Monoclonal antibodies against melanoma

The present invention provides a human monoclonal antibody against melanoma, characterized in that it binds to the gangliosides GM3 and GD3 but essentially does not bind to the gangliosides GM1, GM2, GD1a, GD1b and GD2, the binding of the antibody to the gangliosides having been determined by immune staining after thin layer chromatographic separation of the gangliosides. The present invention also provides a process for the production of human monoclonal antibodies directed against melanoma, wherein, without previous immunization, B-lymphocytes are isolated from a healthy person, the isolated B-lymphocytes are immortalized, antibodies from the immortalized B-lymphocytes are screened by immune-histochemical analysis for binding against melanoma and/or melanoma metastases, the positively reacting B-lymphocytes are selected, cultured and monoclonal antibodies obtained therefrom.

The present invention is concerned with a process for obtaining human 
antibodies which are directed against melanoma, with the antibodies 
obtained by the process according to the present invention and with the 
use thereof. 
Melanoma, a tumour of the skin, is an extremely aggressive tumour. 
Especially metastasing melanoma can scarcely any longer be successfully 
treated by conventional methods. Therefore, there is a great need to find 
new therapeutics which can be used for the treatment of melanomas. 
Dippold et al., (Proc. Natl. Acad. Sci. USA, 77, 6114-6118/1980) have 
reported the production of murine monoclonal antibodies against melanoma. 
One of the antibodies disclosed therein is directed against the 
ganglioside GD3. In J. Biol. Chem., 262, 6802-6807/1987 are also described 
murine monoclonal antibodies against melanoma. These bind equally to GM3 
and GM2 but not to GD3. 
In 1982, Irie et al. (Proc. Natl. Acad. Sci. USA, 79, 5666-5670/1982) 
reported the production of human monoclonal antibodies which react with 
melanoma. The monoclonal antibodies there disclosed react with the 
gangliosides GM2 and GD2, respectively. In Proc. Natl. Acad. Sci. USA, 84, 
2416-2420/1987, are also described human monoclonal antibodies against 
melanoma. These bind strongly to GD3 and GD2 or to GM3 and GD1a, 
respectively. In Cancer Research, 49, 191-196/1989, are also described 
human monoclonal antibodies against melanoma. These bind strongly to GM3 
and GD1a or GD3 and GD2, respectively. The lymphocytes used for the 
production of these monoclonal antibodies originate from melanoma 
patients. 
The monoclonal antibodies described in the literature were obtained either 
from melanoma patients with or without immunisation or by and after 
immunisation of laboratory animals. However, such efforts for obtaining 
antibodies which are also effective against melanoma in vivo display great 
disadvantages. Murine monoclonal antibodies suffer from the disadvantage 
that they are recognised by the human immune system as foreign proteins 
and, therefore, antibodies are formed against these foreign proteins. This 
means that such monoclonal antibodies are eliminated more quickly and thus 
are limited in their effectiveness. However, also in the case of 
antibodies which have been obtained from tumour patients, there is great 
doubt about their effectiveness since, precisely in the case of tumour 
patients, the functionability of the immune system is impaired. 
Furthermore, the antibodies of the prior art recognise by no means all 
primary melanomas and only a small part of melanoma metastases. 
Therefore, it is an object of the present invention to provide antibodies 
against melanoma in the case of which the disadvantages of the prior art 
are at least partially overcome. 
Thus, according to the present invention, there is provided an antibody 
against melanoma, which is characterised in that it binds to the 
gangliosides GM3 and GD3 but essentially not to the gangliosides GM1, GM2, 
GD1a, GD1b and GD2, the binding of the antibody to the gangliosides having 
been determined by immune staining after thin layer chromatographic 
separation of the gangliosides. 
The antibodies according to the present invention, which are human 
antibodies, can be obtained by a process in which, without previous 
immunisation, B-lymphocytes are isolated from healthy subjects, the 
isolated B-lymphocytes are immortalised, antibodies from the immortalised 
B-lymphocytes are screened by immuno-histochemical analysis for binding 
against primary melanoma and/or melanoma metastases, the 
positively-reacting clones are selected and cultured and monoclonal 
antibodies are obtained therefrom. 
The obtaining of antibodies directed against melanoma from healthy subjects 
which do not suffer from melanoma is completely surprising. As donor for 
the B-lymphocytes, there is preferably chosen a healthy person at risk of 
melanoma. By a person at risk of melanoma is to be understood, for 
example, a human with a risk of sunburn, i.e. a fair-haired or 
fair-skinned human, possibly with freckles, who has previously been 
exposed to an intensive ultra-violet radiation, especially preferably over 
a comparatively long period of time. The suitability of the process 
according to the present invention for obtaining human antibodies which 
are directed against and are also effective against melanoma can possibly 
be attributed to the fact that apparently in every person cells 
continuously degenerate, thereby arising melanoma cells which can, 
however, be successfully combated by the body's own immune system. 
The first step of the process according to the present invention is to 
obtain B-lymphocytes from the blood of a healthy person. A "healthy 
person" in the meaning of the present invention is defined as a person who 
displays no symptoms of a melanoma. Obtaining of B-lymphocytes can be 
carried out by known methods. Subsequently, there follows the 
immortalisation of the lymphocytes, various methods being available for 
this purpose. The B-lymphocytes can be fusioned with myeloma cells of 
human or murine origin according to the method of Kohler and Milstein 
(Nature, 256,495/1975). However, heteromyelomas can also be used for the 
fusion. In the same way, it is also possible to transform the 
B-lymphocytes by means of Epstein-Barr virus. Furthermore, there can be 
used the processes described in EP-A-0,093,436 or in EP-A-0,256,512. 
Fusion is thereby carried out with subcellular vesicles which contain a 
transforming DNA. 
From the immortalised B-lymphocytes, those are selected which produce 
antibodies with the desired reactivity against melanoma. The selection of 
the B-lymphocytes for secretion of antibodies which are directed against 
melanoma and are also effective against metastases is carried out, 
according to the present invention, by immune-histochemical analysis for 
binding against melanoma tissue sections, in which case primary melanoma 
and/or melanoma metastases can be used. The immune-histochemical analysis 
of the antibodies is preferably done by binding to melanoma in tissue 
sections by means of an ELISA test with reference to the method of Nielsen 
et al. (Hybridoma, 6, 103-109/1987). In this way, positively determined 
B-lymphocytes are selected, cultured according to conventional processes 
and monoclonal antibodies obtained therefrom according to known methods. 
By means of the process according to the present invention, it is possible 
to obtain antibodies against melanoma which also possess a very high 
reactivity against melanoma metastases. Thus, the present invention also 
concerns human antibodies against melanoma which, in tissue sections, also 
bind at least 70% of the melanoma metastases. 
An antibody according to the present invention is characterised in that it 
binds to the gangliosides GM3 and GD3 but essentially not to the 
gangliosides GM1, GM2, GD1a, GD1b and GD2. This means that the antibodies 
according to the present invention bind to the gangliosides GM1, GM2, 
GD1a, GD1b and GD2 with an affinity of at most about 5% with reference to 
the affinity for the ganglioside GM3 or the ganglioside GD3. The 
determination of the binding ability of the antibody according to the 
present invention to the gangliosides is carried out by immune staining 
after thin layer chromatographic separation of the gangliosides. By means 
of this process, it is possible, to decide with reliability whether a 
specific ganglioside binds to an antibody or not. Less preferred is the 
determination of the affinity of an antibody according to the method of 
the ELISA test. In the case of this method, non-specific binding cannot be 
excluded so that the possibility of falsely positive results exists. These 
falsely positive results would be manifested by a smaller binding affinity 
to gangliosides in the case of which, according to the above-described 
process, no binding is to be ascertained. 
Examples of antibodies which can be obtained by the process according to 
the present invention are the monoclonal antibodies "17" and AH18, 
secreted by the hybridoma cell lines ECACC 90090703 and ECACC 90090701. 
These antibodies are of the class IgM. These cell lines have been 
deposited on Sep. 7, 1990 in compliance with the Budapest Treaty at the 
European Collection of Animal Cell Cultures (ECACC), Porton Down, 
Salisbury, Wiltonshire, England. 
The present invention also provides human monoclonal antibodies capable of 
binding in an equivalent manner to the monoclonal antibodies obtainable 
from the hybridoma cell lines ECACC 90090703 or ECACC 90090701. which have 
been deposited at the European Collection of Animal Cell Cultures (ECACC) 
under the provisions of the Budapest Treaty at Porton Down, Salisbury, 
Wiltshire, England. By the term "antibodies capable of binding in an 
equivalent manner" are to be understood antibodies in the case of which an 
epitope overlapping is detectable with the antibodies in question. The 
epitope overlapping can be detected by a competitive test system. For this 
purpose, for example with the help of an enzyme immunoassay, there is 
examined the extent to which an antibody competes with the known antibody 
for the binding to a defined antigen or special epitope. For this purpose, 
an appropriately immobilised antigen is incubated with the antibody 
according to the present invention in labelled form and an excess of the 
antibody in question. By detection of the bound labelling, there can then 
easily be ascertained the extent to which the antibody in question can 
displace the defined antibody from the binding. If there is a displacement 
of at least 50% in the case of 10.sup.5 fold excess, then an epitope 
overlapping is present. 
Surprisingly, we have also ascertained that antibodies according to the 
present invention bind not only to melanoma but also to other tumour 
tissues, especially lung and mammary carcinoma tissue. An example therefor 
is the antibody "17" according to the present invention. 
The present invention also provides derivatives of antibodies according to 
the present invention which possess the same binding specifity but with 
modifications in the region which is not important for the antigen 
binding. These antibody derivatives can be obtained from antibodies 
according to the present invention by the exchange of one or more constant 
domains and/or linkages with another molecule. Thus, for example, an 
exchange of constant domains for an isotype switch can be carried out 
where, for example, an antibody of class IgM can be converted into an 
antibody of class IgG with maintenance of its antigen specificity. This 
isotype switch can be carried out by cell-biological or 
molecular-biological methods, which are well known (see, for example, P. 
Rothman et al., Mol. Cell. Biol., 10, 1672-1679/1990). However, the 
monoclonal antibodies according to the present invention can also be 
linked with another molecule, especially a label or a toxin, by means of 
which its diagnostic or therapeutic usability is changed. Appropriate 
processes for the linkage of labels, for example enzymes, such as 
peroxidase, or of toxins, for example ricin or cholera toxin, with the 
antibody or radio-active labelling are well known. 
The present invention is also concerned with the use of an antibody 
according to the present invention for the diagnosis or therapy of 
melanoma and especially for the passive and active immunisation of 
melanoma patients. It is thereby preferred to use the antibody "17" 
secreted by the cell line ECACC 90090703 and/or the antibody AH18 secreted 
by the cell line ECACC 90090701. 
Since the monoclonal antibodies obtained by the process according to the 
present invention bind to living melanoma cells and to other tumour cells, 
they can be used for combating these cells in the organism. Thus, the 
present invention also provides a pharmaceutical composition which 
comprises one or more antibodies according to the present invention, 
optionally together with conventional pharmaceutical carrier, adjuvant, 
filling and additive materials. The administration of a medicament 
according to the present invention is possible not only for prevention of 
a tumour but also after the metastasing of a tumour and especially of a 
melanoma. A suitable dosage of the antibody according to the present 
invention for passive immunisation is in the range of from 1 to 200 mg, 
whereby this dosage is possibly to be repeatedly administered. The 
monoclonal antibodies can be administered locally into the melanoma, as 
described by Irie and Morton (Proc. Natl. Acad. Sci. USA, 83, 
8694-8698/1986). However, after metastasing of the melanoma, a systemic 
administration, such as is conventional, is preferred. The antibodies 
according to the present invention are preferably used therapeutically 
alone but can also be used as conjugates with toxins, therapeutics and the 
like. 
Since the antibodies obtained by the process according to the present 
invention are capable of binding melanomas and melanoma metastases, they 
are also outstandingly suitable for the qualitative or quantitative 
detection of melanoma and other tumour cells. The detection thereby takes 
place in known manner by means of an immunological process of 
determination. Processes of this type are well known and do not need to be 
further explained here. The antibodies obtained according to the present 
invention can thereby be used as unlabelled, labelled and/or immobilised 
receptor. 
The antigens or epitopes defined by means of the monoclonal antibodies can 
also be detected in body fluids by immunological processes of 
determination. The monoclonal antibodies according to the present 
invention can thereby be used as labelled and/or immobilised receptor. 
Many variants are known for carrying out the process of determination, all 
of which are suitable. Thus, for example, two, three or more receptors can 
be used and the incubation with the individual receptors can take place in 
various sequences in homogeneous or heterogeneous phase. In each case, 
there is evaluated the signal change due to binding of at least two 
receptors with the substance to be detected in the sample solution. The 
determination according to the present invention takes place either in 
homogeneous phase; for example according to the principle of the 
agglutination test, in the case of which, as receptors, there are used 
coated particles, for example latex particles or erythrocytes, which 
cross-link due to binding with receptors of specific binding capacity and 
the cells to be detected and thereby agglutinate, or in heterogeneous 
phase, preferably as a sandwich immunoassay. In every case, at least two 
receptors R.sub.1 and R.sub.2 are used of which one contains a monoclonal 
antibody according to the present invention, for example "17", or an 
antibody capable of binding in an equivalent manner or a derivative 
thereof, whereas the other receptor contains another antibody according to 
the present invention, for example AH18, or an antibody capable of binding 
in equivalent manner or a derivative thereof. 
In the case of incubation of the body fluid with the two receptors, there 
are formed complexes of R.sub.1, ganglioside and R.sub.2. The receptors 
are chosen in such a way that only complexes in which not only R.sub.1 but 
also R.sub.2 are bound with the ganglioside give a signal change so that, 
in this way, only those gangliosides are detected which are capable of 
binding with both specific antibodies. 
The determination according to the present invention preferably takes place 
as a sandwich immunoassay. For this purpose, receptor R.sub.1 is 
immobilised or made immobilisable and reacted with the sample solution. 
Subsequently, receptor R.sub.2 is added thereto. Complexes are formed of 
the immobilised receptor R.sub.1, the ganglioside to be detected and 
receptor R.sub.2. 
The antibodies according to the present invention can also be used for the 
determination of a prognostic index. After treatment of a melanoma 
patient, it is thereby ascertained whether the antigen in the body fluids 
recognised by the monoclonal antibodies, especially in the serum of the 
patient, appears or disappears after a certain period of time. 
Finally, the present invention also provides a process for the diagnosis or 
therapy of tumours and especially of melanomas, wherein there is 
administered one or a mixture of several antibodies according to the 
present invention, optionally together with conventional pharmaceutical 
carrier, adjuvant, filling and additive materials, preferably in a dosage 
of from 1 to 200 mg. 
The cell lines ECACC 90090703 and ECACC 90090701 mentioned in the present 
invention, which secrete the antibodies "17" and AH18, were deposited on 
the 7th Sep., 1990, at the European Collection of Animal Cell Cultures 
(ECACC), Porton Down, England. 
The following Examples are given for the purpose of illustrating the 
present invention:

EXAMPLE 1 
Selection for Antibodies Against Melanoma 
From healthy persons 20 to 200 ml of blood were removed and the mononuclear 
cells isolated therefrom. Subsequently, these cells were immortalised 
according to conventional methods (Kohler-Milstein or EBV transformation 
or DNA transfection). After 2 to 4 weeks, the culture supernatants of the 
immortalised cells were tested. For this purpose, there was carried out an 
ELISA test on human tissue sections of melanoma. This took place with 
reference to the method of Nielsen et al., (Hybridoma, 6, 103-109/1987). 
Frozen tumour tissues were cut into discs of 3 to 5 .mu.m thickness and 
applied with silane to glass cover plates. The tissue sections can be 
stored at -80.degree. C. or can also be used immediately. For this 
purpose, they were, as required, fixed for 10 minutes at -20.degree. C. in 
acetone. Subsequently, the acetone was allowed to evaporate if still 
present and thereafter the sections were dipped for about 3 minutes into 
PBS (phosphate-buffered saline according to Dulbecco and Vogt, J. Exp. 
Med., 99,167-182/1954). After the PBS had dripped off, 20 to 30 .mu.l of a 
blocking antibody (concentration 40 .mu.g/ml) were applied thereto. This 
blocking antibody was a Fab fragment of a polyclonal sheep serum or of a 
murine monoclonal antibody against human Fc.mu. or Fc.gamma.. The blocking 
antibody was incubated on the sections for 1 to 2 hours or overnight at 
ambient temperature or at 4.degree. C. 
Subsequently, the section was washed three times for about 4 minutes at 
4.degree. C. with PBS. The antibody-containing solution, i.e. the culture 
supernatants of the immortalised cells, was then pipetted thereon. After 
incubation for several hours (usually for 2 hours) at 4.degree. C., the 
antibody-containing solution was rinsed away by washing three times for 
about 4 minutes in PBS. Subsequently, the section was incubated with the 
same antibody as in the case of the blocking except that this time the 
monoclonal or polyclonal antibody used was coupled to peroxidase (2 U/ml). 
Incubation was carried out for 1 hour at 4.degree. C. with subsequent 
washing as described above. The microscope slides were then dipped into 
substrate solution (aminoethylcarbazole in dimethyl sulphoxide/tris HCl 50 
mmol/l, pH 7.3) in the presence of hydrogen peroxide and incubated for 
about 4 to 5 minutes. The microscope slides were subsequently washed for 
about 5 minutes in PBS. 
If desired, the cell nuclei in the tissue sections can be stained with 
Haemalaun (Merck, diluted 1:3 with water). For this purpose, the 
microscope slides were dipped for about 20 seconds in the Haemalaun 
solution and subsequently "blued" in two PBS baths, in each case for about 
5 minutes. 
For storage, the sections were subsequently coated with an aqueous 
embedding agent, for example glycerol-gelatine (Merck) or Krystal Mount 
(Biomeda Corp., Foster City, Calif.). 
Cell lines which produced the desired antibodies were expanded and cloned. 
The clones obtained were also analysed as described above. The clones 
which produced the desired antibodies were further cultured and the 
antibodies produced by these clones were obtained according to known 
methods. 
By means of this process, there could be obtained the antibodies "17" and 
AH18 according to the present invention. 
EXAMPLE 2 
Antibody Specificity Test 
In order to determine the specificity of the human monoclonal antibodies 
obtained according to Example 1 from the immortalised cell lines, a test 
was carried out for the binding to human primary melanoma and to human 
melanoma metastases. The test was carried out in the manner described in 
Example 1. With the antibodies "17" and AH18 according to the present 
invention, the following results were thereby obtained: 
TABLE 1 
______________________________________ 
reactivity with 
human primary 
monoclonal melanoma metastases 
antibody 
Navi (number positive/number tested) 
______________________________________ 
"17" 28/29 48/52 (90%) 54/54 (100%) 
AH18 23/26 48/54 (90%) 43/51 (80%) 
______________________________________ 
EXAMPLE 3 
3.1. Binding Capacity of the Antibody to Normal Tissue 
The monoclonal antibody "17" obtained according to Example 1 was tested for 
its binding capacity with normal tissue. The test was carried out as 
described in Example 1. 
The antibody was tested for reactivity against human tissue from the brain 
(cortex, pons, thalamus, corpus amygdaloideum), retina, skin 
(keratinocytes, melanocytes, Langerhans' cells, endothelial cells, smooth 
musculature, sweat glands, nerve fibres), muscle, mammary glands, uterus, 
urinary bladder, gall bladder, spleen, kidney, adrenals, lungs, parotid 
gland, intestine, erythrocytes (4 commercial test samples and 300 blood 
donors) and parathyroid. No reaction was found of the antibody according 
to the present invention with the above types of tissue. 
3.2. Binding Capacity of the Antibody to Tumours 
In order to determine the further specificity of the human monoclonal 
antibodies obtained from the immortalised cell lines, further tests were 
carried out on human tumour tissues. The test was carried out as described 
in Example 1. With the antibodies according to the present invention, the 
following results were thereby obtained: 
TABLE 2 
______________________________________ 
reactivity with "17" and AH18 
tumour number positive/number tested 
______________________________________ 
melanoma 48/52 
lung carcinoma 10/19 
mammary carcinoma 
4/9 
______________________________________ 
EXAMPLE 4 
Furthermore, the reactivity of the monoclonal antibodies against purified 
gangliosides was investigated in an immunoblot after thin layer 
chromatography of the gangliosides. 
For this purpose, there were used the gangliosides GM3, GM2 and GD1a of 
Boehringer Mannheim GmbH, GD2 and GD3 of Biocarb, Sweden, GM1 of Fidia, 
Italy and GD1b of Pallmann, Munchen. Thin layer plates HPTLC Alu silica 
gel 60 F.sub.254 were obtained from Merck. The HPTLC plate was developed 
with chloroform:methanol:water (0.02% calcium chloride dihydrate) 60:40:9 
v/v/v. As fixing agent for the thin layer plates, there was used high 
molecular weight polyisobutyl acrylate (Aldrich Chemicals) in the form of 
a 0.1% solution in hexane. 
Carrying Out 
The procedure and modifications thereof are well known and are here given 
only by way of example. 
Sample Application 
5 .mu.l of a 1 mg/ml solution of the gangliosides were applied, the 
above-mentioned developing agent being used as solvent. The samples were 
applied successively, for example with a Hamilton syringe, in an about 5 
mm wide strip on the thin layer plate; inbetween, they were always allowed 
to dry well in order that the coating remained as thin as possible. 
Finally, the thin layer plate was again well dried with a hot-air blower. 
The plate was introduced into a chamber saturated with developing agent 
and incubated until the developing agent had moistened about 80% of the 
plate. The plate was subsequently removed and allowed to dry. 
The plate was then rinsed in 0.1% polyisobutyl acrylate solution for about 
1 minute, whereafter the plate was again allowed to dry. For the purpose 
of blocking, the plate was covered with a 1% BSA/PBS solution (bovine 
serum albumin of Boehringer Mannheim GmbH) using a pipette (under avoiding 
any air bubbles!) and left to stand for about 30 minutes at ambient 
temperature. Thereafter, the blocking solution was poured off. 
Subsequently, washing was carried out twice with PBS. For this purpose, the 
plate was immersed in PBS in a dish and, in each case, left for 2 minutes. 
Inbetween, the PBS was sucked off (never apply the PBS directly to the 
plate; do not shake; do not allow the plate to dry out). 
Thereafter, the plate was covered with a solution containing the monoclonal 
antibody (concentration 1 to 10 .mu.g/ml) and left for 1 hour at ambient 
temperature. 
Subsequently, it was washed five times with PBS (as described above). The 
plate was then covered with conjugate (polyclonal, peroxidase-labelled 
sheep Fab fragment against human Fc.mu., concentration as in the case of 
the tissue test in Example 1) and incubated for 1 hour at ambient 
temperature. Subsequently, the plate was washed six times with PBS as 
described above. The substrate was then added thereto and the plate gently 
moved during the development. 
As substrate, there was used TMB/DONS (tetramethylbenzidine (TMB) 12 mg 
+dioctyl sodium sulphosuccinate (DONS) 40 mg dissolved in 10 ml methanol 
and mixed with 10 ml citric acid/phosphate buffer, pH 5.0 (25 ml 0.1M 
citric acid, Merck, +28 ml 0.2M disodium monohydrogen phosphate dihydrate, 
Merck, to 100 ml), as well as with 10 .mu.l 30% hydrogen peroxide. It was 
developed as long as the negative control was not stained and then washed 
several times with distilled water. The plate was dried while protecting 
from light and immediately photographed since the staining with TMB/DONS 
bleaches. 
If the above-described procedure is carried out with MAB "17" or with MAB 
AH18, then there are obtained the results shown in the following Table 3. 
The MAB's "17" and AH18 react with GM3 and GD3 but not with GM1, GM2, 
GD1a, GD1b and GD2. 
For control, the gangliosides were made visible in a parallel experiment by 
staining with resorcinol. For this purpose, the plate was sprayed with 
resorcinol solution (resorcinol from Merck, 400 ml +100 ml water +5 ml 
sulphuric acid) and developed for about 10 minutes in a drying cabinet at 
110.degree. C. This control shows that, of all gangliosides, equal amounts 
had been applied to the thin layer plate. 
TABLE 3 
______________________________________ 
Detection of gangliosides by thin layer chromatography 
ganglioside 
resorcinol 
MAB "17" or MAB AH18 
______________________________________ 
GM1 + - 
GM2 + - 
GM3 + + 
GD1a + - 
GD1b + - 
GD2 + - 
GD3 + + 
______________________________________ 
From Table 3, it can be seen that the antibodies according to the present 
invention only react with certain gangliosides. Of the tested 
gangliosides, GM3 and GD3 show positive signals. No noteworthy reactivity 
(&lt;5%) was found with gangliosides GM1, GM2, GD1a, GD2 and GD1b. 
EXAMPLE 5 
Reactivity of the Antibodies with Cell Lines 
Cells of the cell lines to be investigated were cultured overnight in 
Terasaki plates (obtained from the firm Greiner). The culture supernatant 
was removed from the adherent cells and replaced by the antibody solution 
to be investigated. After incubation for 1 to 2 hours, the antibody 
solution was removed, the cell "lawn" washed several times and the 
monoclonal antibodies bound to the cells were detected. For this purpose, 
after washing with PBS, 100 .mu.l peroxidase-labelled sheep anti-human 
light chain antibody were added thereto and again incubated at ambient 
temperature for 1 to 2 hours. After renewed washing, the enzyme reaction 
was started with a peroxidase substrate (ABTS.RTM.). After 10 to 60 
minutes at ambient temperature, the extinction was determined in a 
photometer at 406 nm. Alternatively thereto, there can also be added a 
peroxidase substrate, such as aminoethylcarbazole, and, after ending of 
the reaction, the brownish precipitate in or on the cells evaluated by 
means of a microscope. 
From the results shown in the following Table 4, it follows that the 
antibody "17" reacts with melanoma SK-MEL 28 cells (ATCC HTB 72), whereas 
no reaction was found with human prepuce fibroblasts (which were 
themselves isolated by known processes). Furthermore, the antibody "17" 
showed reaction with insulinoma cell RIN (obtained from Dr. Eisenbart, 
Josslin Diabetes Center, Boston, Mass. 02215) and the neuroblastoma cells 
IMR 32 (ATCC CCL 127). 
TABLE 4 
______________________________________ 
Antibody reactivity against various cells 
______________________________________ 
IMR32 human 
SK MEL 28 RIN (neuro- 
prepuce 
antibody 
(melanoma) (insulinoma) 
blastoma) 
fibroblasts 
______________________________________ 
"17" ++ + + - 
______________________________________ 
EXAMPLE 6 
Determination of Epitope Overlapping of Antibodies Against Melanoma 
For the detection of the epitope overlapping of an antibody with one of the 
monoclonal antibodies ECACC 90090703 or ECACC 90090701, there was carried 
out a competitive enzyme immunoassay. For this purpose, the gangliosides 
GM3, GM2, GD1a, GD2, GD3, GM1 and GD1b (obtained from Boehringer Mannheim 
GmbH, Biocarb, Pallmann or Fidia; cf. Example 4) were dissolved in 
methanol (10 .mu.g/ml) and, in each case, 100 .mu.l of this solution were 
pipetted into 96-well microtitre plates (Greiner). After evaporation of 
the solution (either overnight at ambient temperature or for 1 hour at 
37.degree. C.), washing was carried out with PBS and then non-specific 
binding positions were blocked with a 1% crotein C solution in PBS 
(incubation at ambient temperature for 1 to 2 hours and washing with 
PBS/0.05% Tween 20). Subsequently, incubation was carried out for 90 
minutes at ambient temperature simultaneously with one of the monoclonal 
antibodies ECACC 90090703 or ECACC 90090701 which had been labelled with 
peroxidase (end concentration 250 mU/ml) and with the antibody to be 
assessed. After washing again four times with PBS/0.05% Tween 20, 
incubation was carried out for 30 minutes with buffer containing the 
enzyme peroxidase substrate ABTS.RTM. in sodium perborate at ambient 
temperature and subsequently the extinction was measured at 405 nm as a 
measure for the amount of the bound, peroxidase-labelled monoclonal 
antibody ECACC 90090703 or ECACC 90090701. This value is compared with the 
extinction which was obtained in the case of incubation with the 
monoclonal antibody ECACC 90090703 or ECACC 90090701 alone (with the 
addition of a corresponding amount of buffer for the compensation of the 
dilution effect arising in the case of the addition of the antibody to be 
assessed). When, with up to a 10.sup.5 fold excess of antibody to be 
assessed with regard to the monoclonal antibody ECACC 90090703 or ECACC 
90090701 enzyme conjugate (250 mU/ml), at least 50% competition is to be 
recognised, then an epitope overlapping is present.