Note: Descriptions are shown in the official language in which they were submitted.
2 ~j C~
FIELD OF THE INVENTION
The present invention relates to human
hybridoma cell lines and to the monoclonal antibodies
produced by these cell lines.
BACKGROUN~ OF THE INVENTION
Malignant gliomas are the most common of all
primary brain tumors. They are also among the most
difficult to manage clinically~ Depending on their
intracranial location, they can grow to a substantial
size before causing any symptoms. Their biological
behavior is notoriously aygressive, although they rarely
metastasize outside of the CNS.
Surgery and radiotherapy alone are seldom
curative; with the best of care, median survival is
under one year. Moreover, desplte major efforts to
introduce new adjunctive therapies, the prognosis for
patients with malignant gliomas has remained essentially
unchanged for the past thirty years.
Many ad;unctlve therapies, including
immunotherapy, are either presently ineffective, or
carry an unacceptable risk of serious morbidity. With
the development of murine hybridomas by Kohler and
Milstein (1975); however, tumor immunodiagnosis and
immunotherapy with monoclonal antibodies (mAb) has
emerged as a promising area of investigation.
Studies with murina mAbs have already
contributed substantially to the understanding of gl~oma
biology and heterogeneity. However, these reagents may
not be ideally suited for clinical application in humans
for a variety of reasons. In contrast, human mAbs offer
the poss1bilities of greater specificity and biological
compatibility than their murine counterparts.
Malignant gliomas are neuroepithelial tumors
which commonly arise in the cerebral hemispheres. As a
group, they include anaplastic astrocytoma, anaplastic
-- 1 --
oligodendroglioma, anaplastic ependymoma, and
glioblastoma multiforme. These tumors all possess
certain microscopic features in common such as dense
cellularity, increased numbars of mitotic figures,
nuclear pleomorphism with hyperchromasia, and cellular
pleomorphism. In glioblastoma multiforme, the above
features are present, as well as necrosis with
pseudopalisades, capillary endothelial proliferation
with pseudorosette formation, and mesenchymal
proliferation. These tumors are heterogeneous and
demonstrate both inter- and intra-tumor variability,
which may well be the major determinant of therapeutic
resistance. Since few markers exist which are selective
for poorly differentiated glioma cells, i.e. stem cells,
it would be highly beneficial to selctively target these
cells within an otherwise heterogeneous population since
failure to deplete a tumor of its stem cell compartment
inevitably results in tumor recurrence.
It is well established that many patients with
malignant gliomas are significantly immunosuppressed,
especially with regards to cell-mediated functions,
although few patients are cachectic. The finding of
immunosuppression in the absence of significant systemic
wasting would suggest that the former is due to tumor-
intrinsic mechanisms, rather than a reflection of
excessive tumor burden, as is usually the case. Humoral
immune mechanlsms in patients with malignant gliomas
also appear to be depressed, albeit they remain intact.
Malignant gliomas possess many antigens in
common with the normal cells from which they were
derived. As a result, it is virtually impossible to
prepare conventional heteroantisera to glioma tissue
without encountering some degree of cross-reactivity
with normal adult brain. The shared antlgens may be
either intracellular, membrane associated, or
extracellular in nature.
-- 2
The role of glycolipids and carbohydrate
structures in cellular interaction, differentiation, and
oncogenesis has received much attention lately. A
subclass of glycolipids known as gangliosides may play
an important role in the regulation of cellular adhesion
and proliferation. When a cell undergoes malignant
transformation, there may be subtle qualitative and
quantitative changes in the chemistry of its surface
gangliosides.
Malignant gliomas are well vascularized
tumors. However, there is brèakdown or total absence of
the blood-brain-barrier that exists in a normal, healthy
brain, in which the brain is normally 'privileged', or
isolated from the effects of the immune system or other
blood-borne agents. Because of this breakdown of the
blood-brain-barrier in patients with these tumors, the
immune system has access to the tumor cells.
BRIEF REFE~ENCE TO THE PRIOR A~T
Studies with murine mAbs raised against glioma
cell lines were reported by Schnegg, et al. (1981b) and
Bourdon, et al. (1983). Both groups immunized mice with
cultured human glioma cell lines, and fused spleen cells
with the mouse myeloma lines P3X63-Ag8 or P3X63-Ag8.653,
respectively. The mAbs BF7, GE2, described by Schnegg
et al (1981b) appear to be relatively glioma-restricted,
whereas antibody CG12 (de Tribolet, et al. 1984) reacts
wlth a spectrum of gliomas, melanomas, and
neuroblastomas. Antibody 81C6 was produced and
characterized by Bourdon, et al. (1983) and found to
react with a glioma-mesenchymal e~tracellular matrix
(GMEM) antigen expressed on gliomas, neuroblastomas,
melanomas, sarcomas, and cultured fibroblasts. In
addition to the above, the GMEM antigen was also found
in normal liver sinusoids, spleen red pulp sinusoids,
kidney medullary tubule interstitium, and glomerular
-- 3 --
mesangium. Whereas absorption of CG12 with normal adult
and fetal brain abolished ~inding activity, neither BF7,
GE2, nor 81C6 were affected by the same treatment.
Reports of human monoclonal antibodies (HmAbs)
from patients with malignant gliomas have appeared in
the literature. However, none of these antibodies have
been well characterized.
Sikora, et al. (1982) reported the production
of HmAbs reactive to 0.25% glutaraldehyde-fixed glioma
cells. These investigators obtained intratumoral
lymphocytes from 12 patients undergoing craniotomy for
malignant glioma and fused them to the E8NA~ 8-
azaguanine-resistant human lymphoblastoid line LICR-LON-
HMy2, which is known to secrete gamma and lambda chains.
A total of 71 hybridomas were obtained from 5 patients.
No subcloning experiments were reported and no
demonstratlon or proof of the monoclonality of the
hybridomas is given. ~his study also reported cross-
reactivity of the HmAbs to other tumor cell-lines. The
disadvantages of the requirement for intratumoral cells
from the human brain as the starting material are clear.
In a subsequent study by Sikora et al. (19~3),
the authors concluded that, for the HmAb singled out for
study, this HmAb was of a low affinity because of the
high concentrations necessary to demonstrate reactivity
with targets.
Other HmAbs cross-reactive with glioma-
associated antigens have been produced and
characterized.
It is an object of the present invention to
provide human monoclonal antibodies to neurological
tumors, useful in immunodiagnosis and with a po~ential
for immunotherapy. It is a further object of the
invention to provide a process for producing such
monoclonal antibodies.
SUMMARY OF THE INVENTION
The present invention is based on the
principle that human patients with neurological tumors
should possess circulating B-lymphocytes with anti-
glioma specificities. It is also based on the principle
that tumor-reactive antibodies derived from glioma
patients should recognize common, non-allelic
determinants in human gliomas which might differ in
specificities and distribution from those recognized by
xenogeneic systems.
In the present invention, human-human
hybridomas are used to prepare human monoclonal
antibodies (HmAbs) to gliomas, the hybridomas being
derlved from fusion of a human myeloma-like line and
lymphocytes obtained from human patients suffering from
gliomas. By appropriate culturing of the resultant
hybridomas, effective HmAbs to human gliomas are
obtained in useful quantities.
The HmAbs and the processes for obtaining them
differ from the work reported by Sikora et al and
referenced earlier, in that, inter alia, Sikora et al
used intratumoral lymphocytes as opposed to peripheral
blood lymphocytes, of glioma-suffering human patients to
form the hybridomas. Also, Sikora et al used human
lymphoblastoid cell lines as the fusion partner as
opposed to human myeloma-like cell line used in the
present invention.
Monoclonal antibodies derived from human cell
lines as in this invention have slgnificant advantage
over those derived from the cells of other mammalian
species, particularly when intended for use in human
subjects. It is believed that human source-derived mAbs
have increased compatibility with and increased
sensitivity of recognition for human target cells.
Thus, the mAbs of the present invention have beneficial
application to their proposed range of uses, including
-- 5
~t, A'l ~ F' i~
their use in diagnostic imaging for the
radiolocalization of neurological tumors, and in
immunotherapy by either direct action or by lin~ing
these mAbs to chemotherapeutics which can then be
targeted directly to the tumor cells.
DESCRIPTION OF THE PREFER~ED EMBODIMENTS
The present invention utilizes hybridomas
derived from the fusion of a hum~n myeloma-like cell
line and a lymphocyte obtained from the circulating
blood of a person having a neuroectodermal tumor. The
preferred myeloma-like cell lines are characterized by
being deficient in the enzyme hypoxanthine-guanosine
phosphoribosyl transferase (HGPRT) and by being an
immunoglobulin non-secretor. The specific, most
preferred such myeloma-like cell line is that designated
TM-H2-SPZ. This is a sub-line of the parent line TM-H2
described by Sullivan et al (1982).
From the myeloma-like cell line TM-H~-SP2
there have been prepared, by cell fusion techniques,
five novel hybridomas, each of which derives from a
different lymphocyte extracted from the blood of a human
patient having a neuroectodermal tumor, fused to TM-H2-
SP2. These novel hybridomas, designated BT27/lA2;
BT27/2A3; BT32/A6; BT34/A5; and BT54/B8, all produce
mAbs to gliomas, in relatively good yield. Viable
samples of these novel hybridomas are in process of
being deposited with the American Type Culture
Collectlon (ATCC), Bethesda, Maryland, in accordance
with the provisions of the Budapest Treaty.
A remarkable characteristic of the mAbs of the
present invention is the high degree of similarity
amongst the five mAbs, derived from the five hybridomas,
despite being derived from cells obtained from four
~`J ~) 2.; ._. ~J ~J i~
different tumor patients, each having a different tumor
type, history, or stage of tumor development.
The human myeloma-like cell line TM-H2-SP2
used in the preferred embodiment of the present
5 invention is the immunoglobulin non-secreting subline of
the parent line, TM-H2, described by Sullivan, et al.
(1982). TM-H2 is a hypoxanthlne guanine phosphoribosyl
transferase (EC 2.4.2.8) deficient (HGPRT-) derivative
of an unknown human myeloma-like line, which was
selected in 0.8% (w/w) methylcellulose for res$stance to
6-thioguanine (6 ~g/ml) and failure to grow in
hypoxanthine-aminopterin-thy~id$ne (HAT) medium
(Littlefield, 1964). TM-H2 produces and secretes up to 3
~g/ml of IgG(K) in supernatant culture fluid when grown
to a concentration of 5 x 10$ cells/ml in alpha-MEM
containing 10% FCS. The subline TM-H2-SP2 produces but
does not secrete immunoglobulin, as determined by
quantitative ELISA. TM-H2-SP2 has a 46, XX karyotype,
and was grown continually in the presence of
6-thioguanine (6 ,ug/ml) to prevent HGPRT revertants. The
line is negative for Epstein-Barr nuclear antigen
(EBNA-), and therefore does not appear to be an
EBV-immortalized derivative of lymphoid cells.
The invention i5 further illustrated in the
following specific examples and experimental protocols
and procedures.
MATERIALS AND METHODS
. . _
Derivation of Human Anti-Glioma Monoclonal Antibodies
from Patients with Neurological Tumors
Clinical Material
Human patients were selected who were
suspected of having a brain tumor on the basis of
history, physical examination, and CT scan. From each
such patient, approximately 40 ml of peripheral venous
blood was withdrawn into four heparinized glass tubes
(Vacutainer), and stored at room temperature until
further use.
Within 2 hrs. of sampling, the lymphocytes
were separated on a Ficoll-Hypaque density gradient
(specific gravity 1.077 g/ml) by carefully layering two
20 ml aliquots of whole blood onto 20 ml of 5% (w/v)
Ficoll-Hypaque and centrifuging at room temperature for
25 minutes at 1000 G. The lymphocytes were harvested
from the interface, washed once with sterile
phosphate-buffered saline (PBS) (8 g NaC1, 1.144 g
Na2 HP04 (anhydrous), 0.2 g KH2 P04, and 0.2 g KCl in 1
liter deionized water and adjusted to pH 7.2), and
re-suspended in 10 ml alpha minimum essential medium
(alpha-MEM) containing 10~ (v/v) fetal calf serum (FCS;
Flow Laboratories, Rockville, MD), 292 mg/L L-glutamine,
44 mg/L L-asparagine, 100 U/ml penicillin, and 100 ,ug/ml
streptomycin.
The lymphocytes were then counted in a
hemacytometer after diluting 0.1 ml of the mixture with
0.9 ml of 2% glacial acetic acid, in order to lyse any
remaining red blood cells. If human myeloma-like
-- 8
~ ~' C`J ~' ~ ''` ~3
TM-H2-SP2 cells in logarithmic growth phase were
avallable, a fuslon procedure would be carried out
immediately (see below); otherwise the lymphocyte
mixture was stored at 4C if it was felt that the
TM-H2-SP2 line would be ready for fusion within the next
24 hrs. If no fusion procedure was to be carried out,
the lymphocyte mlxture was re-suspended in 1 ml alpha-
MEM + 10% FCS, and stored at -70C in a cryotube
~Nunclon, Denmark) containing 0.1 ml sterile dimethyl
10 sulfoxide (DMS0) as a preservatlve.
At the time of surgery, a portion of
autologous tumor (1-5 gm) was removed and placed in
sterile Elliott's solutlon (Abbott Laboratories Inc.)
for preparation of KC1 extracts. After mincing with fine
15 scissors, fresh tumor was placed in a conical test tube
and agitated overnight at 4C with 3 M KCl (1 ml/gm wet
tissue) The crude cell lysate w~s diluted 1:10 with
ddH20, and clarified by high speed centrifugation at
12,000 G for 20 min . at 4C. The extract was then
20 dialyzed and equilibrated to PBS over the next 6 hrs,
and then filter-sterilized with a 0.22 ~m Millipore
filter. The protein concentration of the tumor extract
was determined using the method of Lowry et al. (1951),
and the necessary dilutions made with PBS to bring the
25 final protein concentration to 100 ~g/ml. Aliquots were
stored at -20C until ready for use in the screening
assay (see below).
Human Glioma Cell Lines
The human glioma cell lines used in this study
were obtained from other research institutions.
All cell lines were grown in (alpha-MEM
containing 10~ fetal calf serum (FCS; Flow
35 Laboratories), 292 mg/l L-glutamine, 44 mg/l
L-asparagine (anhydrous), 100 U/ml penicillin, and
_ g _
100 ~g/ml streptomycin in the presence of 5% C02
atmosphere at 37C. The cells were grown to confluence
in standard tissue culture flasks (Nunclon, Denmark),
and passaged in a split ratio of 1:2 using citrated PBS
(Gibco, Grand Island, NY) containing 0. l~ trypsin
(Difco Laboratories, Detroit, MI) to remove the cells,
and FCS to stop the action of the enzyme on the cells.
Fusion Procedure to Prepare Hvbridomas
The method used was a variation of the
original method of ~ohler and Milstein (1975).
Twenty-four hours prior to fusion, the TM-H2-SP2 cell
line culture in mid- to late-logarithmic phase was
dlluted 1:1 with fresh alpha-MEM containing 10% FCS and
6-thioguanine (6 ~g/ml). This was done to ensure a
ma~ority of cells were cycling in growth phase. At the
time of fusion, an aliquot of TM-H2-SP2 cells was
stained with an equal volume of 0.16~ trypan blue, and
the viable cell count was determined in a hemacytometer
by the dye exclusion method (Gorer and O'Gorman, 1956).
If the viable cell count was below B0~ the cells were
not used on that day.
The TM-H2-SP2 cells were then combined
directly with the peripheral lymphocytes in a lymphocyte
to myeloma cell ratio of 4:1, and pelleted together by
centrifugation at 500 G for 5 min. The supernatant was
discarded, and the cells were fused under serum-free
conditions by the gradual addition of 1 ml of 50
polyethylene glycol (m.w. 1450) (Sigma, St. Louis, M0)
diluted 1:1 in serum-free alpha-MEM over 1 minute
(Pontevorco, 1976). This was followed by dilution over
the next 4-5 minutes with lO ml of serum-free alpha-MEM,
of which 2 ml ~ere added with gentle stirring over the
first 2 minutes.
-- 10 --
~PJ~
The fusion mixture was then pelleted by
centrifugation as before, and washed twice with 10 ml of
hypoxanthine-aminopterin-thymidine (HAT) medium
containing 10% FCS, lO- 4 M hypoxanthine, 4 x 10- 7 M
aminopterin, 1.6 x 10-5 M thymidine, 292 mg/l
L-glutamine, 44 mg/l L-asparagine, 100 U/ml penicillin,
and 100 ~g/ml streptomycin. A viable cell count on 0.1
ml of the fusion mixture was performed as before, and
the cells diluted to a concentration of either 1 x lOs
or 2.5 x 105 viable myeloma cells/ml by addition of
fresh HAT medium. The cells were then dispensed in 200
,ul aliquots into 96-microwell trays (Linbro Laboratories
Inc., McLean, VA; 0.28 cm2/well), and incubated at 37C
in 5% CO2 atmosphere.
Culture Conditions
The hybridomas were maintained in HAT medium
for 7 days post-fusion, with exchange of half the
culture media on day 5, followed by re-feeding with
fresh HA~ medium. On day 7, 50~ of the culture media was
again removed, and the hybridomas re-fed with
hypoxanthlne-thymidine (HT) medium (HAT medium minus
aminopterin). This process of exchange feeding with HT
medium was continued every 5 days untll the hybridomas
became microscopically visible (approximately 46 weeks
post-fusion, at which point they were ready for testing
by ELISA. Selected hybridomas were continuously
propagated up to transfer into 25 cmZ culture flasks
(Nunclon, Denmark) in a volume of 2 ml via 24 well trays
(Linbro Laboratories Inc., McLean, VA), then stored at
-70C in 10% FCS and 10% DMSO and alpha-MEM. During
culture, all hybridomas were maintained con~inually in
HT medium.
~ J ~ .; .,/; J 3
Tumor Extract ELISA
The screening method used was a modification
of Douillard and Hoffman (1983). Briefly, 96-well
polyvinyl chloride Microliter ELISA plates (Dynatech,
Alexandria, VA) were incubated for 12-18 hours at 4C
with 200 ~l/well of 3 M KCl autologous tumor extract as
described under "Clinical Material" at a concentration
of 5 ~g proteln/ml (1 ,ug/well) in carbonate-bicarbonate
10 buffer, pH 9.6 (0.8 gm Na2CO3, 1.47 gm NaHC03, 0.1 gm
NaN3 made up to 500 ml with ddH20). The plates were
washed 3 times with PBS containing 0.05% (v/v) Tween-20R
(PBS-Tween; Sigma, St. Louis, M0) Then 50 ~1 of the
hybridoma supernatant to be tested was added to 50 ,ul
15 PBS per well.
Culture supernatant from the parental myeloma
line, TM-H2, containing IgG(K), was used as a
nonspecific immunoglobulin control for the initial 15
fusions, whereas culture supernatant from one of the
20 hybridomas (BT27/2D2) containing 1-2 ~g/ml IgM was used
as a control in subsequent screening assays.
After 2 hours incubation at room temperature,
th~ plates were washed 3 times with PBS-Tween, and 200
~l/well alkaline phosphatase (ALP)-conjugated goat
25 anti-human ~- and gamma-chain plus goat anti human kappa
and lambda immunoglobulin light chains (TAG0,
Burlingame, CA) were added at 1:2000 dilution in 1%
bovine serum albumin (BSA; Fraction V, Sigma, St. Louis,
M0) in PBS. After a further 2 hours incubation at room
30 temperature, the plates were again washed 3 times with
PBS-Tween, and developed with freshly prepared Sigma 104
phosphatase substrate (p-nitrophenyl phosphate disodium;
Sigma, St. Louis, M0), 1 mg/ml in diethanolamine buffer,
pH 9.8 (97 ml diethanolamine, lO0 mg MgCl2.6H20, 0.2 gm
35 NaN3, ddH20 added to bring volume to lL) . The plates
were left at room temperature for 20-30 minutes, then
phosphatase activity was determined as absorbance at 405
nm using a Dynatech Microelisa autoreader (Dynatech,
Alexandria, VA). A hybridoma supernatant was considered
to be 'positive' if phosphatase activity exceeded the
mean background level of wells with control culture
supernatants by greater than 2 standard deviations.
Flxed Cells ELISA
The method used was adapted ~rom Suter, et al.
(1980). Flexible polyvinyl chloride Microliter ELISA
plates were coated with 100 ~l/well freshly prepared
poly-L-lysine (50 ~g/ml in PBS), and allowed to stand at
room temperature for 40 min. The outer rows and columns
were not used because they tended to give false positive
absorbance values for this particular ELISA assay. The
poly-L-lysine was then removed, and the plates washed
once with PBS. Freshly harvested human glioma cells,
which had grown to confluence in alpha-MEM in a 175 cm2
tlssue culture flask, were removed in PBS with a rubber
policeman and seeded into microwells at 105 cells per
well in 100 ~1 PBS. The plates were allowed to incubate
for 45 minutes at room temperature, then gently rinsed 3
times with PBS.
The cells were fixed by the addition o~ 100
~l/well of 0.1% glutaraldehyde in PBS for 3 minutes,
then gently r~nsed 3 times with PBS. To block any
residual glutaraldehyde activity, 100 ~l/well of PBS
containing 0.2% gelatin 275 Bloom (Fisher, Montreal, QC)
with 0.2% NaN3 (PBS-gelatin) was added, and the plates
allowed to incubate at 4C for at least 12 hours before
proceeding further with the assay. In this condition,
the plates could be stored at 4C for up to 6 months.
On the day of the assay, the PBS-gelatin was
removed by inversion of the plate, and lQO ~1 of
hybridoma supernatant was added directly to each
- 13 -
~ J 3 ~ ~ ~ ~
microwell without prior washing. Control supernatants
for this assay were the same as for the autologous tumor
extract assay described in the previous section. The
plates were incubated for 30 minutes at 37C, then
cooled for 15 minutes at room temperature. They were
washed 5 times with freshly prepared PBS containing
0.05% (v/v) Tween-20R and 0.1% (v/v) gelatin
(PBS-Tween-gelatin), and the microwells filled with 100
,ul of ALP-conjugated goat anti-human kappa and goat
anti-human lambda immunoglo~ulin light chalns diluted
l:1,000 each in P~S-Tween-gelatin.
After 30 minutes incubation at 37C and 15
minutes cooling at room temperature, the plates were
washed 3 times with PBS-Tween-gelatin. Phosphatase
activity was developed with the addition of 200 ~l/well
Sigma 104 phosphatase substrate solution (l mg/ml)
freshly made in diethanolamine bu~fer (97 ml
diethanolamine, 800 ml deionized water, 0.2 g NaN3, and
lO0 mg MgCl2.6H20, made up to 1 liter with deionized
water and ad~usted to pH 9.8 with HCl~, and assessed as
absorbance at 405 nm using an automated Dynatek
Microelisa reader. Hybridoma microwells with phosphatase
activity which exceeded mean background levels of ~he
control supernatant wells by greater than 3 standard
deviations were considered 'positive' ir. this assay.
Flow Cvtometry
Confluent phase cultures of the cell lines
were gently detached from tissue culture flasks with a
rubber policeman and collected in ice cold PBS
containing 1% FCS (In general, all cell preparations
were scraped. In some experiments, however, the cells
were recovered by trypsinization, which did not result
in any differences in labelling). Approximately l x 106
cells per sample were aliquoted into 10 ml test tubes,
- 14 ~
~ ~ 3~
and pelleted by centrifugation for 5 min at 500 G. The
PBS-l~ FCS was poured off and 300 ~1 of spent hybrldoma
supernatant was added to each sample. After resuspending
the pellet, the cells were placed on ice for 60 min.
Following 3 washes with 5 ml cold PBS-1% FCS, lO0 ,ul of
FITC-con;ugated goat anti-human IgM (IgG fraction, ~u
chain specific; Cappel Laboratories, Cochranville, PA),
diluted 1:10 in sterile P~S, was added to each sample.
The cells were then placed on ice for 60 min, and washed
3 times w~th PBS-1% F~S as before. After the final wash,
the cell pellet was resuspended in 500 ,ul of PBS-l~ FC~,
and transported to the flow cytometer on ice.
The apparatus used in these experiments was a
FACS-III fluorescence-activated cell sorter
(Beckton-Dickinson FACS Systems, Mountain View, CA)
linked to a custom-built microprocessor-based system
capable of performing realtime correlated acquisition,
storage, and display of multiparameter (3-parameter)
data (Stewart and Price, 1986~. Prior to each
experiment, the FACS-III was calibrated and standardized
for small angle light scatter and fluorescence intensity
with polystyrene latex microspheres and "Fluoresbrite"-
carboxylated microspheres (Polysciences IncA, Warrington
PA). Fluorescence intensity between 530 and 560 nm was
measured on all samples using an excitation wavelength
of 488 nm. Logarithmic small angle scatter,
fluorescence, and 90 scatter data were collected on a
minimum of 2 x 10~ gated cells from each sample, and the
information stored on flexible diskettes. Fluorescein-
labelled preparations are excited at 488 nm, and
fluorescence is measured between 530 nm and 560 nm using
a suitable combination of optical filters. Rhodamine-
labelled preparations were excited at 514 nm, and
fluorescence was measured above 620 nm.
- 15 -
rd ~ . J ~
Freezing and Thawing Procedures
All cells (gliomas, hybridomas, etc.) were
frozen in essentially the same manner. Approximately
107-iO~ cells were pelleted by centrifugation at 500 G
for 5 minutes, and the supernatants were discarded. The
cells were resuspended in 1 ml of the usual tissue
culture media, and pipetted into a 43 x 12.5 mm cryotube
(Nunclon, Denmark) . Sterile DMSO (0.1 ml~ was added to
10 the cryotube, and the cells placed directly into the
-70~C freezer. After 24 hours, the cells could be
removed to liquid nitrogen for long term storage.
Cells were thawed by removing a cryotube from
the -70C freezer and placing it directly in a 50C
15 water bath for 70 seconds. Before the ice pellet was
fully dissolved, the contents of the cryotube were
emptied into a screw-cap conical tube (Sarstedt, W.
Germany) containing 50 ml of tissue culture medium, and
pelleted by centrifugation at 500 G for 5 minutes. The
20 supernatant was then discarded, and the cells
re-suspended in fresh media and plated or placed in
suspension culture according to the cells' requirements.
KarYotyping of_HYbridomas
Metaphase chromosome spreads were prepared by
first culturing =1 x 106 hybridoma cells overnight in 3
ml of HT medium containing 10~ FCS and 0.06 ~g/ml
colcemid (Fisher). The next day, the cells were washed
30 in PBS and pelleted before resuspension in 3 ml 0.07 M
KCl for 20 min at room temperature. Then lO ml of
methanol:acetic acid (3:1) was added, and the cells were
pelleted at 1700 rpm. The cells were washed twice again
with methanol:acetic acid- before re-suspension in 1 ml
35 of methanol:acetic acid. The mixture was dropped from a
height of approximately 2 meters onto pre-warmed glass
- 16 -
slides in order to burst the cells and spread their
chromosomes. The cells were stained for 3 min with
Giemsa (Fisher, Montreal, QC~, followed by mounting with
Permount (Fisher, Montreal, QC). Fifteen to 20 metaphase
spreads were counted to establish the chromosome number
distribution.
Southern Blotting
Genomic DNA was isolated from TM-H2-SP2,
BT27/lA2, BT27/2A3, BT32/A6.5 (a subclone of BT32/A6),
and BT27/2D2 cells according to Maniatis, et al. (1982).
T~n ~g of DNA were digested overnight at 37C with Bam
HI and Hind III restriction enzymes (23 units/~g DNA;
Boehinger-Mannheim, West Germany) according to the
manufacturer's specified conditions. After digestion,
the DNA was subJected to electrophoresis on a 0.8% (w/v)
agarose gel (Maniatis, et al. 1982), and transferred to
a Genescreen P1US membrane (NEN, Boston, MA) by the
usual method. Following prehybridlzation with herring
sperm DNA (80ehingerMannheim, West Germany), the
Southern blot was hybridized (Kaufmann, et al. 1985)
overnight at 65~C with a 3 2 P-labelled JH probe (Oncor
Inc., Gaithersburg, MD), specific activity of 5-6 x
108/~g. The probe includes the entire human JH region,
and totals 5.6 kbp (Revetch, et al. 1981). Following
washing, the blot was exposed to Kodak X-ray film with
intensifying screens at -70C.
Characterization of Human Monoclonal Antibodies to
-
Glioma Cell Surface Antigens
Determination of Immunoqlobulin _Isotype and
Concentration
Immunoglobulin chain class was determined by
coating four sets of 96-well microliter ELISA plates
with goat anti-human Ig (IgA + IgG + IgM) (Cappel
Laboratories, Cochranville, PA ) at 1:4500 dilùtion.
Four replicates of the supernatants to be tested were
added to each of the plates, and the latter incubated at
room temperature for the usual 2 hrs. Spent culture
media from the parental line, TM-H2, served as a source
for kappa and gamma chains, and supernatant from the
human B-lymphocyte line, RPMI 1788 (Huang and Moore,
1969), as a source for lambda and ,u chains. One plate
was then labelled with ALP-con~ugated goat anti-human
kappa chains, another with ALP-conjugated anti-human
lambda chains, the third with ALP-con;ugated anti-human
gamma chains, and the last plate with ALP-conjugated
anti-human ~- chains. All ALP-con~ugated antisera (Tago
Inc., Burlingame, CA) were used at a dilution of 1-2000
in P8S and 1% BSA. The ELISA plates were developed with
Sigma 104 phosphatase substrate in the usual manner, and
O.D. measurements performed at 450 nm.
Measurement of human IgM concentrations in
spent hybridoma culture media from the 3 hybridomas was
performed by quantitative ELISA some 10 months
post-fusion for BT27/lA2 and BT27/2A3, and 9 months
after the fusion date for BT32/A6. From the time of
fusion to the date the ELISA was performed, the
hybridomas were grown continuously in vitro for a total
of 4-6 months.
On the day the ELISA was performed, the
hybridomas were growing in logarithmic phase at a
- 18 -
~sJc.~
concentration of 5 x 105 cells/ml, in culture media
which had been completely replenished 5 days previously.
Concentrations of Ig were determined by quantitative
ELISA.
For each hybridoma supernatant to be tested,
one 96-well ELISA plate was coated wlth goat anti-human
Ig (IgA + IgG + IgM) (Cappel Laboratories, Cochranville,
PA) at 1:4500 dilutlon. Affinity purified human IgM
(Cappel Laboratories, Cochranville, PA) at a
concentration of 5.5 mg/ml was diluted in PBS to a final
concentration of 500 ~g/ml. From the latter solution,
dllutions in PBS were prepared with concentrations of 50
ug/ml, 5 ,ug/ml, 0.5 ~g/ml, and 0.05 ~g/ml.
The IgM standards were then applied to the
ELISA plates, along with the supernatants to be tested.
ALP-con~ugated go~t anti-human IgM (~ chain specific;
Tago, Inc., Burlingame, CA) was added as a second
antibody, and the plates developed with Sigma 104
phosphatase substrate in the usual manner. A standard
curve of IgM concentration versus O.D. was plotted using
a 4-cycle, semi-logarithmic scale, and the mean O.D.
values for the hybridoma supernatants converted to IgM
concentration.
Antibodv Dilution Curves
Spent culture supernatants from each of the
hybridomas were diluted in non-specific control IgM
(BT27/2D2) and reacted in an ELISA assay with the 3 M
KC1 extract of BT-27 (high grade astrocytoma).
Undiluted control supernatant from BT27/2D2 was also
included in each ELISA plate for purposes of compa~ison.
For each ELISA plate, the mean background
(BT27/2D2) O.D. value was subtracted from the mean O.D.
value for each dilution, and the difference (delta O~Do )
plotted as a function of dilution. Mean O.D. values
-- 19 --
which were greater than background were compared with
the latter using Student's t-test for unpaired data.
Antibody Affinity ELISA
The method used is a modification of De
Bernardo and Davies (1987). Briefly, a 96-well ELISA
plate was coated with the 3 M KCl extract of brain tumor
BT-27 (high grade astrocytoma)~ The plate was washed 3
times with PBS containing 0.05% Tween-20R (PBS-Tween),
and the wells filled with 50 ,ul of PBS. Fifty ~l of
spent hybridoma culture supernatant from each of the 5
HmAbs (BT27/lA2, BT27/2A3, BT32/A6, BT34/A5, BT54/B8),
as well as control IgM (BT27/2D2) were added to half of
the wells. Fifty ,ul of PBS was added to the remaining
wells.
After 14 hrs incubation at 4C, 50 ~l of fluid
was withdrawn from the PBS-containing wells, and
hybridoma supernatants added back. The ELISA plate was
then incubated for a further 4 hrs at 4C, after which
it was emptied, and washed 3 times with PBS-Tween.
ALP-conjugated goat anti-human IgM (,u chain specific;
Tago, Inc., Burlingame, CA) diluted 1:2000 in 1~ BSA-PBS
was then added to the wells, and the incubation
continued for a further 2 hrs at room temperature. The
plate was developed with Sigma 104 phosphatase substrate
in the usual manner, and mean O.D. values for each
supernatant and incubation time were calculated.
Statistlcal comparisons between different
supernatants incubated for the same length of time were
carried out using Student's t-test, or the Mann-Whitney
~-test, where two variances differed significantly
according to the F-test (p<0.05). Comparisons of the
same supernatant incubated for different lengths of time
were also made. The results were plotted on a bar graph
as mean differences ~ standard errors.
- 20 -
~ 3,~
Antigen Expression and Glioma Culture Densitv
The human glioma cell line, SK-MG 1, was grown
to confluence in T25 flasks (Nunc, Denmark) under
standard conditions, and removed with trypsin. The
cells were diluted in (alpha-MEM contalning 10% FCS and
re-seeded into T25 flasks at varlable split ratios
ranging from 1:2 to 1:8. After 72 hrs in culture
without any exchanges of media, the old culture media
was poured off, and ice cold PBS containing 1% FCS was
added to the flasks. The cells were then gently scraped
off with the aid of a rubber policeman, and labelled
with either BT27/2A3, or spent culture supernatant from
the parental myeloma line, TM-H2.
Rhodamine isothiocyanate (RITC)-conjugated
goat antihuman IgM (F(ab')2 fraction, ,u chain specific;
Cappel Laboratories, Cochranville, PA) was used as a
second antibody for the BT27/2A3-labelled cells, and
RITC-con~ugated goat anti-human IgG (F(ab')2 fraction,
gamma chain speciflc; Cappel Laboratories, Cochranville,
PA) was used for the TM-H2-labelled cells. Flow
cytometric analysis was then performed as described
above.
PrimarY Tumor Explant Cultures
Sterile surgical biopsy specimens of primary
neuroectodermal tumors were collected in ice cold MEM
containing 20~ FCS. Approximately 0.5-1 g of specimen
was finely minced with sterile scissors and 5-10 pieces
measuring 1 mm3 were placed in T25 flasks containing 10
ml culture media. After 7 days of incubation under
standard conditlons, the media was replenished and the
cultures returned to the incubator. In 2-3 weeks, the
explants formed confluent monolayers which could be
passaged and analyzed by FCM.
- 21 -
~ 3 t,,
Four such explants were labelled with BT27/2A3
and RITC-conjugated goat anti-human IgM (~ chain
specific; Cappel Laboratories, Cochranville, PA). The
percentage of reactive cells was estimated by
overlapping the BT27/2A3 and control (BT27/2D2)
fluorescence distributions (plotted to peak height), and
determining the channel number where the two
distributions crossed. The percentage of cells lying to
the right of this plot for the control distribution was
then subtracted from the BT27/2A3 distribution, to give
a minimal estimate of the number of cells detected as
significantly labelled by the HmAb.
Lipid Dot Blots and Immunochromato~raphy
Total lipid extracts of cell lines to be
analyz d were prepared according to Svennerholm and
Fredman (1980). Briefly, approximately 5 x 108 cells
were grown to confluence in standard 850 cm2 tissue
culture roller bottles, scraped off in PBS, and
homogenized in 3 ml ddH20 at 4 C. The homogenate was
then combined with 10.8 ml methanol, and 5.4 ml
chloroform, which was added dropwise with constant
stirring. After mixin~ for 30 minutes at room
temperature, the remaining unlysed cells and debris were
spun down at 10,000 rpm for 10 min. The remaining
supernatant was placed in a 37C water bath and
evaporated to dryness under a constant stream of N2.
The residue was dissolved in 200 ~1 of 2:1 chloroform :
methanol, clarified by centrifugation in an Eppendorf
microcentrifuge for 10 min, and stored at -20C in a
tightly capped freezer vial (Nunclon, Denmark).
Dot blots were prepared by spotting 2 ,ul of
each cell line to be tested onto Merck HPTLC alumlnum
backed silica gel 60 plates (Applied Analytical
Industries, Wilmington, NC). The plates were then
- 22 -
J ~ J ~
dipped in a solution of 0.05% polyisobutyl-
methylmethacrylate (Polyscience, Inc., Warrrington~ PA)
in hexane for 30-60 seconds, and air dried for 15 min.
After blocking with 1% BSA in Tris-buffered saline (20mM
Tris-HCl, 0.5 M NaCl, pH 7.5; TBS ) for 2 hrs. at 4C,
the blots were exposed to hybridoma supernatants for an
addltional 2 hrs. at 4C, then washed with TBS six
times. Peroxidase conjugated goat anti-human IgM (u
chain specific; Kirkegaard and Perry, Gaithersburg, MD)
10 diluted 1:400 in 1% BSA-TBS was then added for a further
2 hrs. at 4C, and the plates washed once again in TBS.
The peroxidase reaction was developed with 3,3'-diamino-
benzidine tetrahydrochloride (DAB) dissolved in 0.05 M
Tris-HCl (pH 7.5) at a final concentration of 1 mg/ml,
15 in the presence of 0.05% H20z. After rinsing with
ddH20, the plates were dried with a hair dryer and
photographed promptly.
For thin layer lmmunochromatograms, 4 ,ul of
lipid extract was spotted onto each lane of the HPTLC
20 plates and dried under vacuum for at least 4 hrs. The
plates were then chromatographed in chloroform:methanol:
water (60:35:8) and dried under vacuum overnight, at
least 16 hrs. The plates were then coated with
polyisobutylmethylmethacrylate as described above. The
25 plates were blocked with 5% BSA in TBS at 4C for 2 hrs.
The chromatograms were then exposed to hybridoma
supernatants for an additional 2 hrs at 4C and washed
as described above. Peroxidase conJugated gozt anti-
human IgG SH~L chain specific) (BIO/CAN, Toronto, ON)
30 diluted 1:2000 or peroxidase con~ugated goat anti-human
IgM (Fc specific) (BIO/CAN, Toronto, ON) diluted 1:2000
in 1% BSA-TBS was added for a further 2 hrs at 4C.
After washing again, the plates were developed as
d~scribed for the lipid dot blots.
- 23 -
2V3~vJ~
Labelling of Normal Human Astrocytes
Cultured normal human astrocytes were obtained
as a gift from the laboratory of Dr. Jack Antel,
Montreal Neurological Institute, Montreal, PQ. The
cells were derived from patients undergoing craniotomy
for epilepsy and were grown on glass coverslips. They
were labelled on the coverslips using the same general
method (reagents and time) as outlined in the section on
Flow Cytometry with the exception that all washes were
carried out on coverslips using PBS containing 1% FCS
and 0.02% (w/v) NaN3. The coverslips were wet-mounted
using PBS with FCS and azide and viewed with a Leitz
fluorescence microscope.
Cell Sorting
An established culture of SK-MG-l (98th
passage) waQ scraped with a sterile rubber policeman,
resuspended in 1 ml of alpha-MEM ~ 10% FCS, and
transported to the FCM on ice. After the usual
calibration of the FCM, simultaneous fluorescence and
small angle light scatter data were collected on 5 x 104
cells in order to establish 2-dimenslonal gates for the
purpose of sorting. Each area was chosen so as to
correspond to either a S-(small or low small angle light
scatter) cell subpopulation or to a L-(large or high
small angle light scatter) cell population. A total of
2 x 104 cells from each area were sorted and collected
into 1 ml of alpha-MEM + 10% FCS and placed on ice.
Each sample of sorted cells was then diluted
wlth fresh alpha-MEM + 10% FCS to a volume of 40 ml and
distrlbuted into two 96-microwell plate-~, approx. 200 ~1
per well. The cells were fed every 5 days by exchanging
half the old supernatant for fresh media. On the 10th
day following the sort, the total number of colonies
- 24 -
/
~ 3 ~ ' 3 j 3
containing >25 cells were counted in each of the 4
plates. An individual microwell might contain anywhere
from 1 - 6 individually distlnguishable colonies.
Ten S-cell-derived and 10 L-cell-derived
clones from wells with a single colony were then
selected and passaged further into 24-well plates
(Linbro; 2.01 cm2/well) in a volume of 1 ml alpha-MEM +
10% FCS. The S-cell clones were assigned the numbers
S101 - SllO and the L-cell clones were assigned L101-
L110. The clones were later tested for reactivity by
FCM as described above.
Cell Cycle AnalYsis
Confluent cultures of the human glioma cell
line, SK-MG-l, were detached from tissue culture flasks
with a rubber policeman and fixed in ice cold 70~ (v/v)
ethanol for 30-60 min. Approximately 1 x 106 cells per
sample were aliquoted into 10 ml test tubes, pelleted by
centrifugation for 5 min at 500 G, and washed in PBS.
Twenty ,ul of RNase (Boehringer Mannheim, West Germany;
1 mg/ml in 10 mM Tris-HCl, 5 mM NaCl, pH 7.5) was added
to each sample to digest RNA, along with 1 ml of
propidium iodide (50 ,ug/ml in 10 mM Tris-HCl, 5 mM
MgCl2, pH 7.4). After incubating for 60 min at 37~C,
the cells were placed on ice for an additional 30 min,
then transported over to the FACS-III apparatus on ice.
After calibrating for linear small angle light
scatter, red fluorescence, and DNA content using the
human pseudodiploid cell line, HL-60, data was collected
on a minimum of 2 x 104 gated cells and stored on
flexible disks for subsequent analysis.
- 25 -
J ~ J ~j 3
Cell Morphology
After labelling SK-MG-1 for FCM with BT27/2A3
and BT27/2D2 as desribed above, small angle light
scatter vs. fluorescence data were collected on 5 x 10~
cells in order to establish 2-dimensional windows for
the purposes of sorting. The windows were chosen in
order to define morphology of:
1) S-cells which increase in fluorescence after
10 labelling with ~T27/2A3;
2) S-cells which remained unchanged in terms of
fluorescence after labelling with BT27/2A3; and
3) L-cells.
Approximately 1 x 104 cells were sorted from
15 each window and collected in ice cold alpha-MEM + 10~
FCS. Samples from each sorted area, as well as total
unsorted cells, were then centrifuged onto glass slides,
air dried, and stained with Wright's stain (Fisher,
Montreal, PQ). The cytocentrifuge preparations were
20 then viewed through a Leitz microscope and photographed
with Fu~icolor HR film (ASA 400).
Karyoty~g_of SK-MG-1 Sublines
Metaphase chromosome spreads were prepared as
out,ined above. For each cell to be studied, one
confluent T25 flask (Nunc, Denmark) was incubated
overnight in 3 ml alpha-MEM + 10~ FCS and 0.06 ~g/ml
colcemid (Gibco, Grand Island, NY). The remainder of
30 the procedure was followed exactly as outlined above
(see "Karyotyping of Hybridomas").
- 26 -
.'~ ,J ~.~ .,' J
EXAMPLE 1
Cell fusions were performed, to make
hybridomas from human myeloma-like cell line TM-H2-SP2
and peripheral blood lymphocytes (P~L) obtained from
eight different brain tumor patients, coded 8T-24, BT-
27, BT-32, ~T-34, BT-38, BT-39, BT-54 and BT-55. These
patients, both male and female, ranged from 5 to 55
years of age and had a variety of neuroectodermal tumors
including fibrillary astrocytoma, oligoastrocytoma,
primitive neuroectodermal tumor, and glioblastoma. The
fusions were performed as previously described.
Incubation of the cells in the 96-microwell trays was
conducted using fresh tissue culture media and fetal
calf serum (FCS), supplemented with L-glutamine (292
mg/l) and L-asparagine (44 mg/l). The hybridoma
outgrowth, defined as the number of microwells
containing macroscopic colon$es of greater than 50-100
cells (visible to the naked eye) divided by the total
number seeded, and expressed as a percentage, ranged
from 0~19.5%.
The number of PBL in all samples available for
fusion varied from 6.0 x 106 to 5.8 x 107. Although the
ratio of myeloma cells to lymphocytes remained constant
at 1:4 for each fusion, the plating density that was
used ranged from l.0 x 105 to 2.5 x 105 myeloma cells
per ml of fusion mixture. The lower plating density was
chosen in some fusions in attempts to encourage better
initial monoclonality, whereas the higher plating
density seemed to favor improvad hybridoma outgrowth.
The concentration of PEG was 50~ (v/v) in all fusions.
Typically, discernable hybridoma growth
appeared from 4 to 6 weeks after a fusion, and new
growth in microwells often continued to be observed for
several we~ks thereafter. In general, those colonies of
- 27 -
/Js~ 5
hybridomas which appeared earliest tended to bè the most
stable. Not all macroscopic colonies visible at the end
of the 96 microwell (0.28 cm2/well) culture period could
be successfully propagated to amounts suitable for
yrowth in 24 well dishes (2.01 cm2/well). In fusion BT-
54, for example, of the seven colonies which originally
grew in 96 microwells, only one was capable of prolonged
growth in vitro. Instances of hybridoma growth failure
occurring later than three months post-fusion were not
observed.
EXAMPLE 2
The hybridomas were screened for the presence
of human immunoglobulin and reactivity with 3 M KCl
extracts of autologous tumors, or glutaraldehyde-fixed
glioma cell lines.
A total of 1,121 wells containing growth of
putative hybridomas from the fusions described in
Example 1 were screened, of which 162 (14.5%) were ~ound
to react with tumor extracts, or glioma cell lines. Two
of the fusions, namely RT-27 and BT-32, were screened
for reactivity with several glioma cell lines, and
instances were found of individual microwells which
tested positive in multiple ELISA assays.
In the last two fusions, BT-54 and BT-55, the
ELISA assay was modified to detect only those microwells
which contained reactive IgM species. This was
accomplished by substituting culture supernatant from
hybridoma BT27/2D2 for TM-H2 as a control, and adding
alkaline phosphatase conjugated goat anti-human IgM as a
second antibody. The reason for this was that analysis
of immunoglobulin chains present in 34 hybridomas from
fusions BT-24, BT-27, BT-32 and BT-34, indicated that
all 34 contained IgM. Careful study of a few of these
hybridomas indicated that the IgM alone was responsible
- 28 -
for anti-tumor activity. Hybridoma BT27/2D2, derived
from cells of patient BT-27, a hybridoma which was
consistently negative in various ELISA tests of immune
reactivity, was chosen as a non-specific IgM control (1-
4 ,ug/ml).
The fact that all five HmAbs in this study
were of the IgM isotype may be significant. This may be
a result of the immunosuppressive mechanisms, discussed
earlier, present in glioma patients which might prevent
the elaboration of a secondary (IgG) immune response in
these patients. Alternatively, this may simply be a
reflection of the type of antigen which the five HmAbs
recognize, i.e. a carbohydrate.
EXAMPLE 3
All five hybridoma supernatants were found to
contain ,u heavy chains. Only BT34/A5 contained lambda
light chains, the other four HmAbs contained kappa light
chains. Each HmAb was shown to label the cell surface
of human glioma line SK-MG-l using FCM and FITC~
conjugated goat anti-human IgM (IgG fraction, ~ chain
specific; Cappel Laboratories, Cochranville, PA),
indicating that the IgM molecules were binding to the
tumor cell surface membrane (data not shown) . No gamma
chains were detected in any of the hybridoma
supernatants.
After approximately six months of maintenance
in culture, the estimated IgM concentration for BT27/lA2
was 5.0 ,ug/ml; for BT27/2A3, it was 44 ,ug/ml, for
BT32/A6 3.5 ,ug/ml, for BT34/A5 2.4 ~g/ml, and for
BT54/B8 22.4 ~g/ml. After a further 6 months in
continuous culture, IgM production levels were found to
be comparable.
- 29 -
~ )J
EXAMPLE 4
A total of nine high immunoglobulin-producing
hybridomas, from the above fusion series, as described
in Example 1, were screened for reactivity with the
human glioma line SK-MG-l, using the FACS-III Flow
Cytometer. Five supernatants derived from the
hybridomas, designated as BT27/lA2, BT27/2A3, BT32/A6,
BT34/A5 and BT54/B8 were found to label this particular
glioma cell line. All five contained tumor-reactive IgM
species. Thus, from eight fusions and a total of 59
hybridomas which were macroscopically visible at six
weeks, only five (8.4~) reacted with the cell surface of
a glioma cell line, and were capable of sustained growth
in culture. It was noted that BT27/2A3 labelling of SK-
MG-l is enhanced if the latter is maintained at a high
cell density prior to testing.
EXAMPLE 5
Three of the five HmAbs were tested by ELISA
for their relative reactivities. At the initial
concentration of supernatant fluid which was tested, the
order of reactivity was BT27/2A3 > BT27/lA2 > BT32/A6,
which is the same as the order of their respective IgM
concentrations (see Example 3). The final titre which
gave O.D. readings significantly higher than control IgM
baseline was 1:16 for BT27/2A3, 1:4 for BT27/lA2, and
1:2 for BT32/A6. Since this assay involves the addition
of 50 ,ul of hybridoma supernatant to 50 ,ul of PBS in the
initial step, the initial dilution tested in the ELISA
was 1:2. There was no evidence of an initial plateau
phase in any of the dilution curves, indicating that
under the ELISA conditions which were used, the quantity
of tumor extract was not a limiting factor.
- 30 -
~ J 3 r~ v O ( 1
EXAMPLE 6
. _
Flow cytometric analys:Ls of cultured human
cell lines and strains was performed with the cells
grown to confluence and maintained in that state with
exchanges of fresh media at least 24 hrs prior to
labelling. Suspension culture cell lines were maintained
and used from high cell density cultures, i.e. near
saturation. Screening was carried out according to the
method outlined above in parallel on two or more
separate occasions with established positive cell lines
to verify maintenance of immune reactivity of the HmAbs.
Results of FCM screening with human cell lines
are summarized in Tables 1 and 2. Several different
classes of neuroectodermal, and non-neuroectodermal
tumors and tissues were tested. All five HmAbs
demonstrated a similar pattern of reactivity for the 30
cell lines which were studied, with few notable
exceptions. For example, the human epithelial cerYical
carcinoma cell line, ME180, reacted with BT27/lA2 and
BT27/2A3, but not BT32/A6. Antibodies BT34/A5 and
BT54/B8 both failed to react with the glioma cell line
SKI-l, but labelled melanoma line M-4.
Only HmAb BT32/A6 had a singular pattern of
reactivity. Antibodies BT27/lA2 and BT27/2A3 exhibited
one pattern of reactivity, and antibodies BT34/A5 and
BT54/B8 had another pattern of reactivity, which
differed slightly from each other. None of the HmAbs
reacted with any of the hematological cells lines which
were tested.
Despite some minor differences in the pattern
of reactivity against a panel of human tumor cell lines,
all five HmAbs appear to be recognizing similar
molecular substances, both in terms of biochemical
composition and biological distribution even though
- 31 -
~ ~J
T~lo ~: R-act~ty o~ t~ao l~ab~
T~L~ ~Jg~e ~ ~ ~ C12~
Glioma SK-MG-l PoJltlv~b po~ltlvs po~itlve
S~-MG-13 po~Cl~- po~ltl~- po iti~
SXI-l po~ltlv- po~ltlve positiv~
LN-21S n~g~tlv~ n-g~tlv~ negaCive
LN-340 nay~lv- noqatl~- nagatlvo
U-178 posltlvo positiv~ po~itiv~
U-373 po8itlv~ po~ltlvo po~itlve
Melanoma M-4 ney~tlvd n~g-t$v~ neg~tiYe
IGR 37 n~g-tlve n~gativa negative
~GR-39 neg~tivo neg~lv- negativ2
NeuroblaRtoma IMR-32 neg~tlv- n-ga~lv- n~gative
S~-N-MC n-g~tlv~ nngatlva negative
Retlnobla~toma Y-76 nog~tlv- n-gatlvo n~gative
He~tologlcal c~ c n-g~tlv~ negat~v- negaelve
K-562d n-g~tlvo nogatlvo negative
HL-60- n-ga~lv- nng~tlv- negativ~
Other Tumor-~ H-L ~ pos~elv- po~ltlve posittvo
M~l~O~ po~ltlve posi~iv~ negsCive
c-33af n-g~tlvo n~gatlv~ n~gativ~
S~1166g n~g~tivn nagntiva n~g~tiv2
S~1~17g n-g~tlve n~gatlvoe n~gativ~
5~94Eg n g~tl~- nogatlvo negaeiv2
HS-29g ncga~lv- nagatlv- n~gativ~
J32h n-gatlv- nngatlvn negative
Embryonlc HlF nogatlv n~gatlve n~gative
Fibrobla-~t ~1-38 n~gativ8 n~g~tivo negative
~ootaot-~ nhybrldo~, bc~ VQr~ d-t-etod to hav~ bound HmAb
abov- th~ b-e~ground l~vol o~ la~llln~ ~t~ eontrol ~nt$body,
CT-e~ uk8~1a, dehronlc my-loeyt$c l~u~-mla, ~ACUt~ pro~yoloeytic
l~uk-~la, ~opltholtal e-rvlcal e~re~no~ , geolonle ad~noe~reimoma,
htranaltional bla~d-r e-ll carelne~
- 31a -
Tablo 2: R-activity o~ H~Ab~ ~T34/AS and BT54/B8
T1~mor Type Cell Lin8 ;3T34~p5a ~ ~7/2A8
Glioma SK-MG-1 positiveb poaitive po~itive
SKI-1 negative negative po~itive
U-373 positive poYitive positive
Melanoma M-4 positive po~itive negative
Hematological CCRF-CEMC negative negative negative
~-562d negative negative negative
HL-60e negative negative negative
u-937f negative negative N.D.
Rajig negative negative N.D.
Other Tumors HeLah negative negative po~itive
ME180h negative negative negative
C-33Ah negative negative negative
SW1417i negative negative negative
sW1463i negative negative negative
J82i negative negative negative
Embryonic IMR-90 poqitive po9itive N.D.
Fibroblast WI-38 negative negative negative
root~otoa: ahybridoma , bsee Table 4.1, c~ cell leukemia, dchronic
myelocytlc leukemia, eacute promyelocytic leukemia, fhi3tiocytic
lymphoma, monocyte-like, gB cell lymphoma, hepithelial cervical
carcinoma, icolonic adenocarcimoma, itranqitional bladder cell
carcinoma, N.D.: not done.
- 31b -
~ i3
being derived from four different patients with
different tumors.
EXAMPLE 7
Four tumor explants (glioblastoma multiforme,
recurrent meningioma, medulloblastoma, and astrocytoma
grade II) were adapted to tissue culture and studied by
FCM after brief passages in vitro. The greatest
apparent proportion of labelling occured with a
recurrent meningioma (46%), whereas the lowest
proportion of labelling (7%) was found in a low grade
astrocytoma. These results indicate that BT27/2A3 is
capable of reacting with primary glial and non-glial
neurological tumors in the early stages of adaptation to
tissue culture.
EXAMPLE 8
In order to get some indication of relative
antibody affinity, a modification of the method of De
Bernado and Davies (1987) was used. The results of ELISA
testing of the five HmAbs with tumor extract from BT-37
(glioblastoma multiforms) after either a long ~18 hr) or
a short (4 hr) incubation at 4C, are expressed in terms
of O.D. measurements. Comparison of the O.D. readings
for each HmAb at the two timepoints indicates that there
is a statistically significant (p<0.05) increase in O.D.
for BT32/A6 and BT54/B8 with the longer incubation. None
of the other 3 HmAbs (BT27/lA2, BT27/2A3, and BT34/A5)
showed a significant increase in reactivity after a
longer incubation at 4C. These observations would
suggest that BT32/A6 and BT54/B8 behave as a low
affinity antibodies at low temperatures.
After the long incubation period (18 hrs at
4C), all five HmAbs confirmed positi~e (p<0.05) by
- 32 -
F' ~
ELISA when compared to the control. After the short
incubation time (4 hrs at 4C), however, only BT27/lA2,
BT27/2A3, and BT34/A5 were significantly positive;
BT32/A6 and BT54/B8 did not react significantly,
possibly because of their low affinity nature.
EXAMPLE 9
Dot blots of total lipid extracts from
cultured neuroectodermal cell lines were examined.
LN-340 and M-4 are glioma and melanoma cell lines which
have been previously shown to be unreactive with any of
the five ~mAbs (see Example 6) . Compared to control
HnAb BT27/2D2, there was some indication of potential
reactivity for all five HmAbs when tested against
glycolipids from the glioma cell line U~373. As for the
glioma line SK-MG-1, all the HmAbs except for BT54/B8
seemed to exhibit some degree of reactivity to the lipid
extract, although the quality of these dot blots was not
entirely satisfactory.
Immunochromatography on total lipid extract of
SK-MG-l was performed using BT34/A5 and the parental
line, TM-H2, as control. A single specific band with Rf
0.60 was observed. As well, a non-specific band with
Rf = 0.80 was evident in both the BT34/A5 and T~-H2
lanes. In separate experiments, similar results were
obtained for BT27/lA2 and BT27/2A3 compared to BT27/2D2,
i.e. a specific band at Rf = 0.60. Immunochromatography
with HmAb BT54/B8 failed to reveal the presence of any
specific banding pattern, but this result is consistent
with the dot blot experiment.
These results suggest that the HmAbs recognize
a determinant of a glycolipid, or ganglioside, which was
not detected in association with glycoproteins.
-- 33 --
'~'`i3 "'3~,
EXAMPLE 10
Cultured normal human astrocytes were assessed
for labelling with the HmAbs. All reagents were checked
for immune reactivity against an established positive
human glioma cell line (U-373). None of the HmAhs
BT27/lA2, BT27/2A3, and BT32/A6 appeared to label
cultured astrocytes from two different individuals.
Antibodies BT34/A5 and BT54/B8 also failed to label
normal human astrocytes on at least one occasion.
EXAMPLE 11
The chromosomal content for each of the
hybridomas was determined by karyotyping as previously
described. The parental cell line, TM-H2-SP2, was also
studied, and found to have a mean and modal number of 46
chromosomes, as previously reported (Sullivan, et al
1982). All three hybridomas tested were pseudodiploid,
Z0 with BT27/lA2 and BT32/A6 having 2n to 3n number of
chromosomes, and BT27/2A3 slightly less than 2n. The
latter observation was confirmed by DNA flow cytometry.
EXAMPLE 12
Monoclonality was determined by Southern blot
analysis, and revealed that hybridomas BT27/lA2,
BT27/2A3, and BT32/A6.5, each possessed two rearranged
bands bearing homology to the ~H gene region. Hybridoma
BT27/2D2 appears to possess three such bands. The B cell
fusion partner used in these experiments, TM-H2-SP2, has
only one band and an apparent deletion. Furthermore,
there is no evidence for a TM-H2-SP2 type of
rearrangement of the JH region in any of the hybridomas.
As a control, normal PBL DNA, which is composed of
60-70% T cell derived genetic material, was found to
- 3g -
yield a blot profile identical to placental (germline)
DNA. There is also a common low molecular weight band
containing an unrelated homologous sequence present in
each of the lanes.
For conducting the Southern blot analysis,
genomic DNA was isolated from hybridoma cells and
digested with BAM Hl and HIND III restriction enzymes,
then electrophoresed on a 0.8~ (w/v) agarose gel,
hybridized and labelled with a JH region probe (ONCOR
Inc., Gaithersburg, Maryland), as described earlier.
The presence of two JH rearrangements in each of the
three hybridomas is consistent with monoclonality.
The fusion partner chosen according to the
preferred embodiment of this invention thus yields
stable B cell hybridomas secreting IgM antibodies which
react with autologous tumor extracts and glioma cell
lines. Growth of the hybridomas, e.g. by supplementation
with additional quantities of the amino acids
L-glutamine and L-asparagine. This fusion partner TM-
H2-SP2 appears to be superior to any previously reported
fusion partner for normal PBL.
Biological S~nificance Of A Glioma Cell_Subpopulation
Identified By Human Monoclonal Antibodies
EXAMPLE 13
Reco~nition of a Subpopulation of Glioma Cells
Initial studies of the human glioma line,
SK-MG-l, using 2-dimensional FCM revealed two discrete
subpopulations of cells. These consisted of a smaller
subpopulation, both proportionally and also in terms of
small angle light scatter, and a larger subpopulation,
which contained the majority of the cells for this
particular line. These will be referred to as S-cells
and L-cells, respectively.
- 35 -
After reacting SK-MG-1 with either of the
three HmAbs described in the previous chapters, there
appeared to be selective labelling of only the S-cell
subpopulation. This selective labelling was
consistently observed over many experiments, and was
also observed in many other tumor cell lines (e.g. HeLa)
which also contained two discrete subpopulations.
Evidence suggests that the S-cell
subpopulation consists of immature cells representative
of the main stemline of SK-MG-l, and capable of stem
cell-like behaviour. Cell size is often regarded as a
characteristic feature of differentiation or anaplasia.
In many normal tissues, terminally differentiated 'end'
cells tend to be larger than their smaller
undifferentiated precursors.
EXAMPLE_14
Generation of Subpopulation-Derived Sublines from
SK-MG-1
Earlier studies of monoclonally-derived
sublines of SK-MG-1 involved the generation of randomly
selected, unsorted clones. In these experiments,
monoclonally derived sublines were established from
sorted S- and L-cells of SK-MG-1. The flow cytcmetric
properties of the resulting monoclonal S- and L-cell
derived sublines (SlOl-10, and L101-10, respectively)
were then compared to those of the parental line,
SK-MG-1. Three important conclusions were drawn:
1) The colony forming efficiency in vitro for sor~ed S-
and L-cells was equivalent (approximately 1%).
2) All S- and L-cell derived sublines were found to
contain S- and L-cells.
3) The small angle light scatter (optical size) of the
S-cells in the L-cell derived sublines was significantly
lower (P<0.05) than the small angle light scatter of the
- 36 -
2 ~J i ~
S-cells in the parental line, SK-MG-l. In contrast, the
FCM profile of the S-cell derived sublines appeared
identical to that of SK-MG-1.
Table 3 compares the modal small angle light
scatter channel number for both S- and L-cells in
SK-MG-1 with 8 S-cell derived and 5 L-cell derived
sublines. The L-cell peaks for SK-MG-1, S106, and L102
appear to map together, as do ths S-cell peaks for
SK-MG-1 and S106. The S-cell peak for L102, however, is
10 seven channels lower (44.5~ lower after conversion to a
linear scale) than the corresponding S-cell peak of
SK-MG-1.
Typical FCM sortings of S- and L-cells were
centrifuged onto glass slides and stained with Giemsa.
15 Qccasional mitotic figures were observed in FCM sortings
of L-cells, but very rarely among the S-cells.
These results suggest that there exists a high
degree of phenotypic similarity between the S-cell
derived sublines and the parental line, SK-MG-1,
20 compared with the L-cell derived sublines. When total
DNA content and cell cycle state of SK-MG-1 and S- and
L-cell derived sublines was examined, S-cell derived
sublines were more similar to SK-MG-1 than were L-cell
derived sublines.
EXAMPLE 15
Cell Cycle Analysis of SK-MG-1 and Its Sublines
Analysis of sorted S- and L-cells of SK-MG-1
30 demonstrated clearly that the S-cell subpopulation
contained cells which were almost exclusively in the G1
or Go ('resting') portion of the cycle. The L-cell
subpopulation, however, contained cells primarily in the
G1 and S phases, suggesting that they were actively
35 dividing.
- 37 -
Table 3 Small angle light scatter dataa for SK-MG-1 and
its small and large cell d~rived clones
small cells larae cells
SK-MG-1 (parental) 76.75 i .49 95.50 + 1.85
(n= 4) (n- 4)
small cell clones 77.75 ~ .63 95.94 + 1.00
(n= 8) (n= 8)
large cell clones 72.80 + 1.20b 98.00 + .95
(n= 5) (n= ~)
aresults expressed as mean + standard error for modal channel number, 128-channel
1092 scale; n refers to the number of FACS distributions pooled to obtain these values
bp<o.o5, compared with SK-MC,-1 using a two-tailed Student's t-test
- 37a -
No statistically significant differences were
found between the DNA histograms of sorted S-cell
derived clones and the parental line, SK-MG-1 (Table 4).
The S-cell derived clones therefore, again appear to
resemble SK-MG-1 phenotypically. By comparison, the
average DNA content (as estimated from the Go/G1 peak)
of cells in the L-cell derived clones was statistically
greater (p<0.05) than the parental line.
These results suggested that the L-cells may
represent divergent hyperploid sublines of SK-MG-1. To
verify this hypothesis, the karyotypes of SK-MG-l and
its S- and L-cell derived clones were studied.
EXAMPLE 16
Karyotypic AnalYsis Of SK-MG-l and Its Sublines
Metaphase chromosome spreads were prepared for
SK-MG-l, and three different S- and L-cell derived
sublines (Table 5). Statistical analysis revealed that:
1) There was no significant mean chromosomal difference
between SX-MG-1 and its S-cell derived sublines;
however,
2) The variance among S-cell derived sublines was
significantly lower than that of the parental line.
3) L-cell derived sublines had, on average, a higher
ploidy number than SK-MG-l (compatible with the results
of DNA labelling with propidium iodide).
4) One particular L-cell derived subline (Ll08), had a
variance about the mean that was significantly greater
(P<0.05) than for SK-MG-l.
These results suggest that the S-cell
subpopulation of SK-MG-l possess properties resembling
those of a hypothetical tumor stem cell. For example,
the S-cells are phenotypically smaller and more
anaplastic than the L-cells. When S-cells are
individually sorted, they establish sublines which are
- 38 -
Table g DNA contenta of SK MG-1 and its small and
large cell derived clones
smal! cell$ l~rge ce!/s
SK-MG-1 (parental) 5Q.33 i .67 57.33 + .67
(n= 3) (n= 3)
small cell clones 49.20 + .73 55.50 + .72
(n= 5) (n= 6)
large cell clones 57.10 + .78b 63.33 + .g4b
(n= 10) (n= 9)
aethanol-fixed ce11s were stained with propidium iodide; results expressed as mean +
standard error for modal channel number, 128-channel linear scale; n refers to the
number of FACS distributions pooled to obtain these values
bp<o.oo5, compared with SK-MG-1 using a two-tailed Student's t-test
- 38a -
2 ., ~ v ~
Table 5 Metaphase chromosomes of SK MG-1 and its small
and large cell derived clones
mean+SD~ mode ranaQb ~c dfd p~
SK-MG-1 (parental) 57.4 + 4.2 56 46-68
S103 54.5 + .93 51 50-64 -0.66 19 21.009
S104 55.5 + 1.0 55 49-66 -0.44 19 18.109
S110 53.7 + 1.3 50.5 46-68 -0.82 l 9 10.959
L103 73.2 + 5.3 60 46-146 2.32f 38 1.58
L107 66.2 ~ 3.2 60.5 51-100 1.65 38 1.74
L108 96.2 + 10.7 74.5 46-~2D0 3.37~ 19 6.329
aresults expressed as mean + standard error for 20 metaphase spreads
brange represents lhe lowest to highest chromosome number counted
Ccalculated t-statistic
ddegrees of freedom
ecalculated F-statistic
fp~o.oo5, compared with SK-MG-1, using a two-tailed S~udent's t-lest
9p~0.01, compared with SK-MG-1, using a two-tailed Ftest with df = (19,19)
- 38b -
similar in terms of cell size, DNA content, and
chromosoma number to the parental line.
The L-cell subpopulation of SK-MG-1 is also
capable of establishing colonies in vitro after sorting,
a property which would not be expected i* L-cells were
differentiated 'end cells'. The fact that these colonies
also contain S- and L-cell subpopulations may be that
the L-cell derived sublines appear to be from a
different stemline than that which predominates in
SK-MG-l. The L-cell subpopulation may, in fact, include
a mixture of differentiated 'end' cells derived from the
S-cell subpopulation, and divergent minority sublines of
SK-MG-1 with stem cell-like properties. Evidence in
support of this is given by the finding of a
significantly lower chromosomal variance about the mean
among the S-cell derived sublines than among the SK-MG-1
parental line.
- 39 -
