Res. Immunol.
0 INSTITUT PASTEUR/ELSEVIER Paris 1992
1992,
143, 879-891
Human monoclonal IgM antibodies from foetal B-cell hybridomas directed against a surface antigen on human tumour cells J. Bohn (I), 0. Josimovic-Alasevic c2), U. Settmacher (3), S.T. Kiessig (I), A. Lukowsky cl), H.-D. Volk (I), T. Diamantstein c2), R. Von Baehr (I) and S. Jahn (I) (*) (I) Institute of Medical Immunology Universitiit zu Berlin, and Q) Institute
and ‘3’ Clinic for Surgery, Medical School (Charit&), Humboldtof Immunology, Klinikum Steglitz, Freie Universitiit, Berlin (Germany) SUMMARY
In order to assess the existence of B lymphocytes capable of producing anti-tumour antibodies in non-tumour-bearing individuals, human lymphocytes derived from foetuses and adults were fused with the heteromyeloma cell line CB-F7. By indirect immunofluorescence, 29 out of 4,472 IgM-producing hybridomas (from 8 foetuses and 8 adults) were shown to produce antibodies which bind to colon carcinoma lines Co10205 and SW620, Raji lymphoma cells and small cell carcinoma of the lung. In vitro growth of tumour cells recognized by these antibodies was inhibited. The antibodies also mediated complement-dependent cytotoxicity. All antibodies tested recognized a cell surface molecule of 55 kDa. Southern blot hybridization analysis of hybridoma DNA with a human J, probe showed that the hybridomas were derived from clonally unrelated B cells. These results demonstrate that human foetal and adult B cells from non-tumourbearing individuals are able to produce IgM antibodies recognizing defined cell surface molecules expressed on some tumour cells.
Key-words: B lymphocyte, city, Immunosurveillance.
Tumour, IgM; Hybridomas, Surface antigens, Cytotoxi-
INTRODUCTION
It currently remains controversial as to whether antibodies are involved in tumour surveillance. Antibodies reacting with tumour cells were found to be present in several tumour-bearing patients (Cote and Houghton, 1985 ; Pfreundschub et al., 1978). However, it is not clear whether these antibodies belong to the natural repertoire of non-tumour-bearing individuals or
Submitted
October
20, 1992, accepted November
whether they are the consequence of an immune response to the respective tumour. The view that natural antibodies may be involved in the control of tumour growth is supported by results obtained in animal models (Chow and Bennet, 1989; Essex et al., 1971). However, the antigens recognized by such antibodies have not yet been defined. If natural antibodies play a role in tumour
17, 1992.
(‘1 Address reprint requests to: Dr. S. Jahn, Inst. Med. Immunol., Charit& Schumannstrasse 20/21, Berlin O-1040 (Germany).
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surveillance, then in normal individuals, lymphocytes should exist which have the capacity to produce antibodies reacting with surface molecules of tumour cells. In order to prove this possibility foetaI B cells were immortalized and cloned (Grunow et al., 1988). The resulting monoclonal antibodies (r&b) were tested for their capacity to inhibit growth and to mount complementdependent cytotoxicity in a panel of tumour lines. In addition, attempts were made to characterize the antigens recognized by such antibodies. MATERIALS
Human
AND METHODS
hybridomas
Human lymphocytes from foetal liver and spleen and from adult spleen were fused with the HATsensitive human x mouse heteromyeloma line CBF7, as previously described in detail (Grunow et al., 1988). Organ fragments were derived from foetuses after clinically indicated pregnancy terminations between the 16th and 34th week of gestation because of neural crest defect or Potter’s syndrome (Settmacher et al., 1990). Fusions with lymphocytes derived from adult spleen (partial splenectomy after trauma, portale hypertension) have been described in detail (Jahn et al., 1988). Immunoglobulin production by hybridoma cells has been routinely tested by ELISA (supernatant Ig; Jahn et al., 1986) or immunofluorescence (cytoplasmic Ig ; Jahn et al., 1991) using peroxidase or FITC-labelled antibodies to human Ig heavy and light chains, respectively (Medac; Hamburg, Germany). Tumour
ET AL.
Other cell types Peripheral blood lymphocytes were prepared as described (Jahn et al., 1988). Fibroblast and endothelial cell cultures derived from surgical specimens (non-tumour patients) were kindly provided by Dr. G. Pankonin (Central Institute of Cancer Research, Berlin-Buch, Germany). Erythrocytes were prepared from blood group A, B, AB and 0 donors according to routine methods. Immunofluorescence Tumour cell lines (Raji, Colo205, SW620, H69) were grown in flat-bottom 96-well plates (Falcon, Becton-Dickinson, USA) to confluency. Then, the medium was removed and the plates were incubated with cell culture supernatants for 30 min on ice. After washing (culture medium), a FITC-conjugated antihuman Ig antibody (Dakopatts, Glostrup, Denmark) was added for 30 min. Plates were analysed by fluorescence microscopy. As controls, supernatants were tested for binding to fibroblasts, endothelial cells and erythrocytes. Cytofluorometric analyses were carried out using an “Epics C” cytofluometer (Coultier, Krefeld, Germany) as previously described (Falck et al., 1987). 3H-thymidine
incorporation
assay
Tumour cells (104/well) were cultured for 3 days in the presence of human monoclonal IgM in 96-well plates in RPM1 supplemented with 10 % FCS or 10 Vo heat-inactivated human AB serum. The cultures were pulsed with ‘H-thymidine (Amersham, Brunswick, Germany) for 18 h. The ‘H-thymidine incorporation was assessed by liquid scintillation counting (LS 233, Beckman, Berkeley, CA, USA).
ceils
All tumour cell lines studied were obtained from the American Type Culture Collection (ATCC ; Rockville, USA). Cells were cultured in RPMI-1640 supplemented with 10 070 foetal calf serum (FCS) without antibiotics at 37°C in a humidified 5 Vo CO-,containing air atmosphere. As detected by bisbenzimide-H dye (Serva; Heidelberg, Germany) and microbiological tests, the tumour cells were free of mycoplasmas.
CMC analysis was performed as previously described (Bindon et al., 1988). Normal human serum was obtained from defibrinated blood (group AB) of healthy volunteers. Guinea pig serum was obtained from clotted blood. To remove cytotoxic antibodies, sera were absorbed on the respective human tumour cells. “Cr-1abelled tumour cells were incu-
CMC FCS FITC
HAT mAb PBS
= complement-mediated cytotoxicity. = foetal calf serum. = fluorescein isothiocynonate.
Complement-mediated
cytotoxicity
(CM0
= hypoxanthine, aminopterin, thymidine. = monoclonal antibody. = phosphate-buffered saline.
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bated with human IgM at room temperature for 10 min. Then, the diluted human or guinea pig sera were added and the cells were incubated for 3 h at 37”C, followed by counting the activity in the cell pellet and supernatant in a gamma counter (LKB ; Uppsala, Sweden). Maximal lysis was measured in supernatants of 1 070Triton-X100-lysed cells. Spontaneous release was detected in cell cultures containing medium only. The percentage specific lysis was calculated as follows: experimentalrelease- spontaneousrelease x loo. % ;ypzsific= maximal release - spontaneousrelease
Immunoprecipitation
Colon carcinoma SW620 cells were externally iodinated according to the chloroglycoluril method (Markwell and Fox, 1978). Briefly, 2 x 10’ cells resuspended in 1 ml of Dulbecco’s phosphatebuffered saline (PBS) were added to a glass scintillation vial previously coated with 100 pg of 1,3,4,6-tetrachloro-3a,6a-diphenyl-glucoluril (Iodo, GEN, Rotterdam, The Netherlands). Na12’I (18.5 MBq carrier-free, Amersham, Brunswick, Germany) was added, and after 10 min of gentle agitation at room temperature, the cells were transferred to a tube containing 50 pg of tyrosine and were then washed with D-PBS. For solubilization, cells were suspended in 0.5 ml of a lysis buffer (20 mM TRIS-HCl pH 7.7; 20 mM EDTA, 150 mM NaCl, 1 mM phenylmethylsulphonyl fluoride, 100 KIU/ml aprotinin, 5 Kg/ml soybean trypsin inhibitor, 0.02 % NaN, and 0.5 070Nonidet P-40, Sigma-Chemie; Munich, Germany) and incubated for 30 min on ice. The supernatant, centrifuged at 10,000 g for 15 min, was preabsorbed with l/10 (v/v) rabbit anti-human Ig (Dakopatts, Glostrup, Denmark) coupled to Sepharose beads. The rabbit anti-human Ig-coupled Sepharose beads were allowed to react with culture supernatants containing approximately 3 pg/ml of CB-HTl or CB-HT2 and were used for immunoprecipitation by incubation with the preabsorbed extract for 60 min at 4°C. The immunoprecipitates were washed, extracted with SDS-PAGE sample buffer and analysed by SDS-PAGE under reducing conditions as described (Osawa et al., 1982).
Western blot Tumour cell lysis was carried out by 0.5 % NP40 or alternatively with 3 M KC1 (Parham ef al., 1982). The membrane fraction was spun down at 30,000 g for 2 h. Samples were run on SDS-PAGE (Laemmli, 1970) and transferred to nitrocellulose (SchleicherSchiill, Dassel, Germany). Strips were incubated with
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hybridoma culture supernatants (IgM concentration 1 pg/ml) at 4°C overnight. After washing (PBS, Tween 0.1 070) the POD-labelled goat-anti-humanIgM (see ELISA) was added for 1 h at room temperature. After washing, the reaction was developed by adding 3.3 diaminobencidine containing 0.1 % NiCl, as a substrate. A molecular weight standard marker (Biorad, Richmond, VA, USA) was run in parallel. Southern blot DNA samples were prepared from human hybridoma lines by phenol extraction as described (Sambrook et al., 1989). DNA then were digested with Hind11 or EcoRl and 10 11.8were electrophoresed through 1 070agarose gels, blotted onto nitrocellulose filters and hybridized with a J,-probe, kindly provided by Dr. P. Leder (Stanford, CA; Ravetch et a/., 1981). The gene probe was labelled by nick translation using 32P-d-CTP (Amersham, Brunswick, Germany) and 10’ counts per minute were added to each blot. All gels were run using lambda phage DNA digested with Hi&III as size standards. Blots were washed in 2 x SSC with 0.2 % SDS, 15 min at room temperature, followed by a washing with 2 x SSC at 65°C and a final washing with 0.2 x SSC at 65°C.
RESULTS
Occurrence of anti-tumour-reactive antibodies in hybridoma culture supernatants derived from fusions of human lymphocytes from different immune organs
Lymphocytes from human foetal and adult spleen and foetal liver were fused to HATsensitive heteromyeloma CB-F7 cells. After 21 days, culture supernatants from initial hybri-
doma cell lines were analysed for Ig production by ELISA. Furthermore, the reactivity against different
tumour
cell lines was tested by immu-
nofluorescence. Among 4472 IgM-secreting cell lines, 29 hybridomas secreted antibodies reacting with the tumour cells (table I). Hybridomas producing anti-tumour IgM with comparable frequencies were detected in fusions of both foetal and adult lymphocytes. Taking into account a plating density of 1.5 x lo5 unseparated mononuclear cells per well, comprising 20 % of B cells, one may calculate that nearly l/250,000 fusionable B lymphocytes were able to produce
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Table I. The occurrence of human hybridomas producing antibodies reacting with tumour cells in fusions of lymphocytes from adult or foetal immune compartments. Organ source
Fusions n
Wells seeded
5
1,496 2,424 2,380
Foetal liver Foetal spleen Adult spleen
Cultures containing anti-tumour antibodies
IgM-producing hybridomas 547 2245 1680
2 15 12
Tumour cell lines Colo205, SW620, H69 and Raji were immobilized to 96-well culture plates. Undiluted culture supernatants from primary hybridoma lines were incubated. After washing, a FITC-labelled rabbit anti-human Ig antibody was added. Screening was carried out by fluorescence microscopy. Wells were counted as positive when more than 25 ‘70 of the cells were stained by the respective mAb.
Table II. Reactivity with different cells of natural human IgM mAb derived from foetal and adult lymphocytes. Cells Small cell lung carcinoma:
CB-60 H69 H128
Colon carcinoma : Co10205 SW620 Burkitt lymphoma : Raji Daudi Leukaemia cells : K562 Molt 4 Jurkat Cem Normal cells Fibroblasts Endothelial cells Erythrocytes (blood groups A, B, AR 0) Human PBL
CB-HTl
CB-HT2
CB-HT3
CB-HT4
CB-HT5
-
+ +
+ +
-
-
-
-
+ +
+ +
+ +
+ +
+ +
-
+ +
+ +
+ -
+ +
+ -
-
-
-
+ -
-
-
-
-
-
-
-
-
As a control, an isotype-identical human mAb (CB-60) was used. Analyses were carried out on a cytofluometer Epics C (see also fig. 1).
IgM that binds to tumour cells. All human mAb reacting with tumour cells were found to be of the IgM isotype ; 15/29 expressed lambda and 14 expressed kappa light chains. Five hybridomas were studied in more detail : CB-HTl from a fusion of adult spleen, CB-HT2 and CB-HT3 from a foetal liver (34th week of gestation) cell fusion and CB-HT4 and CB-HTS hybridomas established from foetal spleen lymphocytes (35th
week). The reactivity of the IgM antibodies produced by these hybridomas with human tumour and normal cells was tested by immunofluorescence. All 5 IgM antibodies were found to bind to colon carcinoma cells (Colo205, SW620) and to the Raji Burkitt lymphoma line (table II). Two out of five antibodies also bound to lung small cell carcinoma cells (H69, H128) as well as to Daudi cells. One mAb IgM was found to bind
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to the K562 erythroleukaemia line. No binding was seen to T-cell leukaemic (Molt4, Jurkat) and normal cells. The cytofluorometry binding pattern of the adult (CB-HTl) and foetal (CB-HT2) hybridoma IgM to Raji and SW620 cells was shown (fig. 1). Between 50 and 70 Vo of colon carcinoma cells and 20 and 40 % of lung tumour cells were stained by the respective mAb.
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Characterization of the antigen bound by human IgM mAb on the tumour cell surface SW620 cells were surface-iodinated, the 125Jlabelled material was extracted, purified by immunoprecipitation by mAb CB-HTl and CBHT2 that bound to rabbit anti-human IgMcoupled Sepharose and analysed by SDS-PAGE.
SW 620
Raji Control
Control
CB-HTl
CB-HT2
Fluorescence
HYBRIDOMAS
Intensity
(log,
o scale)
Fig. 1. Cytofluorometric analysisof natural antibody binding to tumour cells SW620 and Raji.
An isotype-identicalantibody of the sameconcentration (1 pg/ml) served as a control.
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Under reducing conditions, a major specific band of about 55 kDa was detected as being recognized by both mAb (fig. 2). Two bands with a high MW of 200 and 185 kDa which were clearly visible in lanes CB-HTl and CB-HT2 also
r
N
F
f
:
8
8
8
kDA
200
-
97
-
66
-
When tumour cell lines SW620 and Co10205 were cultured in the presence of tumour cell binding mAb, after 24 h, adherent cells took on an elongated shape and were arranged in distinct clusters and syncytia (fig. 4). This effect was specific, inasmuch as only mAb showing a positive result in immunofluorescence assays were able to affect tumour growth in vitro. As illustrated in figure 5, the mAb inhibited the 3H-thymidine incorporation rate into tumour cells in a dosedependent manner. Time-course experiments showed that inhibition of tumour cell growth reached a maximum on day 3 after addition of antibodies.
4
43
appeared in the control lane (non-binding antibody CB-92), although at much lower intensity barely visible on the photograph. Western blot analyses (fig. 3 right) performed with the same antibodies showed a band only at 55 kDa, so it was assumed that only this band was specific. Furthermore, it was seen that the different human mAb from the foetal and adult repertoire recognized an antigen with the same molecular weight (MW) of about 55 kDa on one particular tumour cell line (SW620; fig. 3 left). An antigen of the same MW was found on 5 different tumour cell lines with antibody CB-HT2 (fig. 3 right).
Inhibition of tumour cell growth in vitro by mAb
-
116
ET AL.
-
Fig. 2. Autoradiog rarn (SDS-PAGE) of antigens on human tumour cell surfaces recognized by natural antibodies. Extracts from ‘2SI-surface-labelled SW620 cells were immunoprecipitated with CB-HTl or CB-HT2 coupled to sepharose beads or with the non-tumour binding antibody CB-92, reactive with ssDNA. Bound material was analysed by SDS-PAGE under reducing conditions.
Antibody-dependent tumour cell lysis
complement-mediated
The ability of human IgM mAb to permit complement-mediated tumour cell lysis was tested using the 51Cr-release test. Human and guinea pig sera were used as complement sources and showed comparable results. Three antibodies which were previously found to react with tumour cells in immunofluorescence assayspermitted their lysis in the presence of complement (table III). Further experiments showed humancomplement-mediated tumour cell lysis to be dose-dependent (fig. 6).
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Fig. 3. Western blot analysisof the binding of natural IgM mAb to different tumour cell lines.
The membranefraction was preparedand run through an agarosegel asdescribed.After blotting to nitrocellulosepaper, (a) different mAb were addedto SW620derived material, or (b) CBHT2 antibody wasincubatedwith nitrocellulosestripswith membranefractions from severalhuman tumour lines.
Immunogenetic analysis of B cells producing natural antibodies reacting with tumour cells
Since several hybridomas which produced antibodies specific for an antigen expressedon the tumour cell surface were derived from the same foetal organ source, we attempted to analyse a possible clonal relationship. HindI and EcoRl-digested hybridoma DNA from 5 cell
lines analysed by Southern blots using the J, probe. Placenta DNA served as a control to show the IgH germline configuration. All five hybridomas exhibited Jn-specific bands of discrete size (fig. 7). Therefore, hybridomas derived from a single fusion (CB-HT5 and CB-HT4 : lanes A and B ; CB-HT3 and -HT2 : lanes C and d) could not be clonally related.
886
J. BOHN
ET AL.
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HYBRIDOMAS
887
10
RAJI
DAUDI
SW 620 COLO 205 K 562
Fig. 5. Inhibition of 3H-thymidine uptake in tumour cell cultures by natural IgM mAb. Three days after seeding,tumour cellswere incubated with various concentrationsof the CBHT2 antibody and pulsedwith 3H-thymidinefor 18h. Mean valuesof four independentexperiments are shown. Bars representSEM. As a control, a non-binding IgM antibody CB-60 wasused. Black column: n control ; cross-hatchedcolumn: q 0.25 pg/ml ; white column: 0 0.50 pg/mI ; hatched column: 69 0.75 yg/ml ; large cross-hatchedcolumn: 1.OOKg/ml.
Table III. Complement-mediated tumour cell lysis induced by human natural IgM. Antibody
Molt4
Colo205
SW620
CB-F7 CB-60 CB-HTI CB-HT2 CB-HT3
3 6 7 2 3
2 7 72 32 54
4 3 40 78 43
Vo Specific lysis: Raji H69 0 0 92 59 62
2 5 32 47 8
HI28
Daudi
K562
1 2 21 33 2
2 2 64 67 4
0 1 1 4 65
Antibody concentrations were adjusted to 0.5 Kg/ml. The CB-60 (IgM) and CB-F7 (conditioned medium derived from the Ig-nonproducing fusion line) served as controls. Data from one representative experiment out of 4 independent experiments using guinea pig serum as a complement source were presented. Percent specific lysis was calculated as described in “Materials and Methods”.
DISCUSSION
Antibodies to antigens on tumour cells were detected in sera from tumour patients (Cote and Houghton, 1985; Pfreundschuh et al., 1978). During immortalization of B lymphocytes deriv-
ed from tumour patients, human mAb which bind to tumour cells were obtained (Houghton et al., 1983 ; Murakami et al., 1985 ; Olsson and Kaplan, 1980; Shoenfeld et al., 1987). The human mAb described were either specific and probably reacted with the lymphocyte donor
888
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20
ET AL.
.-
0 0
0.26
0.80
0.70
1.00 pm/ml
Fig. 6. Complement-mediated lysis of Raji cellssensitizedwith different human mAb.
Lysis wasmeasuredby the 5’Cr-releaseassayusingabsorbedhuman complement.Mean values from three independentexperimentswere shown. Spontaneousreleasewas 3.3 f 2.1 %. Vertical bars indicate SEM. Full line: CB-HTI ; broken line, CB-HT2; dotted line: CB-HT3.
tumour cells only (Cote and Houghton, 1985; Murakami et al., 1985), or else bound to a broad panel of tumour and normal cell lines as well as to different autoantigens (Houghton et al., 1983; Olsson and Kaplan, 1980). The functional role of such antibodies in tumour defence of patients, however, remains unclear (for review, see ref. Sands and Jones, 1980). It is of interest to determine whether antibodies against surface antigens expressed on tumour cells appear as a consequence of an antitumoral immune response in patients or whether they exist in the natural repertoire of non-tumour-bearing donors as well, indicating the possible role of natural antibodies in tumour surveillance in man. The latter hypothesis is supported by data from animal models. In xid mice, a relationship between the absence of natural antibodies and tumour incidence has been described (Chow and Bennet, 1989). Furthermore, Essex et al. (1971) estimated a lower in vivo resistenceto feline-sarcoma-virus-induced tumour growth in animals with a decreased level of natural antibodies. A relationship between natural antibodies of the IgM isotype recogniz-
ing tumour cell carbohydrates and mouse strain resistance to the tumour was established (Gils et al., 1990). If natural antibodies take part in tumour surveillance, then lymphocytes should exist in nontumour-bearing individuals with the capacity to produce antibodies reacting with surface molecules of tumour cells. In order to prove this, human foetal B lymphocytes (Settmacher et al., 1990) were fused with CB-F7 heteromyeloma cells (Grunow et al., 1988). Furthermore, hybridization experiments were carried out with adult spleen cells (Jahn et al., 1986). The high efficiency of the fusion technique enabled us to analyse several thousand primary hybridoma lines. Twenty-nine out of 4,479 IgM-producing hybridomas were found to secrete antibodies against the tumour cells. To study such antibodies in more detail, five hybridoma cell lines were established by repeated limiting dilution cloning : two hybridomas from a foetal spleen, two from a foetal liver and one from an adult spleen. As shown by Southern hybridization of the DNA with a human J, gene probe, these hybridomas
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A
0
889
mldlll
ECORI PI
HYBRIDOMAS
C
D
I
PI
ABCDt
Fig. 7. Southernblot analysisof Hi&III-
andEcoRl-digestedDNA obtainedfrom humanhybridomasproducing natural IgM reacting with tumour cells. DNA samples(10 pg) wereelectrophoresedthrough an agarosegel. Southernhybridization using a J, probe was performed as describedin “Materials and Methods”. A: CB-HT5 ; B: CB-HT4; C : CB-HT3 ; D : CB-HT2 ; E : CB-HTl . P = placenta DNA. Arrowheads indicate the position of HindII-digested lambda phagefragments.
were generated from different maternal B lymphocytes. All antibodies tested reacted with an antigen of 55 kDa expressed on the surface of SW620 cells. Interestingly, an antigen of the same MW could be found on 4 different tumour cell lines with one particular antibody. Although a comparable binding pattern for the human mAb studied was seen on colon carcinoma and lymphoma cell lines, only 2 IgM bound to small cell lung carcinoma cells as well. Only one antibody was seen to bind an antigen on K562 erythroleukaemia cells. This may be explained either by different binding affinities of the antibodies
or by the recognition of different epitopes on the same antigen. No binding of the antibodies to normal cells (fibroblasts, endothelial cells, erythrocytes) could be detected. In conclusion, several findings support the view that such natural antibodies from the foetal repertoire (if also detected in normal individuals) may play a role in tumour defence : i) the antibodies bind to a cell surface molecule common to the tumour cells tested ; ii) binding to such molecules changes the morphology of the tumour cells and inhibits their proliferative capacity ; moreover, iii) in the presence of human
890
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complement, the cells can be lysed. Natural antibodies were at least partially related to the multispecific IgM, reacting with autoantigens and foreign material of a different molecular structure (Dighiero et al., 1985; Nakamura et al., 1988 ; Tron and Bach, 1989). The main role in producing such antibodies is played by CDS-bearing B lymphocytes (Casali and Notkins, 1989 ; Hayakawa et al., 1984). We are currently investigating whether antibodies from the foetal repertoire studied here may bind other antigens. Posner et al. (1990) showed that IgM antibodies produced by EBV-transformed B cells from healthy donors were able to bind to a broad panel of autologous cells and haematopoietic cell lines. However, multispecificity cannot impair the fact that tumour-cell-binding IgM antibodies exist within the natural repertoire of nontumour-bearing individuals. The size of this Bcell pool may only be speculated from fusion data. Therefore, l/250,000 B cells in both the foetal and adult repertoire may produce antibodies reacting with tumour cells. However, this conclusion is of little significance, since only a fraction of the B lymphocytes may be fused, de pending on their in vivo activation state and other unknown factors. Nevertheless, our DNA analysis data suggestthat a heterogeneous B cell pool, rather than a distinct clone, could be responsible for humoral tumour surveillance. Further experiments will be directed toward a more detailed characterization of the antigen bound by mAb from the B-cell repertoire of non-tumour-bearing individuals to human tumour cells. Taken together, present results suggest that, in addition to their antiinfectious (Bos et al., 1989) and autoantigen-clearance (Grabar, 1983) functions, natural antibodies may play a role in tumour surveillance.
Anticorps IgM monoclonaux humains produits par des hybridomes de cellules B faetales et dirigks contre un antigbe de surface de cellules tumorales humaines Dans le but d’ttablir I’existence de lymphocytes B capables de produire des anticorps antitumoraux
ET AL.
chez des sujets non porteurs de tumeur, des lymphocytes humains fcetaux et adultes ont Ctt fusionnts avec la lignie cellulaire CB-F7 httiromyelomateuse. Par immunofluorescence indirecte, 29/4472 hybridomes se sont montres producteurs d’IgM (a partir de 8 f&us et 8 ad&es) se liant aux lignees carcinomateuses intestinales Co10205 et SW620, a la lignee lymphomateuse Raji et aux carcinomes pulmonaires a petites cellules. La croissance in vitro des cellules tumorales reconnues par ces anticorps est inhibee. Ces anticorps provoquent egalement une cytotoxicite dtpendante du complement. Tous les anticorps testes reconnaissent une molecule de surface de 55 kDa. La technique d’hybridation de type Southern avec une sonde J, a permis de montrer que les hybridomes sont derives de cellules B clonales non apparentees. Ces resultats montrent que des cellules B fcetales et adultes de sujets non porteurs de tumeurs, sont capables de produire des IgM reconnaissant des molecules de surface exprimees par certaines cellules tumorales. Mets-cl&: Lymphocyte B, Tumeur, IgM; Hybridomes, Antigenes de surface, Cytotoxicite, Immunosurveillance.
References Bindon, C.L., Hale, G., Brilggemann, M. & Waldman, H. (1988), Human monoclonal IgG isotypes differ in complement activating function at the level of C4 as well as Clq. J. exp. Med., 168, 127-142. Bos, N.A., Meeuwsen, C.G., Wijngarden, P.V. & Benner, R. (1989), B cell repertoire in adult antigen-free and conventional neonatal Balb/c mice. - II. Analysis of antigen-binding capacities in relation to Vu gene usage. Europ. J. Immunol., 19, 1817-1822. Casali, P. & Notkins, A.L. (1989), Probing the human B cell repertoire with EBV : polyreactive antibodies and CDS+ B lymphocytes. Ann. Rev. Immunol., 7, 513-525. Chow, D.A. & Bennet, R.D. (1989), Low natural antibody and low in vivo tumor resistance in xid-bearing B-cell deficient mice. J. Immunol., 142, 3702-3706. Cote, R.J. & Houghton, A.N. (1985), The generation of human monoclonal antibodies and their use in the analysis of the humoral immune response to cancer, in “Human hybridomas and monoclonal antibodies” (Engleman, E.G., Foung, SK., Larrick, J. & Raubitschek, A.) (pp. 189-210). Plenum Press, New York. Dighiero, G., Lymberi, P., Holmberg, D., Lundquist, I., Coutinho, A. & Avrameas, S. (1985), High frequencies of natural antibodies in fusions of newborn mice. J. Immunol., 134, 765-771. Essex, M., Klein, G., Snyder, P.S. & Harrold, J.P. (1971), Correlation between humoral antibody and regression
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of tumors by Feline Sarcoma Virus. Nature (Lond.), 233, 195-196. Falck, P., Volk, H.-D., Kiowski, S., Neuhaus, K. & Von Baehr, R. (1987), Characterization of mononuclear cells with monoclonal antibodies using cytofluometry. Z. Klin. Med., 42, 2281-2284. Gils, J., Alvarez, R., Vinuela, J.E., Ruiz de Moralez, J.G., Bustos, A., De La Concha & Subiza, J.L. (1990), Inhibition of in vivo tumor growth by a monoclonal IgM antibody recognizing tumor cell surface carbohydrates. Cancer Res., 50, 7301-7306. Grabar, P. (1983), Autoantibodies and the physiological role of immunoglobulins. Immunol. Today, 4, 337-340. Grunow, R., Jahn, S., Porstmann, T., Kiessig, S.T., Steinkellner, H., Steindl, F., Mattanovich, D., Giirtler, L., Deinhardt, F., Katinger, H. & Von Baehr, R. (1988), The highly efficient human B-cell immortalizing heteromyeloma CB-F7. J. Immunol. Methods, 106, 257-265. Hayakawa, K., Hardy, R.R., Honda, M., Herzenberg, L.A., Steinberg, A.D. & Herzenberg, L.A. (1984), Ly-1 B cells: functionally distinct lymphocytes that secrete IgM autoantibodies. Proc. nut. Acud. Sci. (Wash.), 81, 2494-2498. Houghton, A.N., Brook, S.H., Cote, R.J., Taormina, M.C., Oettgen, H.F. & Old, L.J. (1983), Detection of cell surface and intracellular antigens by human monoclonal antibodies : Hybrid cell lines derived from lymphocytes of patients with malignant melanoma. J. exp. Med., 158, 53-65. Jahn, S., Kiessig, S.T., Lukowsky, A., Volk, H.-D., Porstmann, T., Grunow, R. & Von Baehr, R. (1986), Pokeweed mitogen induced synthesis of human IgG and IgM in vitro. Biomed. Biochim. Actu, 45, 467-476. Jahn, S., Grunow, R., Kiessig, S.T., Specht, U., Matthes, H., Hiepe, F., Hlinak, A. & Von Baehr, R. (1988), Establishment of human Ig-producing heterohybridomas by fusion of mouse myeloma cells with human lymphocytes derived from peripheral blood, bone marrow, spleen, lymph node, and synovial fluid. J. Immunol. Methods, 107, 59-66. Jahn, S., Schwab, J., Hansen, A., Heider, H., Schroeder, C., Lukowsky, A., Achtman, M., Matthes, H., Kiessig, S.T., Volk, H.-D., Kruger, D.H. & Von Baehr, R. (1991), Human hybridomas derived from CD-5+ B lymphocytes of patients with chronic lymphocytic leukemia (B-CLL) produce multi-specific natural IgM(kappa) antibodies. Clin. exp. Immunol., 84, 413-417. Laemmli, U.K. (1970), Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227, 680-685. Markwell, M.A. & Foy, C.F. (1978), Surface-specific iodination of membrane proteins of viruses and eukaryotic cells. Biochemistry, 17, 4807-4817.
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HYBRIDOMAS
Murakami, H., Hashizume, S., Ohashi, H. (1985), Shinohara, Y., Yasumoto, K., Nomoto, K. & Omura, H. (1985), Human-human hybridoma secreting antibodies specific to human lung carcinoma. In vitro cell. Dev. Biol., 21, 593-595. Nakamura, M., Burastero, S.E., Notkins, A.L. & Casali, P. (1988), Human monoclonal rheumatoid factor like antibodies from CD5 (Leu-1)’ B cells are polyreactive. J. Immunol., 140, 4180-4186. Olsson, L. & Kaplan, H.S. (1980), Human-human hybridomas producing monoclonal antibodies of predefined antigenic specificities. Proc. nut. Acud. Sci. (Wash.), 77, 5429-5431. Osawa, H. & Diamantstein, T. (1984), Partial characterization of the putative rat interleukin 2 receptor. Europ. J. Immunol., 14, 374-376. Parham, P., Androlewicz, M.J., Brodsky, F.M., Holmes, N.J. & Ways, J.P. (1982), Monoclonal antibodies: purification fragmentation and application to structural and functional studies of class I antigens. J. Immunol. Methods, 53, 133-173. Pfreundschuh, M., Shiku, H., Takahashi, T., Ueda, R., Ransohoff, J., Oettgen, H.F. & Old, L.J. (1978), Serological analysis of cell surface antigens of malignant human brain tumors. Proc. nut. Acud. Sci. (Wash.), 75, 5122-5126. Posner, M.R., Elboim, H.S., Tumber, M.B. & Cannon, T.J. (1990), Human monoclonal antibodies reactive with cell surface antigens on human leukemia cell lines : many antibodies are autoantibodies. Hybridomu, 9, 97-110. Ravetch, J.V., Siebenlist, U., Kormeyer, S., Waldmann, T. & Leder , P. (1981), Structure of the human immunoglobulin my locus : characterization of embryonic and rearranged J and D genes. Cell, 27, 583-591. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989), Molecular cloning. Cold Spring Harbor Laboratory Press, New York. Sands, H. 8c Jones, P.L. (1990) Physiology of monoclonal antibody accretion by tumours. Cancer Treat. Res., 51, 97-122. Settmacher, U., Jahn, S., Siodla, V, Volk, H.-D., Neuhaus, K., Adrian, K. & Von Baehr, R. (1990), Polyclonal stimulation of human B lymphocytes derived from fetal liver and spleen cells at different stages of ontogeny. Immunol. Letters, 26, 159-164. Shoenfeld, Y. Hizi, A., Tal, R., Smorodinski, N.I., Lavie, G., Mor, C., Schteren, S., Mammon, Z., Pinkhas, J. & Keydar, T. (1987), Human monoclonal antibodies derived from lymph nodes of a patient with breast cancer react with MuMTV polypeptides. Cuncer, 59, 43-50. Tron, F. & Bach, J.-F. (1989), Molecular and genetic characteristics of pathogenetic autoantibodies. J. Autoimmun., 2, 3 1l-320.
0 INSTITUT PASTEUR/ELSEVIER Paris 1992
1992,
143, 879-891
Human monoclonal IgM antibodies from foetal B-cell hybridomas directed against a surface antigen on human tumour cells J. Bohn (I), 0. Josimovic-Alasevic c2), U. Settmacher (3), S.T. Kiessig (I), A. Lukowsky cl), H.-D. Volk (I), T. Diamantstein c2), R. Von Baehr (I) and S. Jahn (I) (*) (I) Institute of Medical Immunology Universitiit zu Berlin, and Q) Institute
and ‘3’ Clinic for Surgery, Medical School (Charit&), Humboldtof Immunology, Klinikum Steglitz, Freie Universitiit, Berlin (Germany) SUMMARY
In order to assess the existence of B lymphocytes capable of producing anti-tumour antibodies in non-tumour-bearing individuals, human lymphocytes derived from foetuses and adults were fused with the heteromyeloma cell line CB-F7. By indirect immunofluorescence, 29 out of 4,472 IgM-producing hybridomas (from 8 foetuses and 8 adults) were shown to produce antibodies which bind to colon carcinoma lines Co10205 and SW620, Raji lymphoma cells and small cell carcinoma of the lung. In vitro growth of tumour cells recognized by these antibodies was inhibited. The antibodies also mediated complement-dependent cytotoxicity. All antibodies tested recognized a cell surface molecule of 55 kDa. Southern blot hybridization analysis of hybridoma DNA with a human J, probe showed that the hybridomas were derived from clonally unrelated B cells. These results demonstrate that human foetal and adult B cells from non-tumourbearing individuals are able to produce IgM antibodies recognizing defined cell surface molecules expressed on some tumour cells.
Key-words: B lymphocyte, city, Immunosurveillance.
Tumour, IgM; Hybridomas, Surface antigens, Cytotoxi-
INTRODUCTION
It currently remains controversial as to whether antibodies are involved in tumour surveillance. Antibodies reacting with tumour cells were found to be present in several tumour-bearing patients (Cote and Houghton, 1985 ; Pfreundschub et al., 1978). However, it is not clear whether these antibodies belong to the natural repertoire of non-tumour-bearing individuals or
Submitted
October
20, 1992, accepted November
whether they are the consequence of an immune response to the respective tumour. The view that natural antibodies may be involved in the control of tumour growth is supported by results obtained in animal models (Chow and Bennet, 1989; Essex et al., 1971). However, the antigens recognized by such antibodies have not yet been defined. If natural antibodies play a role in tumour
17, 1992.
(‘1 Address reprint requests to: Dr. S. Jahn, Inst. Med. Immunol., Charit& Schumannstrasse 20/21, Berlin O-1040 (Germany).
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surveillance, then in normal individuals, lymphocytes should exist which have the capacity to produce antibodies reacting with surface molecules of tumour cells. In order to prove this possibility foetaI B cells were immortalized and cloned (Grunow et al., 1988). The resulting monoclonal antibodies (r&b) were tested for their capacity to inhibit growth and to mount complementdependent cytotoxicity in a panel of tumour lines. In addition, attempts were made to characterize the antigens recognized by such antibodies. MATERIALS
Human
AND METHODS
hybridomas
Human lymphocytes from foetal liver and spleen and from adult spleen were fused with the HATsensitive human x mouse heteromyeloma line CBF7, as previously described in detail (Grunow et al., 1988). Organ fragments were derived from foetuses after clinically indicated pregnancy terminations between the 16th and 34th week of gestation because of neural crest defect or Potter’s syndrome (Settmacher et al., 1990). Fusions with lymphocytes derived from adult spleen (partial splenectomy after trauma, portale hypertension) have been described in detail (Jahn et al., 1988). Immunoglobulin production by hybridoma cells has been routinely tested by ELISA (supernatant Ig; Jahn et al., 1986) or immunofluorescence (cytoplasmic Ig ; Jahn et al., 1991) using peroxidase or FITC-labelled antibodies to human Ig heavy and light chains, respectively (Medac; Hamburg, Germany). Tumour
ET AL.
Other cell types Peripheral blood lymphocytes were prepared as described (Jahn et al., 1988). Fibroblast and endothelial cell cultures derived from surgical specimens (non-tumour patients) were kindly provided by Dr. G. Pankonin (Central Institute of Cancer Research, Berlin-Buch, Germany). Erythrocytes were prepared from blood group A, B, AB and 0 donors according to routine methods. Immunofluorescence Tumour cell lines (Raji, Colo205, SW620, H69) were grown in flat-bottom 96-well plates (Falcon, Becton-Dickinson, USA) to confluency. Then, the medium was removed and the plates were incubated with cell culture supernatants for 30 min on ice. After washing (culture medium), a FITC-conjugated antihuman Ig antibody (Dakopatts, Glostrup, Denmark) was added for 30 min. Plates were analysed by fluorescence microscopy. As controls, supernatants were tested for binding to fibroblasts, endothelial cells and erythrocytes. Cytofluorometric analyses were carried out using an “Epics C” cytofluometer (Coultier, Krefeld, Germany) as previously described (Falck et al., 1987). 3H-thymidine
incorporation
assay
Tumour cells (104/well) were cultured for 3 days in the presence of human monoclonal IgM in 96-well plates in RPM1 supplemented with 10 % FCS or 10 Vo heat-inactivated human AB serum. The cultures were pulsed with ‘H-thymidine (Amersham, Brunswick, Germany) for 18 h. The ‘H-thymidine incorporation was assessed by liquid scintillation counting (LS 233, Beckman, Berkeley, CA, USA).
ceils
All tumour cell lines studied were obtained from the American Type Culture Collection (ATCC ; Rockville, USA). Cells were cultured in RPMI-1640 supplemented with 10 070 foetal calf serum (FCS) without antibiotics at 37°C in a humidified 5 Vo CO-,containing air atmosphere. As detected by bisbenzimide-H dye (Serva; Heidelberg, Germany) and microbiological tests, the tumour cells were free of mycoplasmas.
CMC analysis was performed as previously described (Bindon et al., 1988). Normal human serum was obtained from defibrinated blood (group AB) of healthy volunteers. Guinea pig serum was obtained from clotted blood. To remove cytotoxic antibodies, sera were absorbed on the respective human tumour cells. “Cr-1abelled tumour cells were incu-
CMC FCS FITC
HAT mAb PBS
= complement-mediated cytotoxicity. = foetal calf serum. = fluorescein isothiocynonate.
Complement-mediated
cytotoxicity
(CM0
= hypoxanthine, aminopterin, thymidine. = monoclonal antibody. = phosphate-buffered saline.
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bated with human IgM at room temperature for 10 min. Then, the diluted human or guinea pig sera were added and the cells were incubated for 3 h at 37”C, followed by counting the activity in the cell pellet and supernatant in a gamma counter (LKB ; Uppsala, Sweden). Maximal lysis was measured in supernatants of 1 070Triton-X100-lysed cells. Spontaneous release was detected in cell cultures containing medium only. The percentage specific lysis was calculated as follows: experimentalrelease- spontaneousrelease x loo. % ;ypzsific= maximal release - spontaneousrelease
Immunoprecipitation
Colon carcinoma SW620 cells were externally iodinated according to the chloroglycoluril method (Markwell and Fox, 1978). Briefly, 2 x 10’ cells resuspended in 1 ml of Dulbecco’s phosphatebuffered saline (PBS) were added to a glass scintillation vial previously coated with 100 pg of 1,3,4,6-tetrachloro-3a,6a-diphenyl-glucoluril (Iodo, GEN, Rotterdam, The Netherlands). Na12’I (18.5 MBq carrier-free, Amersham, Brunswick, Germany) was added, and after 10 min of gentle agitation at room temperature, the cells were transferred to a tube containing 50 pg of tyrosine and were then washed with D-PBS. For solubilization, cells were suspended in 0.5 ml of a lysis buffer (20 mM TRIS-HCl pH 7.7; 20 mM EDTA, 150 mM NaCl, 1 mM phenylmethylsulphonyl fluoride, 100 KIU/ml aprotinin, 5 Kg/ml soybean trypsin inhibitor, 0.02 % NaN, and 0.5 070Nonidet P-40, Sigma-Chemie; Munich, Germany) and incubated for 30 min on ice. The supernatant, centrifuged at 10,000 g for 15 min, was preabsorbed with l/10 (v/v) rabbit anti-human Ig (Dakopatts, Glostrup, Denmark) coupled to Sepharose beads. The rabbit anti-human Ig-coupled Sepharose beads were allowed to react with culture supernatants containing approximately 3 pg/ml of CB-HTl or CB-HT2 and were used for immunoprecipitation by incubation with the preabsorbed extract for 60 min at 4°C. The immunoprecipitates were washed, extracted with SDS-PAGE sample buffer and analysed by SDS-PAGE under reducing conditions as described (Osawa et al., 1982).
Western blot Tumour cell lysis was carried out by 0.5 % NP40 or alternatively with 3 M KC1 (Parham ef al., 1982). The membrane fraction was spun down at 30,000 g for 2 h. Samples were run on SDS-PAGE (Laemmli, 1970) and transferred to nitrocellulose (SchleicherSchiill, Dassel, Germany). Strips were incubated with
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hybridoma culture supernatants (IgM concentration 1 pg/ml) at 4°C overnight. After washing (PBS, Tween 0.1 070) the POD-labelled goat-anti-humanIgM (see ELISA) was added for 1 h at room temperature. After washing, the reaction was developed by adding 3.3 diaminobencidine containing 0.1 % NiCl, as a substrate. A molecular weight standard marker (Biorad, Richmond, VA, USA) was run in parallel. Southern blot DNA samples were prepared from human hybridoma lines by phenol extraction as described (Sambrook et al., 1989). DNA then were digested with Hind11 or EcoRl and 10 11.8were electrophoresed through 1 070agarose gels, blotted onto nitrocellulose filters and hybridized with a J,-probe, kindly provided by Dr. P. Leder (Stanford, CA; Ravetch et a/., 1981). The gene probe was labelled by nick translation using 32P-d-CTP (Amersham, Brunswick, Germany) and 10’ counts per minute were added to each blot. All gels were run using lambda phage DNA digested with Hi&III as size standards. Blots were washed in 2 x SSC with 0.2 % SDS, 15 min at room temperature, followed by a washing with 2 x SSC at 65°C and a final washing with 0.2 x SSC at 65°C.
RESULTS
Occurrence of anti-tumour-reactive antibodies in hybridoma culture supernatants derived from fusions of human lymphocytes from different immune organs
Lymphocytes from human foetal and adult spleen and foetal liver were fused to HATsensitive heteromyeloma CB-F7 cells. After 21 days, culture supernatants from initial hybri-
doma cell lines were analysed for Ig production by ELISA. Furthermore, the reactivity against different
tumour
cell lines was tested by immu-
nofluorescence. Among 4472 IgM-secreting cell lines, 29 hybridomas secreted antibodies reacting with the tumour cells (table I). Hybridomas producing anti-tumour IgM with comparable frequencies were detected in fusions of both foetal and adult lymphocytes. Taking into account a plating density of 1.5 x lo5 unseparated mononuclear cells per well, comprising 20 % of B cells, one may calculate that nearly l/250,000 fusionable B lymphocytes were able to produce
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ET AL.
Table I. The occurrence of human hybridomas producing antibodies reacting with tumour cells in fusions of lymphocytes from adult or foetal immune compartments. Organ source
Fusions n
Wells seeded
5
1,496 2,424 2,380
Foetal liver Foetal spleen Adult spleen
Cultures containing anti-tumour antibodies
IgM-producing hybridomas 547 2245 1680
2 15 12
Tumour cell lines Colo205, SW620, H69 and Raji were immobilized to 96-well culture plates. Undiluted culture supernatants from primary hybridoma lines were incubated. After washing, a FITC-labelled rabbit anti-human Ig antibody was added. Screening was carried out by fluorescence microscopy. Wells were counted as positive when more than 25 ‘70 of the cells were stained by the respective mAb.
Table II. Reactivity with different cells of natural human IgM mAb derived from foetal and adult lymphocytes. Cells Small cell lung carcinoma:
CB-60 H69 H128
Colon carcinoma : Co10205 SW620 Burkitt lymphoma : Raji Daudi Leukaemia cells : K562 Molt 4 Jurkat Cem Normal cells Fibroblasts Endothelial cells Erythrocytes (blood groups A, B, AR 0) Human PBL
CB-HTl
CB-HT2
CB-HT3
CB-HT4
CB-HT5
-
+ +
+ +
-
-
-
-
+ +
+ +
+ +
+ +
+ +
-
+ +
+ +
+ -
+ +
+ -
-
-
-
+ -
-
-
-
-
-
-
-
-
As a control, an isotype-identical human mAb (CB-60) was used. Analyses were carried out on a cytofluometer Epics C (see also fig. 1).
IgM that binds to tumour cells. All human mAb reacting with tumour cells were found to be of the IgM isotype ; 15/29 expressed lambda and 14 expressed kappa light chains. Five hybridomas were studied in more detail : CB-HTl from a fusion of adult spleen, CB-HT2 and CB-HT3 from a foetal liver (34th week of gestation) cell fusion and CB-HT4 and CB-HTS hybridomas established from foetal spleen lymphocytes (35th
week). The reactivity of the IgM antibodies produced by these hybridomas with human tumour and normal cells was tested by immunofluorescence. All 5 IgM antibodies were found to bind to colon carcinoma cells (Colo205, SW620) and to the Raji Burkitt lymphoma line (table II). Two out of five antibodies also bound to lung small cell carcinoma cells (H69, H128) as well as to Daudi cells. One mAb IgM was found to bind
TUMOUR
ANTIBODIES
FROM
to the K562 erythroleukaemia line. No binding was seen to T-cell leukaemic (Molt4, Jurkat) and normal cells. The cytofluorometry binding pattern of the adult (CB-HTl) and foetal (CB-HT2) hybridoma IgM to Raji and SW620 cells was shown (fig. 1). Between 50 and 70 Vo of colon carcinoma cells and 20 and 40 % of lung tumour cells were stained by the respective mAb.
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Characterization of the antigen bound by human IgM mAb on the tumour cell surface SW620 cells were surface-iodinated, the 125Jlabelled material was extracted, purified by immunoprecipitation by mAb CB-HTl and CBHT2 that bound to rabbit anti-human IgMcoupled Sepharose and analysed by SDS-PAGE.
SW 620
Raji Control
Control
CB-HTl
CB-HT2
Fluorescence
HYBRIDOMAS
Intensity
(log,
o scale)
Fig. 1. Cytofluorometric analysisof natural antibody binding to tumour cells SW620 and Raji.
An isotype-identicalantibody of the sameconcentration (1 pg/ml) served as a control.
J. BOHN
884
Under reducing conditions, a major specific band of about 55 kDa was detected as being recognized by both mAb (fig. 2). Two bands with a high MW of 200 and 185 kDa which were clearly visible in lanes CB-HTl and CB-HT2 also
r
N
F
f
:
8
8
8
kDA
200
-
97
-
66
-
When tumour cell lines SW620 and Co10205 were cultured in the presence of tumour cell binding mAb, after 24 h, adherent cells took on an elongated shape and were arranged in distinct clusters and syncytia (fig. 4). This effect was specific, inasmuch as only mAb showing a positive result in immunofluorescence assays were able to affect tumour growth in vitro. As illustrated in figure 5, the mAb inhibited the 3H-thymidine incorporation rate into tumour cells in a dosedependent manner. Time-course experiments showed that inhibition of tumour cell growth reached a maximum on day 3 after addition of antibodies.
4
43
appeared in the control lane (non-binding antibody CB-92), although at much lower intensity barely visible on the photograph. Western blot analyses (fig. 3 right) performed with the same antibodies showed a band only at 55 kDa, so it was assumed that only this band was specific. Furthermore, it was seen that the different human mAb from the foetal and adult repertoire recognized an antigen with the same molecular weight (MW) of about 55 kDa on one particular tumour cell line (SW620; fig. 3 left). An antigen of the same MW was found on 5 different tumour cell lines with antibody CB-HT2 (fig. 3 right).
Inhibition of tumour cell growth in vitro by mAb
-
116
ET AL.
-
Fig. 2. Autoradiog rarn (SDS-PAGE) of antigens on human tumour cell surfaces recognized by natural antibodies. Extracts from ‘2SI-surface-labelled SW620 cells were immunoprecipitated with CB-HTl or CB-HT2 coupled to sepharose beads or with the non-tumour binding antibody CB-92, reactive with ssDNA. Bound material was analysed by SDS-PAGE under reducing conditions.
Antibody-dependent tumour cell lysis
complement-mediated
The ability of human IgM mAb to permit complement-mediated tumour cell lysis was tested using the 51Cr-release test. Human and guinea pig sera were used as complement sources and showed comparable results. Three antibodies which were previously found to react with tumour cells in immunofluorescence assayspermitted their lysis in the presence of complement (table III). Further experiments showed humancomplement-mediated tumour cell lysis to be dose-dependent (fig. 6).
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885
Fig. 3. Western blot analysisof the binding of natural IgM mAb to different tumour cell lines.
The membranefraction was preparedand run through an agarosegel asdescribed.After blotting to nitrocellulosepaper, (a) different mAb were addedto SW620derived material, or (b) CBHT2 antibody wasincubatedwith nitrocellulosestripswith membranefractions from severalhuman tumour lines.
Immunogenetic analysis of B cells producing natural antibodies reacting with tumour cells
Since several hybridomas which produced antibodies specific for an antigen expressedon the tumour cell surface were derived from the same foetal organ source, we attempted to analyse a possible clonal relationship. HindI and EcoRl-digested hybridoma DNA from 5 cell
lines analysed by Southern blots using the J, probe. Placenta DNA served as a control to show the IgH germline configuration. All five hybridomas exhibited Jn-specific bands of discrete size (fig. 7). Therefore, hybridomas derived from a single fusion (CB-HT5 and CB-HT4 : lanes A and B ; CB-HT3 and -HT2 : lanes C and d) could not be clonally related.
886
J. BOHN
ET AL.
TUMOUR ANTIBODIES
FROM FOETAL
HYBRIDOMAS
887
10
RAJI
DAUDI
SW 620 COLO 205 K 562
Fig. 5. Inhibition of 3H-thymidine uptake in tumour cell cultures by natural IgM mAb. Three days after seeding,tumour cellswere incubated with various concentrationsof the CBHT2 antibody and pulsedwith 3H-thymidinefor 18h. Mean valuesof four independentexperiments are shown. Bars representSEM. As a control, a non-binding IgM antibody CB-60 wasused. Black column: n control ; cross-hatchedcolumn: q 0.25 pg/ml ; white column: 0 0.50 pg/mI ; hatched column: 69 0.75 yg/ml ; large cross-hatchedcolumn: 1.OOKg/ml.
Table III. Complement-mediated tumour cell lysis induced by human natural IgM. Antibody
Molt4
Colo205
SW620
CB-F7 CB-60 CB-HTI CB-HT2 CB-HT3
3 6 7 2 3
2 7 72 32 54
4 3 40 78 43
Vo Specific lysis: Raji H69 0 0 92 59 62
2 5 32 47 8
HI28
Daudi
K562
1 2 21 33 2
2 2 64 67 4
0 1 1 4 65
Antibody concentrations were adjusted to 0.5 Kg/ml. The CB-60 (IgM) and CB-F7 (conditioned medium derived from the Ig-nonproducing fusion line) served as controls. Data from one representative experiment out of 4 independent experiments using guinea pig serum as a complement source were presented. Percent specific lysis was calculated as described in “Materials and Methods”.
DISCUSSION
Antibodies to antigens on tumour cells were detected in sera from tumour patients (Cote and Houghton, 1985; Pfreundschuh et al., 1978). During immortalization of B lymphocytes deriv-
ed from tumour patients, human mAb which bind to tumour cells were obtained (Houghton et al., 1983 ; Murakami et al., 1985 ; Olsson and Kaplan, 1980; Shoenfeld et al., 1987). The human mAb described were either specific and probably reacted with the lymphocyte donor
888
J. BOHN
20
ET AL.
.-
0 0
0.26
0.80
0.70
1.00 pm/ml
Fig. 6. Complement-mediated lysis of Raji cellssensitizedwith different human mAb.
Lysis wasmeasuredby the 5’Cr-releaseassayusingabsorbedhuman complement.Mean values from three independentexperimentswere shown. Spontaneousreleasewas 3.3 f 2.1 %. Vertical bars indicate SEM. Full line: CB-HTI ; broken line, CB-HT2; dotted line: CB-HT3.
tumour cells only (Cote and Houghton, 1985; Murakami et al., 1985), or else bound to a broad panel of tumour and normal cell lines as well as to different autoantigens (Houghton et al., 1983; Olsson and Kaplan, 1980). The functional role of such antibodies in tumour defence of patients, however, remains unclear (for review, see ref. Sands and Jones, 1980). It is of interest to determine whether antibodies against surface antigens expressed on tumour cells appear as a consequence of an antitumoral immune response in patients or whether they exist in the natural repertoire of non-tumour-bearing donors as well, indicating the possible role of natural antibodies in tumour surveillance in man. The latter hypothesis is supported by data from animal models. In xid mice, a relationship between the absence of natural antibodies and tumour incidence has been described (Chow and Bennet, 1989). Furthermore, Essex et al. (1971) estimated a lower in vivo resistenceto feline-sarcoma-virus-induced tumour growth in animals with a decreased level of natural antibodies. A relationship between natural antibodies of the IgM isotype recogniz-
ing tumour cell carbohydrates and mouse strain resistance to the tumour was established (Gils et al., 1990). If natural antibodies take part in tumour surveillance, then lymphocytes should exist in nontumour-bearing individuals with the capacity to produce antibodies reacting with surface molecules of tumour cells. In order to prove this, human foetal B lymphocytes (Settmacher et al., 1990) were fused with CB-F7 heteromyeloma cells (Grunow et al., 1988). Furthermore, hybridization experiments were carried out with adult spleen cells (Jahn et al., 1986). The high efficiency of the fusion technique enabled us to analyse several thousand primary hybridoma lines. Twenty-nine out of 4,479 IgM-producing hybridomas were found to secrete antibodies against the tumour cells. To study such antibodies in more detail, five hybridoma cell lines were established by repeated limiting dilution cloning : two hybridomas from a foetal spleen, two from a foetal liver and one from an adult spleen. As shown by Southern hybridization of the DNA with a human J, gene probe, these hybridomas
TUMOUR
ANTIBODIES
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FOETAL
A
0
889
mldlll
ECORI PI
HYBRIDOMAS
C
D
I
PI
ABCDt
Fig. 7. Southernblot analysisof Hi&III-
andEcoRl-digestedDNA obtainedfrom humanhybridomasproducing natural IgM reacting with tumour cells. DNA samples(10 pg) wereelectrophoresedthrough an agarosegel. Southernhybridization using a J, probe was performed as describedin “Materials and Methods”. A: CB-HT5 ; B: CB-HT4; C : CB-HT3 ; D : CB-HT2 ; E : CB-HTl . P = placenta DNA. Arrowheads indicate the position of HindII-digested lambda phagefragments.
were generated from different maternal B lymphocytes. All antibodies tested reacted with an antigen of 55 kDa expressed on the surface of SW620 cells. Interestingly, an antigen of the same MW could be found on 4 different tumour cell lines with one particular antibody. Although a comparable binding pattern for the human mAb studied was seen on colon carcinoma and lymphoma cell lines, only 2 IgM bound to small cell lung carcinoma cells as well. Only one antibody was seen to bind an antigen on K562 erythroleukaemia cells. This may be explained either by different binding affinities of the antibodies
or by the recognition of different epitopes on the same antigen. No binding of the antibodies to normal cells (fibroblasts, endothelial cells, erythrocytes) could be detected. In conclusion, several findings support the view that such natural antibodies from the foetal repertoire (if also detected in normal individuals) may play a role in tumour defence : i) the antibodies bind to a cell surface molecule common to the tumour cells tested ; ii) binding to such molecules changes the morphology of the tumour cells and inhibits their proliferative capacity ; moreover, iii) in the presence of human
890
J. BOHN
complement, the cells can be lysed. Natural antibodies were at least partially related to the multispecific IgM, reacting with autoantigens and foreign material of a different molecular structure (Dighiero et al., 1985; Nakamura et al., 1988 ; Tron and Bach, 1989). The main role in producing such antibodies is played by CDS-bearing B lymphocytes (Casali and Notkins, 1989 ; Hayakawa et al., 1984). We are currently investigating whether antibodies from the foetal repertoire studied here may bind other antigens. Posner et al. (1990) showed that IgM antibodies produced by EBV-transformed B cells from healthy donors were able to bind to a broad panel of autologous cells and haematopoietic cell lines. However, multispecificity cannot impair the fact that tumour-cell-binding IgM antibodies exist within the natural repertoire of nontumour-bearing individuals. The size of this Bcell pool may only be speculated from fusion data. Therefore, l/250,000 B cells in both the foetal and adult repertoire may produce antibodies reacting with tumour cells. However, this conclusion is of little significance, since only a fraction of the B lymphocytes may be fused, de pending on their in vivo activation state and other unknown factors. Nevertheless, our DNA analysis data suggestthat a heterogeneous B cell pool, rather than a distinct clone, could be responsible for humoral tumour surveillance. Further experiments will be directed toward a more detailed characterization of the antigen bound by mAb from the B-cell repertoire of non-tumour-bearing individuals to human tumour cells. Taken together, present results suggest that, in addition to their antiinfectious (Bos et al., 1989) and autoantigen-clearance (Grabar, 1983) functions, natural antibodies may play a role in tumour surveillance.
Anticorps IgM monoclonaux humains produits par des hybridomes de cellules B faetales et dirigks contre un antigbe de surface de cellules tumorales humaines Dans le but d’ttablir I’existence de lymphocytes B capables de produire des anticorps antitumoraux
ET AL.
chez des sujets non porteurs de tumeur, des lymphocytes humains fcetaux et adultes ont Ctt fusionnts avec la lignie cellulaire CB-F7 httiromyelomateuse. Par immunofluorescence indirecte, 29/4472 hybridomes se sont montres producteurs d’IgM (a partir de 8 f&us et 8 ad&es) se liant aux lignees carcinomateuses intestinales Co10205 et SW620, a la lignee lymphomateuse Raji et aux carcinomes pulmonaires a petites cellules. La croissance in vitro des cellules tumorales reconnues par ces anticorps est inhibee. Ces anticorps provoquent egalement une cytotoxicite dtpendante du complement. Tous les anticorps testes reconnaissent une molecule de surface de 55 kDa. La technique d’hybridation de type Southern avec une sonde J, a permis de montrer que les hybridomes sont derives de cellules B clonales non apparentees. Ces resultats montrent que des cellules B fcetales et adultes de sujets non porteurs de tumeurs, sont capables de produire des IgM reconnaissant des molecules de surface exprimees par certaines cellules tumorales. Mets-cl&: Lymphocyte B, Tumeur, IgM; Hybridomes, Antigenes de surface, Cytotoxicite, Immunosurveillance.
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