Note: Descriptions are shown in the official language in which they were submitted.
20~ 1205
1
PCT/EP90/00111 7098 AW/1/ka
A Cell Surface Antigen Associated with Cellular Apoptosis
Cell surface molecules play crucial roles in lymphocyte
growth control. Such molecules may function as receptors
for growth-stimulating cytokines or be associated with
receptors and transmit signals essential for growth
regulation. Receptor blockade or removal of the
stimulating cvtokines can lead to decreased lymphocyte
growth. For example, withdrawal of interleukins slows
human lymphocyte growth and finally leads to a
characteristic form of cell death called "programmed cell
death" or apoptosis. E. Duvall and H. H: Wyllie, Immunol.
Today 7,--=135r'~(-1986). Apoptosis is the most common form of
eukaryotic cell death and occurs in embryogenesis, meta-
morphosis, tissue atrophy, and tumor regression. A. H.
Wyllie, J. F. R. Kerr, A. R. Currie, Int. Rev. Cytol. 68,
251 (1980). It is also induced by cytotoxic T lymphocytes
and natural killer and killer cells; by cytokines such as
tumor necrosis factor (TNF) and lymphotoxin (LT); and by
glucocorticoids. The characteristic signs of apoptosis
are segmentation of the nucleus, condensation of the
cytoplasm, membrane blebbing (zeiosis), and DNA
fragmentation into multimers of about 180 base pairs
(called a "DNA ladder").
Recently, it has been shown that anti-CD3 induces
apoptosis of immature thymocytes in vitro. C. A. Smith et
al., Nature 337, 181 (1989). It has been suggested that
CD3-triggered apoptosis might be responsible for negative
selection of T cells in the thymus.
20712p5
The selective induction of apoptosis in cells, such as di-
seased cells, could prove a useful therapeutic tool.
Monoclonal Antibody-Mediated Tumor Regression by Induction of
Apoptosis is described in Science, Vol. 245, July 1989, pp
301-305.
The present invention relates to a method for inhibiting the
proliferation of a cell infected by the HTLV-1 virus, com-
prising contacting the infected cell with an antibody which
specifically binds to an antigen associated with cellular
apoptosis under conditions appropriate for antibody mediated
cellular apoptosis as well as to a method for killing a sub-
stantial percentage of a population of cells infected by the
HTLV-1 virus, comprising contacting the population of cells
with an antibody which specifically binds to an antigen asso-
_;
ciated with cellular apoptosis under conditions appropriate
for antibody mediated cellular apoptosis. APO-1 is associated
with cellular apoptosis and has a molecular weight of about
52 kD and is expressed by activated and malignant lymphoid
cells. The binding of antibody to APO-1 induces apoptosis and
thus, antibody or analogous binding agents can be used to in-
duce growth inhibition or apoptosis in cells, such as lym-
phoid tumor cells, which carry the APO-1 antigen.
Figure 1 shows an autoradiogram of a SDS polyacrylamide
electrophoretic gel for determination of the molecular
weight of APO-1.
Figure 2 shows the induction of growth inhibition and
apoptosis by anti-APO-1.
Figure 3 illustrates anti-APO-1-induced regression of a
lymphoma in mice.
Figure 4 shows the localization of anti-APO-1 antibody in
xenografts of a lymphoid tumor.
SUBS~i~'E~TE SHEET
3
2~7120
The antigen APO-1 is a cellular membrane antigen having a
molecular weight of appoximately 52kD as determined by
SDS-polyacrylamide gel electrophoresis. APO-1 is
expressed by activated normal human lymphoid cells and
lymphoid tumor cells, including B cell, T cell and HTLV-
1-associated malignant cells such as adult T cell
leukemic cells. The binding of anti-APO-1 antibody to
cells expressing APO-1 results in growth inhibition
and/or apoptosis. This effect is complement independent,
mediated by antibody alone.
APO-1 can be isolated from the cellular membrane of cells
(such as lymphoid cells) which express the-antigen by
conventional =~ech,~iques . Further-., the gene encoding APO-1
can be cloned and expressed to provide the isolated
antigen or portions of it. Isolated APO-1 can be used as
immunogen to raise anti-APO-1 antibody (polyclonal or
monoclonal) or to screen for production of anti-APO-1
antibody by hybridomas, of chimeric anti-APO-1 antibody
by transfected myelomas or of single chain anti-APO-1
antibody by transformed bacterial cells.
Antibodies which bind to APO-1 are useful for inducing
inhibition of cell growth or apoptosis in cells that
express APO-1. For this purpose, monoclonal anti-APO-1
antibodies are preferred. Monoclonal anti-APO-1
antibodies are produced by continuous (immortalized),
stable, antibody-producing cell lines. The preferred
antibody-producing cell lines are hybridoma cell lines.
In principle, however, the cell lines can be derived from
any cells which contain and are capable of expressing
functionally rearranged genes which encode variable
~~.FB~'~'t'~~~~'~ ~=-~= F--
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2071245
regions of the light and/or heavy chains of anti-APO-1
specificity. Preferably, the cell line should have the
capability to assemble the chain (in the case of a single
chain antibody) or chains into functional antibodies or
antibody fragments. Thus, lymphoid cells which naturally
produce immunoglobulin are preferred.
Hybridoma cells which produce monoclonal anti-APO-1 anti-
bodies can be made by the standard somatic cell
hybridization procedure of Kohler and Milstein, Nature
256: 495 (1975). Briefly, a procedure is as follows: the
monoclonal anti-APO-1 antibodies are produced by
immunizing an animal with whole cells bearing APO-1 or
membranes of these cells. Alternatively, the animals can
be immunized with purified or partially purified APO-1 or
peptidic -segments,..carrying one or more immunogenic
epitopes of APO-1. Such peptides can be synthesized and
conjugated to a carrier protein, such as keyhole limpet
hemocyanin, to be used as an immunogen.
The preferred animal for immunization is the mouse.
Various immunization protocols can be used. For example,
mice can be given about 107 APA-1-bearing cells one per
weak over a four-week period by intraperitoneal
injection.
Antibody-producing lymphoid cells (e. g. splenic
lymphocytes) are then obtained from the immunized animal
and fused with immortalizing cells (preferably a myeloma
or heteromyeloma). Many suitable myeloma cell lines are
known in the art. For production of murine hybridomas, an
example is the myeloma P3. X63.Ag8.653. See Kohler and
Milstein, supra. Fusion of the spleen cells and fusion
partner can be carried out in the presence of
~.
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5
2071205
polyethylene glycol according to established methods.
Techniques of electrofusion may also be used.
The resulting hybrid cells are clonally cultured and then
screened for production of anti-APO-1 antibody.
Hybridomas can be screened for secretion of antibodies
which induce apoptosis against a cell line which
expresses the APO-1 antigen. An example of such a cell
line is the malignant human B cell line SKW6.4.
Additional cell lines are set forth in table 1 below.
Purified or partially purified APO-1 can be used to
screen for hvbridomas that secrete antibodies of APO-1
specificity by standard immunoadsorbant assays.
Although animal antibodies can be useful- for human
therapy,--it gay be preferable to convert animal
antibodies to a form which may be better tolerated by a
human. Monoclonal antibodies produced in murine or other
animal systems can be converted to chimeric animal/human
antibodies or to "near human" antibodies by standard
techniques.
APO-1-binding fragments of analogues of anti-APO-1
antibodies can also be produced. For example, antibody
fragments such as F(ab')2, Fab and FV can be produced by
enzyme digestion. In addition, synthetic oligopeptides
representing Fab and FV analogues (single chain
antibodies) can be produced in bacterial cells by genetic
engineering techniques.
The antibodies can be used to induce growth inhibition or
apoptosis in lymphoid cells (normal or malignant) or
other cells bearing APO-1. For example, anti-APO-1 -
antibody can be used to treat tumors bearing the APO-1
6
207120
antigen. As mentioned above, growth inhibition and/or
apoptosis can be induced by antibody in various types of
lymphoid cell malignancies which express APO-1. These
lymphoid cell malignancies include malignancies of B or T
cell lymphocytes. In particular, adult T cell leukemia,
an HTLV-1 associated tumor, can be treated with anti-APO-
1 antibody. In addition nonlymphoid tumors which bear
APO-1 are candidates for the antibody therapy.
The anti-APO-1 antibodies are administered to a patient
afflicted with the tumor in an amount that induces growth
inhibition or apoptosis of APO-1 bearing cells. Effective
anti-tumor dosages and dosage regimens can be determined
for the various types of tumors. Generally, the
antibodies can be given intravenously in- a pharmaceutical
vehicle such :a~s spline.
Lymphoid tumors which express APO-1 can also be treated
extracorporeally. Blood cells or blood leukocytes are
removed from the patient and contacted with anti-APO-1
antibodies in amounts sufficient to reduce or eliminate
tumor cells. After treatment, the blood cells or
leukocytes are returned to the patient.
SUS~TiT'UTE ~:~E~T
7 207 1205
1 antibody is used to detect binding (e. g. if the anti-
APO-1 antibody is a mouse antibody, the second antibody
can be a goat anti-mouse antibody). 'the second antibody
is labelled, preferably with an enzyme or a fluorescent
molecule. After incubation of the cells with the labelled
antibody, the label associated with cell is detected as
indication of the APO-1 expression by the cell.
The invention is illustrated further by the following
examples.
EXAMPLES
Example 1
Methods
Production of Anti-APO-1 Antibodies
BALB/c mice were immunized once per week over a 4-week
period by intraperitoneal injection of 1 x 107 SKW6.4
cells. Four days after the last injection, spleen cells
from immunized animals were fused with the P3.X63.Ag8.653
myeloma [G. Kohler and C. Milstein, Nature 256, 495
(1975)]. Twelve days after fusion culture supernatants
from wells positive for growth of SKW6.4 cells were
tested for their ability to inhibit growth of SKW6.4
cells. Hybridomas that produced blocking monoclonal
antibodies (MAbs) were cloned three times by limiting
dilution at a concentration of 0.5 cells per well. MAbs
were purified from serum-free culture supernatant by
means of a protein A-Diasorb*column (Diagen, Diisseldorf,
FRG). Bound MAbs were eluted with O.1M NaCl and 0.1 M
glycine, pH 2.8, dialyzed against phosphate-buffered
saline and sterilized. The isotype of the MAbs was
*Trademark
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determined by enzyme-linked immunosorbent assay [S.
Kiesel, et al.., Leuk. Res. 11, 1119 (1987)] with isotype-
specific goat anti-mouse Ig that had been conjugated with
horse radish peroxidase (Dunn, Asbach, FRG).
Determination of binding affinity and number of binding
sites
Affinity and number of anti-APO-1 binding sites per cell
were determined by Scatchard analysis as described [I.
von fioegen, W. Falk, G. Kojouharoff, P. H. Krammer, Eur.
J. Immunol. 19, 239 (1989)]. Briefly, MAbs were iodinated
by the IODO-gen*method [P. J. Fraken and J. C. Speck,
Biochem. Biophys. Res. Commun. 80, 849 (1980]. Aliquots
of 5 x 106 cells were resuspended in 200/ul of culture
medium containing 0.1$ NaN3, and different concentrations
of 1251-labelled MAbs. After incubation at 4oC for 4
hours, two 95~u1 portions were removed and centrifuged as
described above by von Hoegen et al.
SDS polyacrylamide gel electrophoresis (SDS-PAGE) for
molecular weight determination
Cells (3 x 106) were labelled with 60 pCi of ~5Se-
labelled methionine (Amersham, Braunsc/hweig, FRG) in 6 ml
of methionine-free culture medium (Biochrom, Berlin) for
48 hours. After washing the cells were incubated in
either control MAb or anti-APO-1 (1 ~g/ml) at 4°C for 45
min. The cells were washed and resuspended in lysis
buffer (tris-buffered saline, pH 7.3, 1~ Nonidet P-40,
1 mM phenylmethylsulfonyl fluoride, 0.1$ aprotinin) at
room temperature for 30 min. The lysates were centrifuged
and supernatants were incubated with protein A-Sepharose*
beads (Pharmacia, Uppsala, Sweden) at 4°C for 1 hour. The
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WO 91/10448 9 PCT/EP90/00111
immune complexes were washed four times with buffer
(tris-buffered saline, pH 7.3, 0.25$ Nonidet P-40) and
resuspended in SDS-PAGE sample buffer containing 5$ SDS
and 5~ 2-mercaptoethanol. The samples were heated to
95°C, centrifuged, and counts per minute of the
supernatants were determined in a ~"-counter. A total of
15,000 cpm were loaded in each lane and analyzed by a 10$
SDS-PAGE (V. K. Laemmli, Nature 277, 680 (1970)). The gel
was dried and subjected to autoradiography. The
autoradiograph is shown in Figure 1; bands represent the
immunoprecipitation of the biosynthetically labelled APO-
1 with either the isotype-matched control MAb (left lane)
or anti-APO-1 (right lane). The numbers on the left
margin indicate the positions of the size markers.
Induction of growth inhibition and apoptosis by anti-APO-
1
(A) The T cell line CCRF-CEM.S2 was cultured in the
presence of purified MAb (1/ug/ml) in a microtiter plate
for 2 hours before photography (left panel of Figure 2,
control MAb 13BI; right panel, anti-APO-1). The CCRF-
CEM.S2 subclone was obtained by cloning cells under
limiting dilution conditions from the CCRF-CEM.S2 T cell
line at one cell per well in 96-well microtiter plates.
CCRF-CEM.S2 was selected because of its high sensitivity
to programmed cell death induced by APO-1 (500 ng/ml) as
measured by microscopic inspection in a 4-hour culture.
(B) CCRF-CEM.S2 cells (106 per milliliter) were incubated
with MAb (1 Nq/ml) in culture medium at 37oC. At various
times, aliquots of 106 cells were removed and DNA was
prepared. In Figure 2B, M, marker; I, control MAb 13BI
for 2 hours; lanes 3 to 7, anti-APO-1 for the times
indicated). (C) SKW6.4 c211s were either incubated with
the isotype matched control MAb FII20 (C7), FII23 (non-
SUBSTI i UTi E S~-.~~T
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binding MAb) (O), or anti-APO-1 (~) in microcultures for
24 hours before labelling with [3H] thymidine for a
further 4 hours. The data represent the mean of duplicate
cultures with a variation of less than 5~. The cells were
cultured in RPMI 1640 medium (Gibco, Grand Island, New
York), supplemented with 2 mM L-glutamine, streptomycin
(100 ~g/ml), penicillin (100 U/ml), 20 mM Hepes buffer pH
7.3, and 10$ heat-inactivated fetal bovine serum (Conco
Lab-Division, Wiesbaden, FRG). For microcultures 1 x 104
per well were cultured in duplicates in flat bottom 96-
well microtiter plates (Tecnomara, Fernwald, FRG) (2001
final volume per well). After 24 hours, the cells wer /e
labelled with 0.5 IuCi of [3H]thymidine (Amersham,
Braunschweig, FRG)/ for 4 hours. Before harvesting, the
microcultures were examined by microscopic inspection.
DNA fragmentation 1 x 108 cells were washed with cold
phosphate-buffered saline and disrupted with NTE buffer,
pH 8 (100 mM NaCl, 10 mM tris, 1 mri EDTA) containing 1~
SDS and proteinase K (0.2 mg/ml). After incubation for 24
hours at 37oC, samples were extracted twice with phenol
plus chloroform (1:1, v/v) and precipitated by ethanol.
The DNA was dissolved in 38 ~1 of NTE buffer and digested
with ribonuclease (1 mg/ml)/for 30 min at 37°C._ To each
sample lO Nl of loading buffer containing 15~ Ficoll* 400
(Pharmacia, Uppsala, Sweden), 0.5$ SDS, 50 mM EDTA, 0.05$
bromophenol blue, 0.05 xylene cyanol in TBE buffer (2 mM
EDTA, 89 mM boric acid, 89 mM tris, pH 8.4) were added.
The mixture was loaded onto a 1~ agarose gel and stained
after electrophoresis with ethidium bromide (0.5/ug/ml).
The size marker was Hind III + ECO RI-digested/~r~DNA.
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WO 91/10448 11 PCT/EP90/00111
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Reactivity of anti-APO-1 with different cells
Aliquots of 108 cells were incubated at 4oC in 100~u1 of
medium with control MAb (FII23 or I3BI) or anti-APO-1 for
30 min. Then the cells were washed and stained with
fluorescein isothiocyanate-coupled goat anti-mouse
IgF(ab')2(70~ug/ml) and analyzed by a cytofluorograph
(Ortho Diagnostic Systems, Westwood, Massachusetts).
For determination of the effect of anti-APO-1 on
tritiated thymidine uptake, cells (104 per well) were
cultured in the presence of MAb (500 ng/ml) for 24 hours
and labelled with [3H)thymidine for 2 hours before
harvest; the data in table 1 represent the mean of
duplicate cultures with a variation of less than 5~.
Leukemic cells from patients were obtained as follows.
Bone marrow cells isolated from the patients were
morphologically >95$ blasts and showed the following
phenotype: pre T-ALL, cytoplasmic CD3+, CD5+, CD7+,
CD34+, Tdt+, CD2 , surface CD3 , CD4 , and CD8 ; T-ALL,
CD2+, cytoplasmic CD3+, CD5+,CD7+ and Tdt+, surface CD3 ,
CD4 , CD8 and CD34 ; common-ALL CD10+, CD19+, CD22+,
CD24+, CD20 . The effect of anti-APO-1 on these leukemic
cells was not tested, because they died under normal
culture conditions.
Normal human lymphocytes were obtained as follows.
Peripheral blood mononuclear cells (PBMC) from healthy
volunteers were isolated by Ficoll Paque (Pharmacia Inc.,
Uppsala, Sweden) density centrifugation.Adherent cells
were removed by adherence to plastic culture vessels
overnight. T cells were isolated from PBMC by rosetting
with 2 amino-ethylisothyouronium-bromide (AET)-treated
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2071205
sheep red blood cells as described (20). Freshly prepared
resting T cells (2 x 106 per milliliter; 96~ OKTII+, 1~
'1'ac+) were activated with pliytohemagglutinin-M (50
~g/ml)) and PMA (10 Ilg/ml) (Sigma Chemical Co., Munich,
,FRG). Two, 7 and 12 days later the T cells were fed with
20 to 30 U/ml or recombinant human interleukin-2 (20 to
30 U/ml). T cells (5 x 105 per milliliter) activated for
12 days (90~ OKTII+; 60~ Tac+) were cultured in the
presence of FII23 or anti-APO-1 (1/ug/ml) in triplicates
for 24 hours and then labelled with (3H]thymidine for a
further 17 hours (see above).
Resting B cells (35.8 CD19~) were isolated by two rounds
of rosetting as above, followed by separation via a
Sephadex*G-10 column as described. T.R. Jerrells, J.H.
Dean, G.L. Richardson, D.B.Herberman, J. Immunol. Methods
32,11 (1980). For activated B cells, PBMC were adjusted
to 2 x 106 cells per milliliter and cultured in the
presence of pokeweed mitogen at l0~ug/ml (Serva,
Heidelberg, FRG) for 6 days. Dead cells and T cells were
then eliminated by rosetting with AET-treated sheep red
blood cells and subsequent centrifugation over Ficoll
Paque. The interphase cells were used as activated B
cells (84~ sIgM+).
Effect of anti-APO-1 on tumor growth in vivo
BJAB cells (4 x 107) were injected subcutaneously into
the left flank of ~u/rZ,u mice. After 5 weeks (day 0) the
mice were injected with 5001ug of MAb into the tail vein.
The results are shown graphically in Figure 3. Control
MAb FII20 (C1); FII23 (O); I3BI (d); and anti-APO-1 (~).
Fourteen days later the size of the tumors was measured
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2071205
at the base of the tumor; the tumors from individual mice
are represented by dots.
Localization of anti-APO-1 in tumor xenografts
Localization of anti-APO-1 in xenografts of BJAB in ~tY/~,u
mice and induction of apoptosis of the tumor is
illustrated in Figure 4. Figure lA, upper row, shows the
uptake of 1251-labelled MAb anti-APO-1 (50 fig, 50,uCi per
mouse) in the tumor at 12 hours (1), 48 hours (2), and
96 hours (3) after intravenous injection of the MAb. The
lower row shows the uptake of 1251-labelled MAb
anti-APO-1 500 fig; as used for therapy; 50~uCi per mouse)
(4) and FII20 (control MAb, binding to BJAB; 50~ug, 50
N Ci per mouse) (5) and 1251-labelled FII23 (control MAb,
nonbinding to BJAB; 50~ug, 50~uCi per mouse) (6) in 48
hours, respectively.
Ten days after intravenous injection of 500~ug of MAb per
mouse the remaining tumor tissue was removed and fixed
with formalin. Paraffin*sections of the tumor were
stained with h aematoxylin/eosin.
In Figure 4B, the left panel shows tumor after treatment
with control MAb FII20; the right panel shows tumor after
treatment with anti-APO-1. Arrows indicate host vessels.
Final magnification, x 92. MAbs were radioiodinated
according to the IODO-Gen method (see above). Labelled
MAbs were injected into the tail vein and animals were
killed by ether anesthesia at the predetermined time
points. The tumors were excised and embedded in methyl-
cellulose and 20~um cryotome sections were prepared.
Lyophilized sections were placed on a Kodak X-omat*AR
film for autoradiography.
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Results and discussion
A MAb (anti-APO-1) was identified that blocks growth and
induces apoptosis of SKW6.4 cells. The anti-APO-1 (IgGG3,
x', KD = 1.9 x 10 10) bound to approximately 4 x 104 sites
on the surface of SKW6.4 cells. The antibody specifically
immunoprecipitated an endogenously synthesized protein
antigen (APO-1) from SKW6.4 cells which, under reducing
conditions, was observed on SDS-polyacrylamid gel
electrophoresis (SDS-PAGE) as a main band of 52 kD
(Figure 1). Apart from actin (43 kD), which was
nonspecifically precipitated with IgG3, anti-APO-1
specifically immunoprecipitated a minor band of 25 kD.
This 25-kD protein might either represent a degradation
product or be noncovalently associated with the 52-kD
protein.
There are two major modes of death in nucleated
eukaryotic cells. Necrosis as a result, for example, of
complement attack is characterized by swelling of the
cells and rupture of the plasma membrane caused by an
increase in permeability. Cells that undergo apoptosis,
however, show a different biochemical and morphological
pattern. This pattern corresponds to the one induced by
anti-APO-1: condensation of the cytoplasm, membrane
blabbing (Figure 2a), and endonuclease-induced DNA
fragmentation (A. H. Wyllie, Nature 284, 555 (1980)) into
multimers of approximately 180 by (Figure 2b). Affinity-
purified anti-APO-1 induced growth retardation and cell
death (Figure 2c), which was not observed with either an
isotype-matched, control MAb (FII20) [anti-MHC(major
histocompatability complex) class I antigens] or the
nonbinding MAb FII23. Abrogation of [3H]thymidine
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incorporation along with increased trypan blue uptake
into dead cells were observed, and growth of 104 SKW6.4
cells in 2001u1 cultures was blocked by more than 95$ by
an anti-APO-1 concentration of only 10 ng/ml (Figure 2c).
The specificity of cell death induced by anti-APO-1
becomes evident from the fact that the following
additional control MAbs were inactive for induction of
apoptosis: 18 nonbinding and 9 binding MAbs of the IgG3
isotype (tested by immunofluorescence on SKW6.4 cells)
and a panel of MAbs directed against known antigens on
the cell surface of SKW6.4 cells including CD19, CD20,
CD22, MHC class II, IgM (immunoglobulin M), and the
SKW6.4 idiotype. (Monoclonal anti-CD19 (HD37) and anti-
CD22 (HD39) were kindly provided by B. Dorken (Polyclinic
of the University, Heidelberg " FRG) and monoclonal anti-
CD20 by G. Moldenhauer (IV Leukocyte typing workshop and
conference, Vienna, Austria, 1989), respectively. The 18
nonbinding and 9 binding MAbs of the IgG3 isotype (tested
by immunofluorescence on SKW6.4 cells) and the MAbs
directed against MHC class II, IgM, and SKW6.4Ig
idiotypes were raised in our own laboratory).
Cell death induced by anti-APO-1 was complement-
independent and occured under serum-free culture
conditions or in culture medium plus serum inactivated at
56°C for 30 min. It differed from death mediated by
complement-dependent lysis by: (i) morphology and
formation of a DNA ladder (Figure 2, a and b), (ii)
exogenous Ca2+ independence and (iii) delayed 5lCr
release from radiolabelled target cells. The kinetics of
membrane blebbing induced by anti-APO-1 (within 30 min;
Figure 2a) was not influenced by the presence of lOmM
EDTA or EGTA. In addition, endonuclease-mediated DNA
fragmentation induced by anti-APO-1 was not inhibited by
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WO 91/10448 16 PCT/EP90/00111
the Ca2+ channel blockers Furamicin (50~uM) or Nifedipin
(50 ~M). When 5lCr-labelled SKW6.4 cells were incubated
with anti-APO-1 (1 ~g/ml) for 2, 4, 8 and 24 hours, the
specific 5lCr release [R. C. Duke, R. Chervenak, J.J.
Cohen, Proc. Natl. Acad. Sci. U.S.A. 80, 6361 (1983)] was
found to be 2.9$, 7.6$, 21.3 and 32.5$, respectively.
Trypan blue uptake was measured at the same time points:
2.5~, 4.7~, 10.6$ and 73.6, respectively, of the cells
were trypan blue positive. In contrast, 2 hours after the
addition of MAbs, plus complement the specific 5lCr
release was 108.7 and 92.7 of the cells stained with
trypan blue. These experiments indicate that cell death
induced by anti-APO-1 is fundamentally different from
antibody- and complement-dependent cell lysis.
To assess the specificity of anti-APO-1, a restricted
panel of tumor cell lines was screened for expression of
APO-1 and susceptibility to growth inhibition and
apoptosis. APO-1 was expressed on various human lymphoid
B and T cell lines and was not found on a gibbon or mouse
T cell line or a human monocytic cell line (Table 1).
Anti-APO-1 blocked proliferation of the APO-1-positive
cell lines listed in Table 1 via induction of apoptosis,
and formation of a DNA ladder was observed in each case.
Two hours after addition of the MAbs (1/ug/ml) the
genomic DNA of each tumor line was isolated and analyzed
on agarose gels as described above. Inhibition of
[3H)thymidine uptake by anti-APO-1 was paralleled by
fragmentation of the genomic DNA. This was not observed
after treatment with control MAb (I3BI).
Expression of APO-1 was not restricted to cell lines in
vitro but could be found on leukemic cells freshly
isolated from patients (Table 1). Since APO-1 was not
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zo~mo~
found on all leukemic cells anti-APO-1 may define a
subpopulation of leukemias.
We also screened human B and T cells for expression of
APO-1. We did not detect APO-1 on resting B cells.
However, APO-1 was expressed on activated B cells (Table
1) and IgM secretion was reduced approximately fourfold
by 3 days of treatment with anti-APO-1. (Activated B
cells (106 per milliliter) were incubated in the presence
of MAb FII23 or anti-APO-1 at 1 ug/ml. After 3 days the
culture supernatants were collected and the IgM
concentration measured with a human IgM-specific ELISA
containing HRPO-conjugated goat anti-human IgM (Medac,
Hamburg, FRG). IgM secretion after treatment with FII23
or anti-APO-1 was 2100 and 550 ng/ml, respectively).
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SUBSTITUTE
SHEET
WO 91/10448 19 PCT/EP90/00111
20~120~
Peripheral resting T cells did not express APO-1.
Activated T cells, however, expressed APO-1 and anti-APO-
1 induced apoptosis and growth inhibition of these cells
(Table 1). Thus, our data suggest that APO-1 is a
species-specific antigen expressed on activated or
malignant lymphocytes.
The striking effect of anti-APO-1 in vitro prompted us to
test its effect on tumor growth in vivo. Although the
Epstein-Barr virus (EBV)-negative, Burkitt-like lymphoma
BJAB was the least sensitive to anti-APO-1 of the B cell
panel in Table 1 and expressed only approximately 1.5 x
104 APO-1 epitopes per cell (4), we selected BJAB for our
in vivo experiments. The reason for this choice was that
only BJAB grew to large tumor masses in unirradiated
r~p/r~~u mice. Five weeks after injection of BJAB cells the
r~~u/r~~u mice carried tumors with a diameter of appoximately
1.0 to 2.5 cm (Figure 3). These mice were injected
intravenously with purified anti-APO-1 (500 Jug per mouse)
or the same quantities of various isotype-matched control
antibodies (FII20, anti-MHC class I antigens, recognizing
5.8 x 105 sites per cell; or one of the two nonbinding
MAbs FII23 and I3BI). As a control we also injected anti-
APO-1 ( 500 ~g per mouse ) into three I~~u/r~ mice carrying
the APO-1-negative B cell tumor OCI.LYI with tumor
diameters of 1.5, 1.8, and 3.4 cm, respectively (OCI.LYI
was obtained from H. Messner, Ontario Cancer Institute,
Toronto, Canada) (see also Table 1). Two days after anti-
APO-1 injection, a whitish discoloration of the BJAB
tumors was observed that was followed by rapid tumor
regression. Macroscopic tumor regression was seen in 10
of 11 treated mice within less than 14 days. The control
antibodies had no effect (Figure 3). In addition, no
tumor regression was observed in the mice carrying
SUBSTITUTE SHEET
Zo 2071205
OCI.LYI, as expected.
To demonstrate proper localization and enrichment of the
injected antibodies, labelled MAbs were visualized by
autoradiography of sections of the BJAB tumor tissue
(Figure 4a). These autoradiographs showed a pronounced
binding of anti-APO-1 in the periphery but only sparse
accumulation in the center of the tumor. The binding
control MAb FII20 showed a qualitatively similar binding
pattern. There was no localization of the nonbinding
control MAb FII23 above background. Furthermore, paired
label. experiments (D. Pressman at al., Cancer Res. 17,
845 (1957)) with labelled anti-APO-1 and FII23 revealed
that the specific enrichment of anti-APO-1 over FII23 in
the tumor was four- and sixfold after 48 and 96 hours,
respectively.
The main purpose of our experiments was to assess whether
anti-APO-1 can also act in vivo. Therefore, the tumor-
bearing mice only received one intravenous injection of
anti-APO-1. at a dose in the range used in MAb therapies.
In other therapy schedules, however, Mobs are injected
repeatedly (see e.g. S.L. Brown et al., Blood 73, 651
(1989). In our experiments regrowth of the BJAB tumor was
observed in three of the ten mice in which tumor
regression had been observed (Figure 3). Regrowth was
observed at the margin of the original tumor
approximately 3 months after the initial macroscopic
tumor regression. One of these tumors was removed and
found to express APO-1 by immunofluorescence and to be
sensitive to anti-APO-1 in vitro at a MAb concentration
similar to the original in vitro BJAB tumor cell line
(Table 1).
;a
WO 91/10448 21 PCT/EP90/0011 l
2a71~0~
To determine the histology of the regressing BJAB tumors
we prepared thin sections of tumors from MAb-treated
r~u/rt~u mice . Ten days after intravenous injection of
FII20, BJAB appeared as a solid tumor composed of densely
packed large blasts with numerous mitoses, some tumor
giant cells, and rare apoptotic figures (Figure 4b, left
panel). The tumor was penetrated by host vessels. In
contrast, almost all remaining BJAB cells of mice treated
with anti-APO-1 (Figure 4b, right panel) showed severe
cytopathic changes including nuclear pycnosis and
cellular enema most pronounced in perivascular
microareas. These morphological changes are
characteristic of apoptosis.
Taken together, these data strongly suggest that
apoptosis is induced by anti-APO-1 and is the mechanism
of death and regression of BJAB tumor cells in vivo. In
fact that FII20, which strongly binds to the cell surface
of BJAB tumor cells, did not cause regression of BJAB
also precludes the possibility that killer cells or
complement that might have bound to anti-APO-1 may have
been involved in the growth inhibition and tumor
regression.
Example 2
Materials and Methods
Cell culture medium and reagents:
All cells were cultured in RPMI 1640 supplemented with L-
glutamine (2 mM final concentration), streptomycin
(100~ug/ml), penicillin (100 U/ml), HEPES (25 mM final
concentration) and 10~ fetal calf serum (FCS, Advanced
SUBSTITUTE SHEET
WO 91 /10448 2 2 PCT/EP90/00111
Biotechnologies Inc., Silver Spring, MD). Recombinant IL-
2 (final concentration 20 U/ml) and recombinant IL-4
(final concentration of 5 ng/ml) were purchased from
Boeringer Mannheim, FRG. The cultures were kept at 37°C
in 95$ air, 5~ C02 at 90~ relative humidity.
Cell lines and leukemic cells from ATL patients:
Various HTLV-1 transformed T cell lines were
investigated. C91/P1 is a cord blood T cell line
transformed by HTLV-I and continuously kept in culture in
the presence of IL-2. All other T cell lines used were
originally derived from patients with ATL:JGCL and DCL
are IL-2 dependent cell lines. MJCL and MT1 are IL-2
independent cell lines which still respond to IL-2 with
enhanced proliferation. CRII2 and HUT102 are the
prototype HTLV-I positive leukemic cell lines in which
HTLV-1 was originally described. Growth of these cell
lines does not depend on IL-2 in the culture medium.
As a prototype APO-1 positive and anti-APO-1 sensitive
cell line, B cell line SKW6.4 was included as a control
in all experiments. Trauth B.C., Klas C., Peters A.M.J.,
Matzku S., Moller P., Falk W., Debatin K-M, Drammer PH.
"Monoclonal antibody-induced tumor regression by
activation of an endogenous suicide programme." Science
1989; 245: 301-305.
Thawed leukemic cells from five patients with ATL were
used for the experiments. The cells from patients with a
high leukemic cell count were frozen and stored in liquid
nitrogen in medium containing 20~ FCS and 10$ DMSO. After
careful thawing there was a considerable loss of cells
which appears to be characteristic for ATL cells. Viable
WO 91/10448 2 3 PGT/EP90/0(Il l l
20'~12Q5
cells were isolated after thawing by density gradient
centrifugation (LSM, Organon Technika Corp., Durham,
N.C.) and cultured in vitro for further experiments.
Immunofluorescence analysis
Immunofluorescence staining was determined by flow
cytometry with the following antibodies: anti-Tac-
antibody was used at lJug/106 cells. Anti-APO-1 (IgG3, )
and an isotype matched control non-binding antibody were
used as a 10~ dilution of the original hybridoma
supernatant. Antibodies against CD3, CD4, CD8 were
purchased from Becton Dickinson (Mountain View, CA.) and
used according to the manufacturer's instructions. For
cell surface staining 1 x 106 cells in 100~u1 medium were
incubated with the appropriate dilutions of the
antibodies in phosphate buffered saline (PBS) containing
1$ FCS and 0.3$ Na-azide. Prior to the addition of
antibodies human IgG was added to a final concentration
of 100 ~ug/ml. After incubation for 30 m, cells were
washed and incubated with a second FITC labelled goat-
anti-mouse Ig antibody (TAGO Burlingame, Ca.).
Cell proliferation
Cell proliferation at the cell concentrations indicated
was determined in 96 well flat bottom plates (Costar,
Cambridge, Mass.). After the time indicated O.lluCi of
[3H]thymidine (3H-TdR, Dupont NEN, Boston, Mass.) was
added to each culture well. Proliferation was assessed by
harvesting the plates and 3H-TdR uptake was determined in
a liquid scintillation counter.
SUBSTITUTE SHEET
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Cell death
After incubation of cells with the anti-APO-1 antibody
viability and cell death were determined by the trypan
blue dye exclusion method.
Results
APO-1 expression on HTLV-I positive T cell lines
We first investigated the expression of the APO-1 antigen
on various T cell lines originally established from
patients with ATL. By immunofluorescence staining all
cell lines used displayed a characteristic mature T cell
phenotype (CD4+, CD8 , Tac+). Only the JGCL cell line was
CD4 , CD8+, Tac+. Strong expression of the APO-1 antigen
was found on all cell lines. The intensity of APO-1
expression was comparable to the expression found on
activated normal T cells or on APO-1 positive B and T
cell lines.
Inhibition of proliferation and anti-APO-1 induced
apoptosis of HTLV-I positive T cell lines
To assess growth inhibition and apoptosis of HTLV-I
positive T cell lines by anti-APO-1 the cell lines were
cultured in vitro for two days in the presence of various
concentrations of anti-APO-1. As a control parallel
cultures were incubated with 10 pg/ml of an isotype-
matched nonbinding control antibody. The proliferation of
all T cell lines tested was inhibited by anti-APO-1
antibody. As a positive control, SKW6.4, the highly anti-
APO-1 sensitive cell line, a B lymphoblastoid line
against which anti-APO-1 was originally raised (see
SUBSTITUTE SHEET
WO 91/10448 2 5 PCT/EP90/00111
20'~12~~
example 1) was includes in each experiment. Growth
inhibition of various cell lines, e.g. JGCL and MJCL, was
quantitatively comparable to growth inhibition of the
SKW6.4 cell line.
All highly sensitive cell lines showed the characteristic
morphological features of apoptosis (membrane blebbing,
condensation of nucleus and cytoplasm) after incubation
with anti-APO-1. After two days of incubation with anti-
APO-1 50-98~ of the cells were found to be dead by the
trypan blue dye exclusion method.
Expression of APO-1 on cultured ATL cells
After we had observed APO-1 expression and induction of
apoptosis by anti-APO-1 on the HTLV-1 positive T cell
lines, we tested whether APO-1 is also expressed on the
leukemic cells from patients with ATL. Thawed cells from
frozen peripheral blood cells (more than 50~ malignant T
cells) were investigated. The recovery of ATL cells after
thawing is usually low. After thawing recovery ranged
from 2 - 35~. These cells showed low Tac and variable
APO-1 expression (3 - 15~ and 1 - 53~ positive cells,
respectively). For further studies, the cells were
cultured in medium supplemental with IL-2 for 5 days.
Under these conditions ATL cells increased APO-1 and Tac
expression. Again the intensity of APO-1 expression was
comparable to the one on HTLV-1 transformed T cell lines
and on other sensitive cells such as activated T cells or
malignant T or B cell lines.
SUBSTITUTE SHEET
WO 91/10448 ~~~~ 2 6 PCT/EP90/00111
Effect of anti-APO-1 on proliferation of ATL cells in
vitro
Since APO-1 was expressed on cultured ATL cells we tested
whether anti-APO-1 inhibited proliferation of these cells
in vitro. ATL cells were cultured either in medium alone
or in medium plus IL-2 and IL-4. The reason for culturing
the cells in IL-2 or IL-4 was that in some cases ATL
cells showed a proliferative response to IL-2 or IL-4
without prior activation. Arima N., Daitoku T., Ohgaki S.
et al., "Autocrine growth of interleukin-2 producing
cells in a patient with adult T cell leukemia", Blood
1986; 68: 779-782; Uchiyama T., Kamio M., Kodaka T. et
al., "Leukemic cells from some adult T cell leukemia
patients proliferate in response to interleukin-4", Blood
1988; 72: 1182-1186. Table 1 shows that ATL cells from
patients 1 and 4 already exhibited a high spontaneous
proliferation. In cells from patients 3, 4 and 5
proliferation was augmented by addition of IL-2 or IL-4.
Addition of anti-APO-1 to the cultures greatly inhibited
the proliferation of the malignant cells. Incubation with
a control antibody did not have any effect.
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Table 2. Effect of Anti-APO-1 on proliferation of ATL
cells in vitro
cells from incubation proliferation growth inhibition
patient with of cells in by anti-APO-1 ($)
No. medium (cpm)
1 medium 29682 8?_
IL-2 18872 73
IL-4 99343 61
2 medium n.d. -
IL-2 110167 99
IL-4 43679 99
3 medium 920 18
IL-2 3700 89
IL-4 ~ 5253 97
4 medium 36672 48
IL-2 46716 52
IL-4 61503 60
medium 629 10
IL-2 1724 71
IL-4 2110 81
After thawing 2x105 cells/well were cultured in a 96 well
flat bottom plate. Where indicated IL-2 (20 U/ml) or IL-4
(5ng/ml) were added. The cells were cultured for 3 days
in the presence of medium alone or with 1 ~ag/ml anti-APO-
1 or 1 ,ug/ml isotype matched control antibody. 0.5~Ci of
H-TdR were added for the last 8 h of culture /.
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WO 91/10448 b ~s'~ 2 8 PCT/EP90/00111
,
Proliferation was measured by determining H-TdR uptake.
Data are given as the absolute cpm (mean) for culture in
medium alone (cpm control) and the percentage of
inhibition of H-TdR uptake by anti-APO-1. The standard
deviation for triplicate cultures was less than 10~.
Incubation with the isotype matched control antibody had
no effect compared to culture in medium alone (not
shown).
Induction of apoptosis of cultured ATL cells by anti-APO-
1 treatment in vitro
To investigate the induction of apoptosis of ATL cells by
anti-APO-1 thawed ATL cells were first cultured for five
days in the presence of IL-2 (20 U/ml). During this time
no net gain in cell numbers during culture was observed.
The cultured cells were then incubated for 48 hours with
1 ~ug/ml anti-APO-1 or control antibody, respectively.
Under these conditions 75 - 100 of ATL cells were dead
after anti-APO-1 treatment.
Conclusion
All HTLV-I positive T cell lines, originally established
from patients with ATL, were found to express APO-1.
Incubation of these cells with anti-APO-1 led to
inhibition of proliferation via induction of apoptosis
similar to activated T cells. The sensitivity of HTLV-I
positive T cell lines for anti-APO-1 mediated apoptosis
varied between different cell lines (antibody
concentration necessary for 50$ of growth inhibition: 70
- 50 ng/ml <JGCL and MJCL> and 1000 - 2000 ng/ml <HUT102
and C91/P1>). The most sensitive cell lines (JGCL and
MJCL) were as sensitive as the most sensitive cell line
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WO 91/10448 2 g PCT/EP90/00111
(SKW6.4) found in previous experiments and against which
the antibody was originally raised. Interestingly,
proliferation of JGCL and MJCL appeared to depend on
exogenous IL-2. This may suggest a connection between IL-
2 responsiveness and sensitivity for anti-APO-1 induced
apoptosis.
Expression of APO-1 was also found on cultured cells from
the peripheral blood of patients with ATL. Thawed cells
from such patients could not directly be studied since
the viability was too low after thawing. However, when
cultured in vitro in medium alone or in the presence of
cytokines (IL-2 or IL-4) these cells were found to
express APO-1. Treatment of these cells in vitro with
anti-APO-1 strongly inhibited the spontaneous
proliferation and the IL-2 or IL-4 augmented
proliferative responses. Finally, the inhibition of
proliferation was accompanied by the induction of
apoptotic cell death. These data on cultured ATL are in
contrast to the data obtained with normal T cells. In
normal T cells APO-1 expression needs prior activation
with mitogens and anti-APO-1 treatment has no effect on
short term proliferation.
Taken together we have demonstrated that expression of
APO-1 is a characteristic feature of HTLV-I transformed T
cell lines and cultured malignant T cells from patients
with ATL. Incubation of these cells and cell lines with
the anti-APO-1 antibody induces apoptosis.
SnJ~s i i ~ U'~~ ~"r~~'~
