CA1217737A

CA1217737A - Process for obtaining monoclonal antibodies directed against tumour antigens and pharmaceutical compositions containing them

Info

Publication number: CA1217737A
Application number: CA000454046A
Authority: CA
Inventors: Walter Birchmeier; Peter Vollmers
Original assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften
Current assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften
Priority date: 1983-05-31
Filing date: 1984-05-10
Publication date: 1987-02-10
Also published as: AU2805884A; AU546797B2; JPS606198A; IL71877A0; EP0127173A2; DE3319751A1; IL71877A; EP0127173A3

Abstract

ABSTRACT

Process for obtaining monoclonal antibodies directed against tumour antigens The present invention provides a process for obtaining monoclonal antibodies directed against tumour antigens, wherein irradiated tumour cells are injected under immunising conditions into inbred experimental animals, which cells are derived from the same inbred strain, whereafter spleen cells of the so immunised animals are isolated and, with the use of the hybridoma technique, are fused with myeloma cells, 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 and change their morphology.
The present invention also provides pharma-ceutical compositions containing the monoclonal antibodies obtained by this process.

Description

1~1773'7

-2-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 121773~

-3-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

-4-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 12~7~37

-5-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.

... .. .. .

-6-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

-7-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

-8-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 g 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 ~2~'7737 ~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 + 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 ~ 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

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for obtaining monoclonal antibodies directed against tumour antigens, wherein irradiated tumour cells are injected under immunising conditions into inbred experimental animals, said cells being derived from the same inbred strain, whereafter spleen cells of the so immunised animals are isolated and fused with myeloma cells to form hybridoma cells, culturing the hybridoma cells and obtaining monoclonal antibodies from the culture supernatant or from ascites fluid of those hybridoma clones which in vitro inhibit the adhesion of living tumour cells and change their morphology.

2. Monoclonal antibodies, obtained by the process according to claim 1.

3. A process for producing a hybridoma for the production of monoclonal antibodies directed against tumour antigens, comprising injecting ir-radiated tumour cells under immunising conditions into inbred experimental animals, said cells being derived from the same inbred strain, isolating spleen cells of the thus immunised animals and fusing the spleen cells with myeloma cells to from a hybridoma.

4. A process according to claim 3, wherein said tumour cells are B16 melanoma cells.

5. A process according to claim 3 or 4 wherein said myeloma cells are NS-1 myeloma cells.