Note: Descriptions are shown in the official language in which they were submitted.
1~1773'7
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The present invention is concerned with a process
for obtaining monoclonal antibodies directed against
tumour antigens and with pharmaceutical compositions
containing these antibodies.
Monoclonal antibodies are substances which react
highly specifically and only react with a particular
antigenic determinant. Monoclonal antibodies, which
are specific for tumour antigens and which are absent
from the non-transformed base cells of the tumour,
open up new po~sibilities for the therapy and diagnosis
of tumour cells (transfonmed cells). The utility of
such monoclonal antibodies, especially for cancer
therapy, thereby depends upon two factors:
1. whether the antibodies are specific for the tumour
cell 9 and are not absorbed by normal tissue and
2. whether the antibodies also interfere functionally
with the tumour development: for example, an
antibody which is selected merely for its binding
ability to tumour cells must not nececsarily also
be functionally active.
Within the scope of functional effectiveness, the
mechani~m of metastasis formation is also of especial
importance. The formation of metastases i9 a critical
and, at the same time, also a complex occurrence in
cancerogenesis. ~t can be divided up into several
different ~tages, for example the release of some
transfonmed cells from the primary tumour, their
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migration in the blood stream of the host organism,
adhesion of the drifting metastasing tumour cells to
the endothelia of the target ~ites, their penetration
and growth in the stroma.
It is an object of the present invention to
provide monoclonal antibodies which, in particular,
are specifically able to prevent the adhesion or
growth of the metastasing tumour cells in the target
organs.
Thus, according to the pre~ent invention, there
is provided a process for obtaining monoclonal anti-
bodie~ directed against tumour antigens, wherein
irradiated tumour cells are injected under immunising
condition~ into inbred experimental animals, whi.ch
cells are derived from the same inbred strain, where-
after spleen cell~ of the 50 immunised animals are
i~olated and fu~ed with myeloma cells with the use of
the hybridoma technique, the hybridoma cells obtained
are cultured and the monoclonal antibodies are obtained
from the culture supernatant or from ascites fluid of
those hybridoma clones which, in vitro, inhibit the
adhesion of living tumour cells or change their
morphology.
The present invention is based upon the recog-
nition that an immunisation of inbred experimental
animals with irradiated tumour cells which originate
from the same inbxed strain (~yngenic tumour cells)
12~7'737
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lead to the activation of spleen cell3 which only
form antibodies against the surface antigens of the
tumour cells. These are not contained in the non-
transfonmed base cells~ By the preparation of cell
S clones from such spleen cells according to methods
of the hybridoma technique, monoclonal antibodies
are obtainable which cross-react strongly with human
tumour cells. Thuq, the process depends upon the - ~
fact that inbred experimental animals are immuni~ed
with their "own" syngenic tumour cells and, with the
spleen cells of these animals, hybridomas are formed
and the clones cultured from these are selected as to
wh~ther they form monoclonal antibodies which, in
vitro, change the adhesion and the morphology of the
corresponding tumour cells and, ~n vivo, prevent the
formation and growth of experimental metasta~es in
the experimental animals. I_ i8 important for the
present invention that the killed tumour cells used
for the immunisation are syngenic for the inbred
experimental animals used. An example of such syngenic
tumour cells are ~he B16 melanoma cells with regard to
C57 BL/6 mice~ B16 Melanoma cells are tumour-forming
mutants of C57 BL/6 melanocytes.
The killing of the syngenic tumour cells used
can take place by means of the methods known for this
purpose, for example by the action of X-rays in
appropriate dosage. The only thing which is important
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is that the surface antigens of the tumour cells
hereby remain intact.
The injection of the syngenic tumour cells
takes place by means of the methods known for
immunisation for the purpose of antiserum fonmation.
Thus, as a rule, there is carried out a multiple
immunisation at intervals of Qeveral weeXs. In the
ca~e of the use of the mice and melanoma cells
mentioned above by way of example, an interval of two
to four weeks between the injections proves to be
sufficient, good re~ults having been obtained even
with only two injections. In carrying out the
immuni~ation, a conventional adjuvant is preferably
employed, for example complete or incomplete Freund's
adjuvant. The use of complete Freund's adjuvant is
preferred for the first immunisation and of incomplete
Freund' adjuvant for the ~econd and possibly further
immunisation. A few days after the last immunisation,
the ~pleen cells of the experimental animals are
recovered. In this case, a period of time of three
to five days proves to be well suited.
~ he recovery of the spleen cells can take place
operatively without sacrificing the experimental
animals. Alternatively, the experimental animals are
sacrificed, the spleen is removed and the cells ~re
separated by known methods.
... .. .. .
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Thus, by means of the syngenic immunisation,
spleen cells are obtained which produce increased
amounts of antibodies against the tumour component
of the tumour cells u~ed for the immunisation. In
order to make the cells obtained permanently cultur-
able and differentiatable with regard to the mono-
clonal antibodies produced by them, they are fused
with myeloma cells according to the known hybridoma - ~
technique (see Kohler and Mil~tein, ~ature, 256,
495-497/1975)~ The hybridoma cells so obtained are
allowed to grow in an appropriate nutrient medium,
with the formation of clones, and the supernatant
nutrient liquid, containing the monoclonal antibodies
formed by each clone, is tested for its effectivenes~
in vitro for preventing the adhesion and/or changing
the morphology of living tumour cells and those
clones are selected for the antibody production which
give the be~t results. For these ~ests, there are
usually employed living tumour cells of the same kind
of tumour which is also used for the immunisation,
thus, for example melanoma cells when immunisation
was carried out with melanoma cells. It has been
ascertained that human tumour cells are also suitable
for this selectioning of the hybridomas suitable for
the cell culture and for the production of antibodie~.
The adhesion test (Test 1) employed according
to the present invention consists in that suspended
~2~7737
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tumour cells are incubated with culture supernatant
of the hybridoma cell clones for about 0.5 to 1 hour,
if possible under physiological conditions. The
evaluation is carried out microscopically by compar-
i~on with control batche~ in which, instead of theclone culture supernatant, there is used regular
culture medium or supernatant of the myeloma cells
used for the hybridisation. By visual detenmination
(counting), there is then ascertained the percentage
of the adhering cells in the hybridoma supernatant in
comparison with the control medium.
In comparison with conventional binding tests
with monoclonal antibodies, the adhesion test used
according to the present invention has two advantages.
First, it is very senqitive and, secondly, it is
functional. As has already been explained above,
meta~tasing tumour cell~ flow in the blood to foreign
organs, there adhere to the endothelia, penetrate them
and grow in the stroma. The first stage of this pro-
cedureO namely the adhesion of the tumour cells tothe target organs, is well ~imulated in the in vitro
adhesion te~t. In vivo investigations have shown
that a considerabl~ part of the antibodies which, in
this manner, are active in vitro, are also active
in vivo.
The morphology test ~Test 2) employed according
to the present invention consists in that adherent
t ~2~7737
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t~our cells are incubated for 1 to 4 hours with
culture supernatant of the hybridoma cell clones, if
possible under physiological ~onditions. Changes of
the cell morphology are detenmined microscopically by
5 comparison with control batches, as in the case of
Test 1.
From cell biological studies, it is known that
there is a relationship between cell morphology and
cell growth. Thus, b~ means of Test 2, antibodies
10 are found which determine the growth of the tumour
cells ~n vivo. These antibodies can act upon primaxy
tumours or can be used for the treatment of already
established metastases.
Investigation of the antibodies obtained accord-
15 ing to the present invention showed that they react
with human tumour cells as strongly as or more strongly
than with the syngenic tumour cells used for the
immunisation. Therefore, the antibodies according to
the present invention can be used for the prophylaxis
20 and therapy of tumours and, especially in the case of
the surgical removal of tumours, are able sub3tantially
to reduce the risk of the occurrence of metastases by
tumour cells hereby floated off.
Consequently, the present invention also provides
25 pharmaceutical compositions containing, as active
material, monoclonal antibodies obtained according to
the proces- of the present invention, together with
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conventional pharmaceutical additives and diluents.
The following Examples are given for the purpose
of illu~trating the present invention:-
Example 1.
mmuni~ation and_production of the hYbridoma~
5 x 106 to 107 B16 mouse melanoma cells,
irradiated with 3500 Rontgen, were injected intra-
peritoneally at intervals of 3 weeks into ~yngenic
C57 BL/6 mice. For the first immuni~ation, the cells
were mixed with 0.5 ml. of complete Freund' 5 adjuvant
and for the second immunisation with incomplete
adjuvant. 3 to 4 days after the last injection, the
animals were sacrificed and the spleen cell~ were
fused with ~S-l myeloma cells according to the method
of Rohler and Milstein (Nature, 256, 495-497/1975).
After 10 to 14 days, culture supernatant was
removed from the growing hybridomas and tested in
vitro in functional tests for antibody activity.
In vitro adhesion test
B16 Melanoma cells were treated for one minute
with trypsin-EDTA, the reaction was stopped by the
addition of excess medium which contained 10% of
serum and the cells were centrifuged off at 200 g.
Batche~ of 300 to S00 of the centrifuged-off cells
were each incubated with 50 ~1. hybridoma culture
supernatant for 30 to 45 minutes at 37C. and 5%
carbon dioxide. As negative control, there was used
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~S-l supernatant or regular culture medium (Dulbecco' 5
modified Eagle's medium with l~/o oetal calf serum).
After tne incubation, the tissue culture dishe3 used
were washed with phosphate buffer-containing
physiological sodium chloride solution, the adhering
cells were fixed with 3~O formaldehyde solution and
counted in an optical microscope, the percentage of
adherent cells in the hybridoma supernatant being
determined in compari~on with the control medium.
In vitro mor~holoq~ te t
Trypsinised tumour cells were cultured for at
least 4 hours on Teraski plates (3000 ~o 5000 per
hole). To the propagated cells were added, in each
ca~e, 30 ~1. of hybridoma supernatant (or correspond-
ing control medium, see above) and incubated for 1 to4 hours under physiological conditions. The plates
were investigated by inverted microscopy for cells
with changed morphology. For storage, the plates
were fixed with ~/O formaldehyde solution.
Test for experimental metastases
a) The hybridoma supernatants of 8 clones contain-
ing the antibodies according to the present invention,
which have been ascertained as being effective in the
adhesion test in the manner described above, were
incubated in vitro with B16 melanoma cells and the
cells then administered intravenously to syngenic mice
according to the method of Fidler, Poste and ~icolson.
~2~7737
After 10 to 12 days, the lungs of the mice were
investigated for B16 metastases. It was thereby
found that 4~/~ of the introduced antibodies, in com-
parison with the corresponding controls, reduced the
number of metastasic colonieq by more than 9~/O.
b) Ascites fluid of the appropriate hybridomaq
were injected intravenously or intraperitoneally into
C57BL/6 mice 3 to 4 hours before the B16 tumour cells.
As control, there was used ascites from ~S-1 hybridoma
cells or from antibodies only effective in vitro. The
lungs of the animals were investigated for meta~tase-q
in the manner described above. The animals treated
with antibodies showed up to 80Yo le~ experimental
metastases.
c) 3 Days after intravenously administered B16
melanoma cells, ascites fluid of the antibodies with
changed morphology was injected intraperitoneally into
C57BL/6 mice. After 10 to 12 days, the lungs of the
mice were investigated for B16 metaqtases. It was
thereby found that 2~/o Of the antibodie~ u~ed reduced
the lung metastases by 80 to 9~/O.
Exam~le 2.
The monoclonal antibodie~ produced as described
in Example 1 were tested for their activity in the in
vitro adhesion test towards various human melanoma
cell lines. The human melanoma cell lines and the
results obtained in comparison with the control sample
are set out in the following Table 1.
~ ;Z 1773'7
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+ I + I 1 ~ 1 I
o
o _l ~ + I I I I I I
U _ _ - _
_~ ++ + ~ I I I I I C
. ~ ~ ,~
.~ s~ ++ + I + I + + +
O P- + +
~ + + + + + ++ + + ,C
~ O H O =
t9 U u~ + + + + + + + + 1:
~ ~ + + ~ + + + + + O
E~ ~ ~ + + ~ + + + .,,
. ~ ++
O _ ~C
~ + + + + + + + + I~Oe ~ ~ 0~ 0
~ ~q
O O O O O
~1 1-')
U~ I O O O O O
a~ ~1 ~ + ~ + + + + + 0 u~
~ ~ ++ + ++ ,~ $ + + + 111111~111
r~ + + + ~ + + + + + + + +
U~ $ + ++ ++ ~ ~ $ $ +
~ ~ ~ + ~ + + + +
_
~ ~ ` 0
~2~7737
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~ he antibodies according to the present invention,
which have been produced with the use of melanoma
cells, cross-react not only with human melanoma cells
but also with other human tumour cells. HoweYer~ no
cross-reaction takes place with non-transfonmed human
cell~ (embryonal lung fibroblasts MRC-5).
The tumour cells used and the results obtained in
the adhesion te~t are given in the following Table 2.
1~3LR 2
Adhesion inhibition in comParison with the control
Anti-HeLa ¦TuWil)A549 )A4313)Tagli- MRC 54)
body bliaostOma
. . . . .
1 ++~ ++++ +f~+ +++++++
~5 2 ~++ +f+ ++++ ~t ~++
3 ++ ++ +++ ++ .+~
4 ++++ ++ +++ ~+ ++
++ ++ +++ ++ ++
6 +++ + +++ ++ +++
2~ 7 + + + + ++
8 _ ~ ~ ~ ~ _ _
Evaluation took place as in Table 1
1) Wilms tumour
2) lung carcin~ma
3) vulva carcinoma
4) normal embryonic lung Eibroblast
