Clin. exp. Immunol. (1990) 80, 100-108

Monoclonal antibodies to early pregnancy factor perturb

tumour cell growth

K. A. QUINN, S. ATHANASAS-PLATSIS, T.-Y. WONG, B.E. ROLFE, A. C. CAVANAGH & H. Department of Surgery, University of Queensland, Royal Brisbane Hospital, Brisbane, Australia

MORTON

(Acceptedfor publication 30 October 1989)

SUMMARY The pregnancy-associated substance early pregnancy factor (EPF) has previously been reported as a product of tumours of germ cell origin. More recently EPF (or an EPF-related substance, tEPF) has also been detected in the serum of patients bearing tumours of non-germ cell origin. We report here the production of tEPF by a variety of cultured transformed and tumour cell lines, of both germ and non-germ cell origin. Antibodies specific for EPF remove all tEPF activity from tumour cell conditioned medium. tEPF production is found to be associated with cell division; tEPF is no longer detected after growth arrest or differentiation. Co-culture of tumour cells with increasing doses of anti-EPF monoclonal antibodies resulted in a significant, dose-dependent decrease in rate of cell growth and viability. Similar anti-EPF concentrations had no effect on the concanavalin A induced proliferation of mouse spleen cells. These studies suggest, therefore, that tEPF is a growth-regulated product of cultured tumour and transformed cells. These cells are also dependent upon tEPF for continued growth, i.e. tEPF is acting in the autocrine mode. Keywords early

pregnancy

factor tumour product monoclonal antibodies anti-proliferative

INTRODUCTION The dedifferentiation and reversion to an embryonic state of normal adult cells after neoplastic transformation is well documented (Rosen, Weintraub & Aaronson, 1980, Gupta & Morton, 1983; Fishman, 1987). It has been postulated that the repertoire of genes which give the cancer cell the ability to divide rapidly and to migrate to new tissue sites and prosper there is the same collection of genes that operates under regulatory and differentiation controls of the conceptus (Fishman, 1987). One molecule for which an oncodevelopmental role has been proposed (Whyte & Heap, 1983) is early pregnancy factor (EPF). This suggestion comes from consideration of the conditions in which EPF has been detected in serum; it is present from shortly after fertilization until the later stages of pregnancy (Morton, Hegh & Clunie, 1976; Morton et al., 1977; Morton, Morton & Ellendorf, 1983; Rolfe, 1982) and, in addition, has been found in patients with choriocarcinoma (Mehta & Shahani, 1987) and germ cell tumours of the testis (Rolfe et al., 1983). More recent studies in our laboratory have detected EPF (or an EPF-like substance, tEPF) in the serum of patients with non-germ cell tumours, including carcinoma of the breast and melanoma, suggesting a more generalized linkage of EPF production with the neoplastic state. The expression of the EPF

gene by tumour cells may represent the inappropriate later expression of a molecule of no consequence to the survival of the cell. Alternatively, it may be one further component of the complex and poorly understood system by which tumour cells procure a selective growth advantage over their regulated counterparts (Todaro et al., 1979). In this study we investigated the production of tEPF by a variety of transformed and tumour cell lines in vitro and determined the effect of neutralization of tEPF by specific monoclonal antibodies on the growth pattern of these cells. The monoclonal antibodies used in this investigation have been characterized for their specificity and capacity to neutralize mouse pregnancy EPF in vitro (Athanasas-Platsis et al., 1989). In addition, administration of these antibodies to mice after fertile mating demonstrated that EPF is essential for embryonic survival. Here the same anti-EPF monoclonal antibodies are used to demonstrate a role for tEPF in tumour cell growth. Unique problems encountered in the production of anti-EPF monoclonal antibodies, suggestive of a role for tEPF in hybridoma cell growth, are also described. MATERIALS AND METHODS Purification of EPF Mouse EPF (mEPF) was produced in vitro by culture of oviducts and ovaries from oestrous mice, stimulated to produce EPF by the addition of prolactin (PRL-3; NIH, Bethesda, MD) and medium conditioned by mouse embryos (Cavanagh, 1984). EPF was purified from the culture medium by immunoabsorp-

Correspondence: K. A. Quinn, Department of Surgery, University of Queensland, Clinical Sciences Building, Royal Brisbane Hospital, Herston Qld 4029, Brisbane, Australia.

100

Anti-EPFperturbs tumour cell growth tion, preparative electrofocusing and gel filtration; the purified product appeared homogeneous when analysed by high-performance gel-permeation chromatography. Human EPF (hEPF) was purified from medium conditioned by the choriocarcinoma cell line BeWo (ATCC CCL 98; Cavanagh, 1985). The steps used in the purification were immunoabsorption, gel filtration and reversed-phase high performance liquid chromatography. By SDS-PAGE, the isolated material appeared to be homogenous, running as a single polypeptide of apparent Mr of about 12 000. Preparation of immunoabsorbent Mature, outbred rabbits were injected subcutaneously with four doses, at monthly intervals, of 1-5 pg mEPF emulsified in an equal volume of Freund's adjuvant (complete for the first injection, followed thereafter with incomplete; CSL, Melbourne, Australia). On days 7, 10 and 12 after the final boost, serum was collected, IgG isolated by standard methods (Johnstone & Thorpe, 1987) and coupled to CNBr-Sepharose (5 mg IgG/ml gel; according to the manufacturer's instructions; Pharmacia, Uppsala, Sweden). As a negative control, preimmune rabbit IgG was also coupled to gel. The anti-mEPFcoupled gel (0- 1 ml) was able to remove EPF activity from 0-1 ml of both pregnant mouse and human serum; no alteration in this activity was found after absorption of sera with control gel. Assay for EPF-the rosette inhibition test This assay is dependent on the original finding of Bach & Antoine (1968) that an immunosuppressive anti-lymphocyte serum (ALS) can inhibit rosette formation between lymphocytes and heterologous red blood cells. A modification of the assay was introduced to detect EPF after it was demonstrated that lymphocytes, pre-incubated in EPF, give a significantly higher rosette inhibition titre (RIT) with an ALS than do lymphocytes from the same donor without EPF (Morton et al., 1976). The assay described in this study was performed with spleen cells from outbred, male Quackenbush mice (Central Animal Breeding House; CABH), rabbit anti-mouse ALS and human red blood cells (hRBC); one batch of ALS was used throughout the experiments (Rolfe et al., 1984; Morton, Rolfe & Cavanagh, 1987). For each test, 1 8 x 107 freshly isolated spleen cells were incubated at 370C for 30 min with 0 2 ml test sample, diluted in HBSS containing 0.01% (w/v) bovine serum albumin (BSA). After incubation, the cells were washed twice in HBSS, reconstituted to 1 0 ml in HBSS and used to estimate the RIT of the ALS. A positive (purified mEPF, 5 ng/ml in HBSS/0-01 % BSA) and a negative (HBSS/0-01 % BSA) control were included with each set of tests. With each test sample, the RIT was recorded as the highest dilution of ALS in which the number of rosettes formed between mouse lymphocytes and hRBC is less than 75% of the number formed in the absence of ALS. This dilution is expressed as log2 (reciprocal dilution of ALS x 10-3). In this study an RIT 2 16 was positive for EPF and a RIT < 16 was negative for EPF (see Morton et al., 1987). Cell culture A variety of transformed and tumour cell lines (listed in Table 1) was used in the following studies. Except where indicated, they were cultured under standard conditions in basal medium, either RPMI 1640 (Flow Laboratories, Irvine, UK) or Dul-

101

becco's modification of Eagle's medium (DMEM, Flow Laboratories), supplemented with 10% fetal calf serum (FCS; Flow Laboratories), 20 mm glutamine (Flow Laboratories) and antibiotics (100 yg/ml streptomycin, Sigma, St Louis, MO 100 U/ml penicillin, CSL), at 37°C in an atmosphere of 5% CO2 in air. Monolayers were dissociated, after washing in serum-free medium, by a short exposure to trypsin-versene solution (CSL). All FCS was pretested to ensure the absence of EPF. Samples of conditioned medium (CM), obtained from cells in log phase, were tested for activity in the rosette inhibition test. To verify that the activity detected in CM is initiated by an EPFrelated molecule (tEPF), I-ml aliquots of CM were absorbed by mixing for 30 min at room temperature with an equal volume of immunobilized rabbit anti-mEPF IgG or pre-immune IgG as a control. Ten-fold dilutions of CM, before and after absorption, were prepared and 0-2 ml of each tested in the rosette inhibition test. Results are expressed as the highest dilution of CM giving a positive response in this assay. Time course of tEPF production in relation to differentiation and growth arrest The rat myoblast cell line L6 (see Table 1) differentiates in vitro in response to nutrient depletion and confluency, to form multinucleated and striated muscle fibres (Yaffe, 1968). These changes in morphology can be observed 7- 10 days after plating; a non-fusing variant L6 line was used as a control in these studies. Both the fusing and non-fusing L6 lines were seeded at 105 cells/ml in 30 ml DMEM + 10% FCS, and confluence was reached within 1-2 days. From this time, aliquots of medium were removed every 24 h and tested in the rosette inhibition test to determine the effect of differentiation on the production of tEPF by these cells. The effect of mitomycin C-induced growth arrest on the production of tEPF by the bovine kidney cell line MDBK (see Table 1) was determined. MDBK cells (104) were seeded in 2 ml DMEM + 10% FCS alone or containing 2 5 ug/ml mitomycin C, and incubated overnight at 37°C (Freshney, 1983). Five cultures in each group were set up. After treatment, all cultures were washed three times with warm DMEM and further incubated for 72 h fresh in DMEM + 10% FCS. Two cultures from each group were pulsed for the last 16 h of the culture period, with 0-5 ,Ci methyl-3H-thymidine 5'-triphosphate (3Hthymidine; Amersham International, Amersham, UK) to ensure that growth in the mitomycin C cultures was arrested. CM was removed from the remaining cultures (triplicate treated and untreated), viability of cells assessed by trypan blue exclusion and CM tested in the rosette inhibition test.

Preparation of monoclonal antibodies Immunization. Adult male BALB/c mice aged 5-6 weeks (CABH) were immunized as follows for the preparation of antiEPF monoclonal antibodies: Schedule 1: Initially mice were injected intraperitoneally monthly for 3 months, with 50-100 ng hEPF in Freund's adjuvant. A final booster shot was administered to each mouse intravenously in saline, 1 month later, 3 days prior to fusion (Galfre & Milstein, 1981). Schedule 2: As more EPF became available, mice were immunized with more realistic amounts of antigen (5-20 yg/ injection) until antibody titres were obtained such that a 1/1000 dilution of serum gave significant binding to hEPF in a solid-

K. A. Quinn et al.

102

Table 1. Titres of tEPF detected, by the rosette inhibition test, in tumour and transformed cell conditioned medium (CM) tEPF titre

Origin/type

Untreated

Absorbed with rabbit anti-mEPF

BeWo MOLT 4 L6 L6 HeLa T24 MDBK CHO-KI

Human choriocarcinoma Human T cell leukaemia Rat myoblast fusing Rat myoblast non-fusing Ca, cervix Human bladder ca Bovine kidney Chinese hamster ovary

108 108 106 106 108 108 108 104

Neg. Neg. Neg. NT Neg. NT Neg. NT

P3X63-Ag8

106

NT

106

Neg.

ATCC CRL158D

106

NT

ATCC TIB18

SP2/0-Ag-14 BALENTL-3

Mouse plasmacytoma, IgG I -secreting Mouse plasmacytoma, non-secreting Mouse plasmacytoma, non-secreting Mouse plasmacytoma Mouse T lymphoma

ATCC CCL98 ATCC CRL1582* ATCC CRL1458* variant of ATCC CRL1458* ATCC CCL2* ATCC HT84 ATCC CCL22 gift from J. R. E. Wells ATCC CCL61 Department of Biochemistry, Adelaide University, Australia ATCC TIB9

106 108

Neg. Neg.

MCA-2

Mouse fibrosarcoma

108

Neg.

B16

Mouse melanoma

108

Neg.

F9 NG2

Mouse embryonal ca Mouse embryonal ca Human melanoma Human fibrosarcoma Human ca, oesophagus

106

NT NT NT NT NT

ATCC CRL1581 Gift from A. Harris, Walter & Eliza Hall Institute, Melbourne, Australia Koppi & Halliday, 1983 Gift W.J. Halliday, Department of Microbiology, University of Qld, Australia Fidler, 1970 Gift, Department of Microbiology, Flinders University, Adelaide, Australia Strickland & Mahdavi, 1978* Cotton, 1982* Medium conditioned by these cell lines Obtained from S. Rosen, NIH, Bethesda, Maryland, U.S.A. Rosen et al., 1980

Cell

P3X63-Ag8.653

P3/NS/l-Ag4-1

> 10l > 10l > 10l > 10l

Source of cell line

tEPF titre, highest dilution of CM giving positive in rosette inhibition test, i.e. RIT 2 16. > 10', CM tested 1/10. * Gift from J. Wiedeman, CSIRO Meat Research Laboratory, Brisbane, Australia. EPF, early pregnancy factor; ca, carcinoma; Neg., negative; NT, not tested.

phase radioimmunoassay (RIA) (see below). Spleens from mice meeting these criteria were fused with myeloma cells as described below, 3 days after a final i.v. boost. Fusion. Spleen cells (108) from selected mice were fused with 107 mouse myeloma cells (P3X63-Ag8.653) following the standard method of Galfre & Milstein (1981) and Lane, Crissman & Ginn (1986). The fusing solution used was 50% polyethylene glycol (mol.wt 4000; Merck, Darmstadt, FRG) w/v in RPMI. In the hope of increasing the stability of anti-EPF-producing hybridomas, fused cells from the second immunization schedule were seeded into selection medium HAT (Flow Laboratories), supplemented with 50% medium conditioned for 48 h by the parent myeloma cells; as well as producing tEPF, these cells are also a rich source of IL-6 (Sugasawara, 1988). Hybridoma supernatants treated to remove EPF. Preliminary experiments demonstrated production of tEPF by both myeloma and hybridoma cell lines including those producing antiEPF antibodies. In these experiments, supernatant medium (I 0 ml) was acidified to pH 2 5 by the addition of 25 ,ul 1 M HCl

(to separate antigen-antibody complexes), mixed at 4°C for 30 min and passed through a C,8 Sep-pak cartridge (Waters Assoc.), activated according to manufacturer's instructions. The flow-through fraction, containing immunoglobulin, was collected and pH raised to 8-0 with solid Tris. EPF bound to the cartridge was eluted with 5 ml 80% acetonitrile (v/v) in PBS. Both bound and unbound fractions were dialysed against PBS overnight at 4°C, then tested, with the supernatant before treatment, for tEPF in the rosette inhibition test and for immunoglobulin by RIA. As a result of the findings in these preliminary experiments, all supernatants were treated to remove any tEPF, free or bound to antibody, before testing immunoglobulin for anti-EPF specificity. Screening hybridomas for immunoglobulin production. Mouse immunoglobulin was detected by solid-phase RIA using the streptavidin-biotin system as described by the manufacturer (Amersham). Supernatants giving counts > 5 times that obtained with culture medium alone were selected for further testing.

103

Anti-EPFperturbs tumour cell growth Screening hybridomas for anti-EPF specificity. Treated hybridoma supernatants were initially screened for capacity to neutralize mouse pregnancy EPF in the rosette inhibition test (Athanasas-Platsis et al., 1989). The hEPF affinity of antibodies in selected supernatants, from the first and second immunization schedule, were compared by RIA. Immunoradiometric assay (IRMA). Due to low affinity of anti-EPF monoclonals obtained from the first immunization schedule, a two-site modification of IRMA (Johnstone & Thorpe, 1987) was used to confirm specificity of selected positive wells. Immunoglobulin in treated supernatants (0 2 ml) was bound to polystyrene tubes, precoated with sheep anti-mouse immunoglobulin (Silenus, Melbourne, Australia), then incubated overnight at 40C with hEPF (0 1 ml, 1 0 Mg/ml in 0 05 M sodium phosphate buffer, pH 7 4/0-2% w/v gelatin; diluting buffer). Next morning, rabbit anti-mEPF IgG (as described in preparation of immunoabsorbant; 0 1 ml, 10 pg/ml in diluting buffer) was added to the washed tubes (3 h, 37 C), followed by donkey anti-rabbit IgG, F(ab')2biotin/'251-streptavidin (Amersham), according to the manufacturer's instructions. Control tests consisted of anti-EPF mouse serum from immune spleen donors (1/1000 in diluting buffer; positive) and blank CM (negative). A result > 2-5 times that with culture medium alone was considered positive for anti-EPF (Goding, 1986). Solid-phase RIA. Hybridomas from the second, more rigorous immunization schedule produced higher affinity anti-EPF monoclonals which could be detected in a solid-phase RIA (Johnstone & Thorpe 1987), with hEPF on the solid phase. Tubes were coated with hEPF (1 ug/ml in 0-05 M sodium carbonate/bicarbonate buffer, pH 9.6) overnight at 4°C, followed by treated supernatants (0-2 ml). After 3 h incubation at 37°C, bound mouse immunoglobulin was detected with sheep anti-mouse IgG, F(ab')2 biotin/'251-streptavidin (Amersham) following the manufacturer's instructions. Controls as in IRMA were included. A result > 5 times that of culture medium alone was considered positive for anti-EPF. Screening selected clones for immunoglobulin class and subclass. Immunoglobulin with anti-EPF specificity was tested for class and sub-class by solid-phase RIA as previously described, with various class and sub-class antisera (Nordic, Tilburg, The Netherlands) on the solid phase. Production of monoclonal antibodies.Following identification of positive wells, the selected hybridomas were cloned to monoclonality by limiting dilution. Stable cell lines were then grown in vivo as ascitic tumours in BALB/c mice (CABH) for the production of antibodies (Goding, 1986). Purification ofmonoclonal IgM. Ascitic fluid was adjusted to pH 2-5 with HCl and applied to a C18 Sep-pak (see above); IgM was then precipitated from the flow-through fraction during dialysis against 5 mm Tris-HCI buffer 7.5, at 4°C overnight (Goding, 1986). Purity of the antibody preparations was monitored by SDS-PAGE (Gorg et al., 1981) and protein concentration determined (Lowry et al., 1951). Before use in cell culture experiments, the antibody concentration was adjusted to 1 mg/ml and the preparation extensively diaysed against DMEM.

Co-culture of cells with anti-EPF monoclonal antibody Two murine tumour lines, a fibrosarcoma (MCA-2) and the B 16 melanoma (see Table I), in addition to the rat L6 myoblast line, were studied. The cells MCA-2 (103), B16 (0-5 x 103) or L6 (103)

were seeded in triplicate, in 0-2 ml culture medium (DMEM +

10% heat-inactivated FCS) containing doses of anti-EPF monoclonals 7/342 or 5/341, or control antibody 7/331, in the range 50-500 ,g/ml (final concentration). Cells were similarly seeded into medium containing no antibody. Four sets of cultures were established and one set examined each 24 h; viability was assessed by trypan blue exclusion and uptake of 3H-thymidine was used as a monitor ofcell division (as follows). After each 24 h period, 0 5 yCi 3H-thymidine was added to each well of one complete set. After a further 16-h incubation, 20 p1 0 1% trypan blue (v/v) in PBS was added to the pulsed wells and the viability of the cells determined as the percentage of cells excluding the dye. The supernatant medium was then removed, the cells washed twice with ice-cold DMEM and precipitated with 250 pl ice-cold 5% TCA (Plate, 1974). The precipitate was washed twice with TCA and solubilized in 0 3 ml 0 25 N NaOH; 0-25 ml of this preparation was mixed with 2 ml scintillation cocktail (Optifluor, Packard) and ct/min incorporated into acid precipitable material were determined for each well. Relative 3H-thymidine uptake for each antibody dose was calculated by expressing the mean ct/min incorporated (from triplicate wells) as a percentage of the average ct/min incorporated in the wells containing no antibody. Mouse spleen cells (5 x 105) were seeded in triplicate in medium (0 2 ml RPMI + 5%heat inactivated FCS) (i) alone; or (ii) containing 12.5 Mg/ml concanavalin A (Con A; Boehringer, Mannheim, FRG); or (iii) 125pg/ml Con A+350 pg/ml antiEPF 7/342. After 72 h incubation at 37°C, each well was pulsed for 6 h with 0 5 pCi 3H-thymidine and cells were harvested onto filters, from which the uptake of isotope was determined by liquid scintillation counting. Data are expressed as the mean ct/min incorporated by the triplicate wells of each test group. RESULTS tEPF is a product of neoplastic cells in vitro tEPF was detected in CM from all transformed and tumour lines investigated (Table 1). This tEPF, like EPF in pregnant mouse serum, was completely removed from CM by absorption with immunobilized rabbit anti-mEPF IgG. The production of tEPF by myeloma cells and its continued production after hybridoma formation proved to be of major significance in the production and screening of the anti-EPF producing hybridomas.

Production ceases after differentiation and growth arrest Eight days after plating, the cells in the fusing L6 cultures were aligned in parallel, with many fused cells evident. After this time differentiation proceeded rapidly with large striated muscle fibres appearing within 24 h. With the progression of differentiation there was a rapid drop in tEPF titre (Fig. 1). No differentiation occurred in the non-fusing cultures which maintained tEPF titres of 106 throughout the culture period. The production of tEPF by the MDBK cell line ceased after inhibition of DNA synthesis by treatment with mitomycin C. The tEPF titre of CM from triplicate cultures of growing cells (mean 3H-thymidine uptake 46 000 ct/min, viability > 95%) was 108. CM from triplicate cultures of mitomycin C treated cells (mean 3H-thymidine uptake 626 ct/min, viability >95%) all tested negative for tEPF in the rosette inhibition test.

K. A. Quinn et al.

104

Table 2. Separation of tEPF from mouse immunoglobulin (mlg) in hybridoma supernatants

mIg titret

tEPF titre*

Sep-Pak fraction

myeloma

Starting material Sep-pak unbound Sep-pak bound

Neg. 106

106

hybridoma 7/342 5/341

7/331

myeloma

Neg. Neg. Neg.

104 Neg.

104

106

Neg.

Neg.

lO,

lo,

lo,

hybridoma 7/342 5/341

7/331

103

103 103

lo,

104 104

Neg.

Neg.

Neg.

* Highest dilution giving positive in rosette inhibition test, i.e. giving RIT 2 16. t Highest dilution giving positive in mIg-specific RIA, i.e. giving > 5 x background ct/min.

a_

Lu

Differentiation complete

neg.

2

3

4

5 6 7 8 9 Days after plating

10

11

12

13

Fig. 1. With the progression of differentiation in the L6 fusing cultures (0) there was a rapid drop in tEPF titre (0, undiluted). No differentiation occurred in the non-fusing cultures (A) and the tEPF was constant throughout the experiment. The experiment was carried out in triplicate. (Neg., negative).

Screening and preparation of monoclonal antibodies Results of testing hybridoma supernatant for tEPF and immunoglobulin, before and after treatment designed to separate tEPF (bound to mouse immunoglobulin and free in solution) from mouse immunoglobulin are shown in Table 2. Before treatment supernatants were positive for free tEPF (starting material) in the rosette inhibition test, but after passage through the Sep-pak no free tEPF remained in the flow-through fraction; tEPF was eluted from the cartridge by 80% acetonitrile. Immunoglobulin did not bind to the Sep-pak; mouse immunoglobulin titre of the flow-through fraction was identical to that of the starting material and no mouse immunoglobulin was detected in bound fractions. From these results the Sep-pak technique was deemed to be an efficient way of separating immunoglobulin from tEPF, enabling the hybridoma supernatants to be screened for anti-EPF specificity. Initially, seven fusions were carried out using the first immunization schedule and approximately 18% of the wells were found to be positive for anti-EPF immunoglobulin by the rosette inhibition test. The hybrid cells in positive wells were cloned, assayed and recloned; active anti-EPF producing clones were identified and all were found to produce immunoglobulin of class IgM. Selected clones were checked for specificity by testing against hEPF in the IRMA (Fig. 2). Two clones, 7/342 and 5/341, from different fusions, were established as stable cell lines. A third clone, 7/331, producing an irrelevant antibody, not of anti-EPF specificity (control IgM), was also cloned to a stable cell line.

rOOO

6s 500 F

I II

I II

Control Medium

2

3

4

5

l

*

II

6 7 5/341

8

9 7/342

Fig. 2. Treated hybridoma supernatants, derived from the first immunization schedule, were screened for anti-EPF immunoglobulin by IRMA. *Supernatants with ct/min > 2 5 times culture medium were considered positive for anti-EPF immunoglobulin. Each value is the mean of triplicate results.

The second immunization schedule resulted in a higher specific fusion efficiency. After one fusion, supernatants from 86% of the initial 48 wells into which the fused cells were seeded tested positive for anti-EPF in the rosette inhibition test. As shown in Fig. 3, only four of these showed significant binding to hEPF in a RIA. These were found to be producing IgG. Attempts to establish these hybridomas as stable monoclonal

105

Anti-EPFperturbs tumour cell growth 3000

(-a)

120

I

2000 H

.E

100

i

60

20

I

m

m

2B 2 2A

V 1

-

40 _i

*

1

-

80 _-,

-

lI

50

m

m

I

m

100

200 250 Monoclonal antibody (,ug/ml)

500

m

D 3 2A 3 2A 4 2B 4 Donor mouse

Q)

serum

Fig. 3. Treated hybridoma supernatants, derived from the second immunization schedule, were screened for anti-EPF immunoglobulin by solid-phase RIA. *Supernatants with ct/min > 5 times those obtained with culture medium were considered positive for anti-EPF immunoglobulin. Each value is the mean of triplicate results.

a) 52)

cell lines have failed; the cloning efficiency of these hybridomas ranged between I and 4% on the first cloning and, to our dismay, immunoglobulin production in clones from all four ceased soon after. Subsequent fusions have yielded similar results. More recent immunization schedules with increased amounts of EPF have resulted in much higher serum anti-EPF titres in the spleen donor. However, the increased affinity and quantity of immunoglobulin produced by the newly fused hybridomas was associated with decreased stability. To date no anti-EPF IgGproducing hybridomas have been established. Tumour cell growth is perturbed by co-culture with anti-EPF monoclonal antibodies Incubation of MCA-2, B 16 and L6 cells in increasing concentrations of anti-EPF monoclonal antibodies resulted in substantial inhibition of cell division and increased cell death (Fig. 4). After 24 h of incubation the rate of cell division was significantly reduced with 50% inhibition of maximal 3H-thymidine uptake (i.e. uptake of cells incubating in the absence of antibody) occurring at 0 16 gM for both anti-EPF monoclonal antibodies with MCA-2 cells, at 0-2 gm for 7/342 anti-EPF and 0 5 4UM for 5/341 anti-EPF with B16 cells and at 0 4 lM for 5/341 with L6 cells. After 96 h, there was a dramatic change in the morphology of the cells incubated with the higher anti-EPF concentrations: the majority of the cells were detached and clumping, as shown in Fig. 5a, which contrasts the appearance of the MCA-2 cells after 96 h incubation with anti-EPF 7/342 with cells incubated in the control antibody 7/331. This change was reflected in a decrease in cell viability and rate of 3H-thymidine uptake (Fig. Sb). The deleterious effect of tEPF neutralisation on tumour cell growth has been very reproducible; similar results have been obtained on three separate occasions with three different preparations of both anti-EPF monoclonal antibodies.

Monoclonal antibody

50

(,ug/ml )

100 200 250 Monoclonal antibody

(,ug/ml) Fig. 4. Anti-EPF monoclonal antibodies inhibit the growth of tumour cells in vitro. The proliferation of the tumour cell lines (a) MCA-2, (b) B16 and (c) L6 was inhibited in a dose-dependent fashion after 24 h incubation in the anti-EPF monoclonals 5/341 (-) and 7/342 (0). Similar concentrations of the control antibody 7/331 (X) had no effect. Each point represents the mean ( ± s.d.)of triplicate wells. Proliferation was assessed by the uptake of 3H-thymidine by the cells, expressed as percentage of 3H-thymidine uptake without antibody.

In contrast, the addition of a tumour growth inhibitory dose of anti-EPF had no effect on the Con A induced proliferation of primary spleen cells. The proliferative response, monitored by 3H-thymidine uptake, in the presence of 500 Mg/ml anti-EPF 7/342 (mean uptake 128 715 + 857 ct/min), was not significantly

.&M:F------.......,.=.,..,.":.Q.:.

120

120

00

100

*' 80 CDC 4

80

ET

t60=

-__60

40t

-40 20

120

50

100

150 200 250

500

Monoclonal antibody (p g/mI) Fig. 5. (Top) The appearance of MCA-2, cells, incubated in increasing concentrations (from top, 100, 250 and 500 pg/ml) of anti-EPF 7/ 342 (left) for 96 h, is shown in contrast to those incubated in the control antibody 7/331 (right). In the diagram, this change is reflected in a decrease in cell viability (U) and proliferation (0). Each point represents mean + s.d. of triplicate wells. Proliferation was assessed by the uptake of 3H-thymidine by cells, expressed as percentage of 3H-thymidine uptake without antibody.

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Anti-EPF perturbs tumour cell growth different from that of spleen cells cultured without antibody (mean uptake 128 533 + 13 000 ct/min). In the absence ofCon A, the proliferation of the cells cultured either with or without antibody was not significant (mean uptake 870 + 132 ct/min). DISCUSSION EPF was first defined as a pregnancy associated substance present in the serum of mice within 6 h of fertile mating (Morton et al., 1976) and subsequent studies detected EPF in a wide range of species within 48 h of both fertile mating and embryo transfer (reviewed by Morton et al., 1987). Studies presented here have confirmed that EPF production is not confined to pregnancy and that a wide variety of tumour and transformed cell lines in culture produce an EPF-like substance, tEPF. The two molecules tEPF and pregnancy EPF are apparently closely related, with a number of similarities evident. The most esoteric of these is the ability of both these molecules, upon binding to a subset of lymphocytes, to stimulate the production of suppressor factors by which the effect of EPF in the rosette inhibition test is mediated (Rolfe et al., 1988, 1989). That the two molecules share common determinants is suggested by the finding that immobilized rabbit anti-mEPF IgG, able to remove EPF from both human and mouse pregnancy serum, absorbed tEPF from CM. The most convincing evidence that tEPF and the EPF detected in mammalian pregnancy are closely related emerges from experiments of Athanasas-Platsis et al. (1989) with the two anti-EPF monoclonal antibodies (7/342 and 5/ 341), found here to inhibit tumour cell proliferation. These experiments showed that administration of the antibodies to female mice after fertile mating resulted in the failure of the mice to maintain their pregnancies. Studies are currently underway to characterize at the molecular level the true relationship of tumour and pregnancy-derived EPF and the species bound by the two monoclonal antibodies. Consistent with the hypothesis that EPF is a product of dividing, primitive cells, tEPF production ceased after the myogenic cell line L6 underwent differentiation and after the growth of MDBK cells was arrested by mitomycin C. The antiproliferative effect ofculturing the cell lines MCA-2, B 16 and L6 in increasing doses of the anti-EPF monoclonals 7/342 and 5/341 is evidence that these cells have an active requirement for the continued presence of tEPF. In this situation it appears tEPF is acting in the autocrine mode, i.e. it is being produced by cells which are simultaneously capable of responding to the same molecule. What the response of these cells is to tEPF has yet to be determined, but these studies have established that there is a requirement. Autocrine mechanisms of growth stimulation in transformed cells have been well documented. They are thought responsible for the relaxed cell cycle control and growth factor independence of these cells (Heldin & Westermark, 1984). Some of the identified transforming growth factors also play a role in the regulation of growth of normal diploid cells but the action of these are strictly regulated by exogenous controls. Whether tEPF is involved in the growth of normal cells remains to be clarified. Certainly culture of Con Astimulated spleen cells, in the presence of a tumour growth inhibitory concentration of anti-EPF, had no effect on the proliferation of these primary cells. Much additional work is needed to elucidate the role of tEPF during normal cell division and propagation.

The effectiveness of the two IgM anti-EPF antibodies in limiting the proliferation of the three tumour lines investigated suggests a reason for the difficulty encountered in the maintenance of hybridomas producing high affinity anti-EPF monoclonals. The production of tEPF by hybridoma cells has been confirmed. The production of antibody specific for a molecule intrinsically linked to the cell's proliferation would create a stressed environment in which the cessation of production would be of ultimate advantage to the survival of the cell population. This may be the reason for the increased instability of the anti-EPF-producing hybridomas. Attempts were made to stabilize these hybridomas by providing an exogenous source of both tEPF and the hybridoma growth factor IL-6 (Sugasawara, 1988) in the form of myeloma CM. However, this strategy was largely unsuccessful, presumably due to insufficient amounts of tEPF. Anti-EPF-producing hybridomas were no longer present after cloning and the long-term culture of uncloned hybridomas resulted in the overgrowth of non-producing cells. Strategies such as this, requiring a substantial amount of tEPF, may have to wait until recombinant material is available. In this respect, the success of Matsuda, Hirano & Kishimoto (1988) in maintaining anti-IL-6-producing hybridomas by supplementation with recombinant IL-6 is encouraging. Taken together, the preceding studies have established that the growth of tumour and transformed cells in culture is dependent upon tEPF. However, in vivo, tEPF may have a further role. As mentioned previously tEPF, like pregnancyderived EPF, is able to bind to lymphocytes and stimulate production of suppressor factors that mediate response in the rosette inhibition test. These suppressor factors, shown to suppress cellular immune responses such as the adoptive transfer of contact sensitivity in mice, have been detected in the serum of mice bearing transplantable tumours (unpublished data). It is interesting to speculate the consequences of induction of these suppressor factors to the tumour-bearing animal. In an analogous situation to pregnancy and the concept of the fetal allograft, these factors may be facilitating the survival of the neoplasm by mechanisms which disarm or modify the host's immunological capacity to recognise and eradicate tumour antigens. The induction of suppressor factors by tEPF, in combination with its apparent role in cell growth, reinforces the notion that tEPF production by the neoplasm is of intrinsic importance to its survival.

ACKNOWLEDGMENT This work was funded by the Queensland Cancer Fund.

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CAVANAGH, A.C. (1985) Purification and partial characterisation of EPF. In Early Pregnancy Factors (ed. by F. Ellendorf & E. Koch) p. 179. Perinatology Press, New York.

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Monoclonal antibodies to early pregnancy factor perturb tumour cell growth.

The pregnancy-associated substance early pregnancy factor (EPF) has previously been reported as a product of tumours of germ cell origin. More recentl...
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