Gl
phase of the cell cycle are distinct from the factors or
1338781
requirements for transit into the S phase. This model is
based principally on studies showing that agents such as low
doses of anti-Ig B-cell activation factors , or anti-Bp35
alone have little or no effect on B-cell proliferation. Yet,
these same agents can drive B-cells to a point in cell
activation where they are susceptible to growth factors. In
contrast, growth factors such as BCGF or IL-2 alone have no
effect on resting B-cells but do augment growth of activated
B-cells.
While the present invention is not to be limited to any
theory or explanation, the results presented herein provide
additional support for a model of distinct regulation of B-
cell activation and growth steps. Here we have shown that
activation and proliferation signals in human B-cells may be
transmitted through distinct cell surface structures.
Although anti-Bp35 mAb activated B-cells to enter the G
phase of the cell cycle, alone, it induced little or no
proliferation. Anti-Bp50 mAb had the opposite effect: it
could not activate B-cells, but when added even as late as
12-24 hours after activation could induce B-cell growth.
The Bp50 molecule presumably could normally function as
either a receptor for a ligand such as a soluble growth
factor or for a signal mediated through cell-cell contact
(i.e., a ligand found on the surface of another cell).
Previous studies have identified several T cell-derived BCGFs
that, like anti-Bp50, augment B-cell proliferation. Both
high and low molecular weight forms of B-cell growth factors
have been identified and different types have been shown to
have additive effects (Kehrl, et al., 1984, Immunol. Rev.
18:75-96; ~ishimoto, 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.
- 36 - 1338781
Invest. 75:732-739; Ambrus, 1985, J. Exp. Med. 162:1319-
1335). Thus, Bp50 might 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 mAb AB-1 reacts with a B-cell
marker expressed only on activated B-cells and blocks BCGF-
dependent proliferation, and thus might recognize the BCGF
receptor or a related structure. Bp50 appears to be distinct
from the AB-l marker since the AB-l mAb does not block the
binding of the G28-5 anti-Bp50 antibody, and unlike the G28-5
mAb, reacts only with activated B-cells (Jung, et al., 1984,
J. Exp. Med. 160:1919-1924). Bp50 is on all B-cells, which
based on absorption analysis and direct binding assays
appears not to be the case for BCGF receptors. Our current
data indicate that Bp50 and the receptor for low molecular
weight BCGF are distinct structures. Using a rabbit
heteroantiserum, Wang and coworkers (Wang, 1979, J. Exp. Med.
149:1424-1433) previously described a 54-kDa qlycoprotein,
gP54, that like Bp50 is expressed on all B-cells but at lower
levels on blood B-cells than tonsillar B-cells. It is
possible, but unlikely, that the rabbit heteroantiserum and
anti-Bp50 recognize the same or related structures: unlike
anti-Bp50 mAb, the rabbit antiserum to gp54 alone was
sufficient to stimulate B-cell proliferation.
Anti-Bp35 alone, unlike anti-Bp50, can activate B-cells
from Go to Gl and thus can be referred to as an nactivationn
signal. Whether or not Bp35 functions only in early B-cell
activation is not yet clear since anti-Bp35 antibodies can
stimulate some B-cells to divide (Clark et al., 1985, Proc.
Nat. Acad. Sci. USA 82:1766-1770). Similarly, Bp50 may not
strictly function only as a ~growthn signal: anti-Bp50
antibodies together with activation signals (anti-Bp35 or
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anti-u) not only augment proliferation but also augment the
total number of B-cells entering G1 (Table 2). In other
words, anti-Bp50 as costimulant acts to promote the
progression of both the activation (Go to Gl) and growth
(G to S) phases of the cell cycle. The BCGF used in these
studies also had similar activity (Fig. 6C). Thus, anti-Bp35
and anti-BpS0 (or BCGF) appear to be most analogous to the
~competence~ and ~progression~ factors described in studies
of fibroblast growth regulation. How B-cells respond to
anti-Bp35 or anti-BpS0 clearly may depend on their state of
differentiation or activation.
Here we have shown that two mAb, anti-Bp35 (a
~competencen signal) and anti-Bp50 (a ~progression~ signal),
together can induce substantial proliferation of highly
lS purified B-cells in the absence of antigen or other known
factors. The natural ligands for these structures are not
yet known. However, since mAb to appropriate epitopes can
mimic both soluble factors and signals mediated by cell-cell
interactions, it may be possible to use appropriate
combinations of mAb to direct and regulate human B-cell
proliferation or differentiation. This, in turn, will help
in devising strategies in vivo for the control of human
diseases such as B-cell malignancies, immunodeficiencies and
certain autoimmune diseases.
The new monoclonal antibody, G28-S, that reacts with a
sinqle-chain polypeptide of approximately 50 Kd expressed on
the surface of human B-cells is but a particular embodiment
of the ligands of the present invention which can augment the
proliferation of activated B-cells. Since human B-cell -
proliferation can be augmented similarly 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
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BCGF. Anti-Bp50 G28-5 and BCGF (low) were very similar in
that they were costimulatory with the same activation agents
(anti-Ig, anti-Bp35 and TPA) but were not costimulatory with
each other or with IL-l or IL-2. Furthermore, the activity
of anti-Bp50 G28-S was not dependent on its Fc domain since
F(ab')2 fragments of G28-S were functionally active. This
suggests that soluble anti-BpS0, like soluble BCGF, does not
require Fc-receptor-bearing accessory cells to exert an
effect. Furthermore, both anti-Bp50 and BCGF are effective
only in the presence of an activation stimulus. In other
words, anti-Bp50 and BCGF are not ncompetence~ factors, but
rather promote the ~progressionN of B-cells through the cell
cycle.
While it is possible that Bp50 may function as receptor
for a ligand such as a B-cell growth factor, several results
suggest that Bp50 is not the receptor at least for the BCGF
(low) used in this study: it is expressed on blood B-cells
while BCGF (low) receptors apparently are not. Candidate
structures for the BCGF (low) receptor, unlike Bp50, are also
expressed only on activated B-cells. Furthermore, both
normal and malignant B-cell populations differ in their
responsiveness to anti-Bp50 versus BCGF (low) (Table 5 and
Figure 10). For instance, some B lymphomas proliferate 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 together induced more proliferation
than either one alone. Anti-Bp50 mimics the activity of
other BCGF, such as BCGF (high) that are co-stimulatory 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
(high).
~ 3~ ~ 1338781
Although BpS0 may be a receptor for a soluble ligand,
alternatively, Bp50 may function as a receptor for a cell-
cell mediated slgnal that regulates BCGF receptor levels
and/or autocrine production. Precedence for differentiation
antigens serving as amplifiers of an autocrine-receptor
pathway comes from studies with T cells. MAb to the Lp220
common leukocyte antigen augments proliferation by elevating
IL-2 receptor expression on activated T cells (Ledbetter, et
al., 1985, J. Immunol. 135:1819). An analogous mechanism may
be operating with anti-Bp50 and expression of certain BCGF
receptors. Bp50 and BCGF (low) apparently are under some
coordinate control since, like IL-l and IL-2 receptors, BCGF
augments expression of Bp50 on certain leukemic cells. The
Bp50 molecule also shares similarities with the Tpq4 molecule
that functions to influence IL-2 production. We and others
have shown that the 9.3 anti-Tp44 antibody augments
proliferation 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). Similarly, anti-Bp50 augments
the proliferation 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
presumably could function in an analogous manner by
stimulating B-cell autocrine production (Gordon, et al.,
19~4, Nature, Lond. 310:145).
5.4.2. MODIFIED LIGANDS ~SED FOR IMMUNOSUPPRESSION
OR TREATMENT OF MALIGNANACIES
According to this embodiment, the ligand of the present
invention can be modified by the attachment of an
antiproliferative agent so that the resulting molecule can
be used to kill cells which express the Bp50 antigen. Such
modified ligands may be used in the treatment of autoimmune
disease in order to supress the proliferation of B-cells and
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1338781
thereby 6uppress the autoimmune response. These modified
ligands can also be used to immunosuppress a transplant
patient to prevent re~ection of a graft. Accordingly,
cytotoxic agents which are used for the suppression of
immune responses can be attached to the liqands of the
invention. When using ligands which augment the
proliferation of B-cells, an increased effect should result
because the drug will be directed to proliferating B-cells.
In another embodiment, the ligands of the present
invention which are modified by the attachment of an
antiproliferative agent can be used to treat malignancies in
which tumors or cells express the Bp50 antigen. Attachment
of these chemotherapeutic agents to the ligands of the
invention should result in a greater specificity of the drug
for the malignant cells. Moreover, a particular advantage
~hould be obtained when treating a B-cell malignancy with a
ligand coupled to a cytotoxin which is more effective in
killing proliferating cells than non-proliferating cells;
treatment with such a ligand should result in a potentiation
of the action of the cytotoxin.
Accordingly, the chemotherapeutic agents or
antiproliferative agents which can be coupled to the ligands
of the present invention include but are not limited to t~e
agents listed in Table 6 below which is derived from Goodman
and Gilman, The Pharmacological Basis of Therapeutics, Sixth
Edition, MacMillan Publishing Co., Inc, New York, pp. 1249-
1313, 1980,
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Table 6
Chemotherapeutic Agents Which Can be Coupled
to Anti-Bp50 Ligands
Class Tvpe Agent
Alkylating Nitrogen Mechlorethamine
Agent Mustard Cyclophosphamide
Melphalan
Uracil Mustard
Chlorambucil
Ethylenimine Thiotepa
Derivatives
Alkyl Sulfonates Busulfan
Nitrosoureas Carmustine
Lomustine
Semustine
Streptozocin
Triazenres Dacarbazine
Antimetabolites Folic Acid Methotrexate
Analogs
Pyrimidine Fluorouracil
Analogs Cytarabine
Azaribine
Purine Mercaptopurine
Anlogs Thioguanine
Natural Vinca Vinblastine
Products Alkaloids Vincristine
Antibiotics Dactinomycin
Daunorubicin
Doxorubicin
Bleomycin
Mithramycin
Mitomycin
Enzymes L-Asparaginase
- 42 -
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Miscellaneous Platinum Cisplatin
Agents Coordinated
Complexes
Substituted Hydroxyurea
Urea
Methyl Procarbazine
Hydrazine
Derivative
Adrenocortical Mitotane
Suppressant
10 Hormones and Adrenocorti- Prednisone
Antagonists costeroids
Progestins Hydroxyprogesterone
caproate
Medroprogesterone
acetate
Megestrol acetate
Estrogens Diethylstilbestrol
Ethinyl estradiol
Antiestrogen Tamoxifen
Androgens Testosterone
propionate
Fluoxymesterone
Radioactive Phosphorous So~um phosphate
Isotopes p
Iodine So~m Iodide
Any method known in the art can be used to couple the
ligand to the chemotherapeutic or antiproliferative agent.
Examples of such methods have been enumerated previously
(see Section 5, supra).
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5.4.3. OTHER USES OF LIGANDS AND Bp50
In addition to the therapeutic applications the ligands
and BpS0 itself have other applications in both in vitro and
in viro diagnostic assays, separation schemes, etc.
The Bp50 receptor can be used to manufacture and/or
design the ligands of the invention. Bp50 can also be used
with the ligands of the invention in assays in vitro which
require a standard to quantify the amount of Bp50 detected
in a sample. Ultimately, BpS0, itself may be useful as a
soluble factor which mediates immunity, e.~. a lymphokine.
In addition to therapeutic treatment and diagnostic
assays, the ligands of the present invention could be used
for identifying or separating cells which express the Bp50
antigen. In addition, if an appropriate radiolabel or
radio-opaque compound is linked to the ligand, the ligand
could be used for in vivo imaging of tumors which express
the Bp50 antigen. Other uses should become apparent to
those skilled in the art from the foregoing description.
6. DEPOSIT OF CELL LINES
The following hybridoma has been deposited with the
American Type Culture Collection, Rockville, MD, and has
been assigned the listed accession number:
Hybridoma ATCC Accession Number
G28-5 HB9110
The present invention is not to be limited in scope by
the hybridoma deposited since the deposited embodiment is
intended as a single illustration of one aspect of the
invention and any cell lines which are functionally
equivalent are within the ~cope of this invention. Indeed
various modifications of the invention in addition to those
shown and described herein will become apparent to those
1338781
skilled in the art from the foregoing description and
accompanying drawings. Such modifications are intended to
fall within the scope of the appended claims.
