THIS INVENTION relates to antagonists and antibodies to growth factor receptors and is particularly concerned with antagonists and antibodies to bombesin receptors. The amphibian tetradecapeptide bombesin (6) and mammalian peptides structurally related to bombesin, which include gastric releasing peptide (GRP) and the neuromedins (7 to 12) are growth factors which are believed to be implicated in the control of cell proliferation. Bombesin-like peptides are present in high concentrations in small cell lung carcinoma (18 to 21) where they could act as autocrine growth factors (22) . The bombesin group of peptides interact in the cell with receptors but while a certain amount is known about the cheπiistry of the bombesin group of peptides, very little is Known about the chemistry of the receptors of the bombesin-like peptides,. In view of the involvement of the bombesin-like peptides in cell growth and the implications on cell growth of the presence or absence of bombesin/receptor interactions, a detailed study of the receptors is clearly of importance.

We have, now developed methods that have enabled us to identify certain receptors to certain peptides of the bombesin family which enables them to be characterised as new compounds and it is with such receptors and,

additionally, to antagonists and antibodies to such receptors, that the present invention is directed. The bombesin family of peptides do have structural differences from one another but also have a common 7-amino acid sequence and we believe that the receptor we have identified is capable of acting as receptor to various -members of the bombesin-family, regardless of the species of origin of the bombesin-like peptide.

In one aspect, the present invention provides a polypeptide having the following characteristics:

1. It is a single chain glycopolypeptide, having at least two mannose side chains.

2. It binds selectively with polypeptides of the bombesin type. 3. It has a molecular weight of 75 to 85 Kilodaltons (Kd) .

4. L. -'It has an isoelectric point of 6.4 to 6.9.

5. Its core protein, obtained using endo-beta-N- glucosaminidase from Flavobacterium meningosepticu , has a molecular weight of about 42Kd.

6. It binds with Antagonist A and Antagonist D, both as hereinafter defined.

In a further aspect, we provide antagonists to the glycopolypeptide receptors as defined above. These antagonists are substances which are structurally quite different to bombesin and the bombesin-like peptides but

which can bind to the bombesin receptor, and inhibit the effects of bombesin, but we believe, not by occupying binding sites that would otherwise be occupied by the bombesin-like peptides. Since bombesin-like peptides have been identified as present in high concentration in small cell lung carcinoma (18-21) , and may act as autocrine growth factors (22), bombesin antagonists ^" "a e of interest in providing a means of interfering with the receptor/bombesin peptide interaction and hence cell growth patterns influenced by the bombesin-like growth factor. We have now shown that an antagonist of bombesin that we have identified can inhibit the growth of human small cell lung cancer (SCLC) lines and is therefore of interest as a promising therapeutic entity. Antibodies against the receptor are of similar interest in that they too can occupy sites on the receptor that might otherwise be occupied by bombesin-like peptides and similarly, may be able to influence the cell growth pattern. So-called Substance P is an 11-mer neuropeptide, of interest in studies in pain transmission, which has the formula:

Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH ₂

We have found that a commercially available structural variant of Substance P, known as [D-Arg , D-Pro 2, D- Trp7'9- Leu11] Substance P and hence of the formula:

)-Arg-D-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-Leu-Leu-NH ₂

which we call Antagonist A and which is also known as

2

[D-Pro jspantide, is, surprisingly, able to act as a bombesin antagonist. Antagonist A is therefore of interest, according to the present invention, in the modification of cell growth influenced by the presence of bombesin-like peptides.

We have also found that a further commercially available structural variant of Substance P, which we call

5 Antagonist D, which is also known as [D-Phe ] spantide and which has"the formula:

D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Leu- H ₂

is even more potent as a bombesin antagonist than Antagonist A and hence is also of-great interest for the modification of cell growth influenced by the presence of bombesin-like peptides.

Our -studies of Antagonists A and D indicate to us the importance of structural variation at amino acid

position 5 in compounds of this type to produce improved bombesin antagonists and the present invention extends to new compounds which are amino acid position 5 variants on Antagonist A and Antagonist D and to such position 5-variants of Antagonist A and Antagonist D for use in method of treatment of the human or animal body by therapy or in a method of diagnosis carried out on the human or animal body.

Suitable position 5 variants are:-

D-Trp, D-Tyr and Me-Phe

A further feature of the present invention comprises antibodies against the bombesin receptors. The antibodies can be raised against the receptor by conventional methods which include the step of injecting a vertebrate with the polypeptide receptor of the invention as immunαgen and can be polyclonal antibodies or monoclonal antibodies.

Polyclonal antibodies against the receptor can be raised by conventional methods involving immunisation of an animal with the receptor molecule in immunogenic form followed by recovery of polyclonal antibodies from blood fragments of the immunised animal.

Monoclonal antibodies against the receptor can be generated by conventional procedures involving the

immunisation of test animals such as mice with the receptor in im unogenic form followed by hydridisation of spleen cells from the immunised mice with myeloma cells to give hydridoma cell lines secreting monoclonal antibodies against the receptor.

The polyclonal or monoclonal antibodies generated in these ways can be purified by affinity chromatography against a solid phase carrying receptor bearing cells, for example, by a procedure which include the steps of bringing the antibody into contact with a solid phase bearing a polypeptide receptor of the invention or bearing Swiss 3T3 cells carrying on their surface a polypeptide receptor of the invention to form an antibody/antigen conjugate and releasing purified antibody from the conjugate.

The antagonists or antibodies of the invention may be formulated with pharmaceutically acceptable carriers"-or diluents e.g. conventional parenteral carriers so that the antagonists-^or antibodies can be administered parenterally where they are of interest for use in a method of treatment of the human or animal body for diagnosis or therapy, and more specifically in the diagnosis or therapy of cancers where uncontrolled cell growth is associated with disorders of proteins of the bombesin family. The antagonists and antibodies are also of interest for use in the production of a medicament for the treatment of uncontrolled cell proliferation.

The antagonists or antibodies of the invention are also of interest for use in the _in vitro diagnosis of uncontrolled cell proliferation by a method which includes the step of bringing the antagonist or antibody into contact with a body sample from a host suspected of suffering from uncontrolled cell proliferation. The method is*particularly useful in the diagnosis of cancers by histology or serum assay.

The isolation and molecular characterisation of the receptors of the invention requires a procedure for their identification and we have developed a system involving the use of certain bifunctional cross-linking reagents which have already been used to identify membrane receptors in other systems (23 to 28) . We have used the cross-linking agent ethylene glycol bis(succinimidyl succinate) to link covalently 125I labelled gastrin

"\ releasin ''peptide ( 125I-GRP) to a surface protein in

Swiss 3T3 cells. This surface protein, when isolated from the Swiss 3T3 cells, displays the characte istics of a specific receptor for the peptides of the bombesin family. This protein was not present in other cell lines which do not exhibit receptor properties for the bombesin-like^ peptides.

More specifically, the polypeptide receptor of the invention can be isolated by a process which comprises incubating a culture of Swiss 3T3 cells in a culture

medium including 125I labelled gast in releasing peptide

( ¹²⁵ I-GRP) , further incubating the ¹²⁵ I-GRP treated Swiss

3T3 cells in the presence of a bifunctional crosslinking reagent and solubilising the resulting 125I-GRP/ crosslinking reagent/polypeptide conjugate to release the polypeptide from the cell surface.

Swiss 3T3 cells are widely available for experimental use and are available from the American Type

Culture Collection in Rockville, Maryland, U.S.A., under the Deposit No. ATCC-CCL92.

The following Examples are given to illustrate the isolation and characterisation of the receptor of the present invention.

^• 't

EXAMPLE.1_

Materials and Methods

Materials

Bombesin and litorin were obtained from Sigma. GRP, the 14-27 βmino acid fragment of GRP and ¹ ] substance P were obtained from Bachem Fine Chemicals (Saf rom lalden, IJ.Σ.) and the 1-16 fragment of GRP and nβuromβdin B were from Peninsula Laboratories (San Carlos, CA). Highly purified platelet-derived growth factor (PDGP) was obtained from Bioprocββsing. Ethylene glycol bis (βuccinimidylsuccinate) (EGS) , diβuccinimidyl βuberate (DSS), dithio-bis (βuccinimidylpropionate) (DSP) and bis [2-(BUccinimidoozycarbomylozy)ethyl] aulphone (BSCOES) were purchased

125 from Pierce Chemical Co. I-GRP (2000 Ci/mmol; ICi « 57 GBq) was obtained from the Radiochemical Centre (Amersham, ϋ.E.) or was prepared by radiolabelling GRP with 12"5"I using the soluble lactoperoxidaβe method (29,30). The labelled peptide was separated

^* 125 125 from unreacted Na I as described (8). I-GRP exhibited mitogenic activity within a similar concentration range to that observed with the unlabelled peptide. All other reagents used were of the highest grade available.

125 Chemical cross-linking of I-GRP to receptors

Confluent and quiescent cultures of Swiss ST3 cells were incubated at 15*C in 1ml of sodium consisting of 0.14 M NaCL, 5 aM ICO., 0.01 U

Na _ft HP0,. 1.8 mM ∑H _o P0_, 1.8 mM Cad.., 1 mM Mgd_. _f 25 mil 2 4 2 4 4-(2-iydroxyβthyl)-1-piperazine ethane sulphonic acid (binding medium)

pH 7.0, supplemented with 0.1% ESA and the appropriate concentration

125 of I-GRP in the presence or abβence of a 500-fold excess of unlabellβd GRP. After 2.5 h the cells were washed three times at 15 ^* C with phosphate buffered saline (PBS) and then incubated for 15 sin at 15 ^* C in 1 al of binding medium, pH 7.4 in the presence of the appropriate cross-linking agent at the concentration indicated. The cross-linking agents (EGS, DSS, DSP and BSCOES) were dissolved in - ^ dimethyl sulphoxidβ immediately prior to use and were added to medium to give a final concentration of dimethylsulphoxide of 1-2%. The cultures were rapidly rinsed twice with PBS at 4"C and solubilized in 0.1ml of 2 x sample buffer 0.2 M Triβ-HCl, pH 6.8, 10% (w/v) glycerol, 6% sodium dodecyl sulphate (SDS) (w/v) 4% β-βercaptoethanol (v/v) and 2 mM ethylenediaminetetraacetic acid. Samples were immediately heated at 100 ^* C for 3-5 min and analysed by either one or two-dimensional gel elβctrophoreβis.

SDS-polyacrylafflide eel electro-phoresis

Slab gel electrophoreβis was performed using 7.5% aerylamide in the separating gel and 5% in the stacking gel, and 0.1% SDS (31).

After βlectrophoresis gels were stained, dβstained and dried down onto paper for autoradiography with Fuji X-ray film (Fuji Kioto Film Co.

Ltd. Japan). Dried gels were exposed to film for 4—8 days.

Two-dimensional gel electrophoresis was performed as described by

O'Farrell (32) using isoelectric focusing in the 1st dimension and

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (8% polyacrylamide) in the second dimension. The samples prepared for isoelectric focusing containing 1.4% LΣB ampholytes, pH 5-7, plus 0.6% LED

ampbolyteβ, pH,3.5-10, 6M urea, and 2% Honidet P-40. The Mr 75000-85000 band from autoradiogra s was scanned using a Joyce-Loebl double beam densitometer and the areaβ under specific peaks were measured with a Hewlett-Packard digitizer. Cell culture procedure (33), assays of DNA synthesis by

^S H-thymidine incorporation (34) and I-GRP binding to intact cells (13) were performed as described previously.

Results and Discussion

When cultures of confluent and quiescent Swiss ST3 cells were

125 incubated with I-GRP at 15 ^* C cell-associated radioactivity reached a maximum after 2.5 h (Fig. 1A) and was considerably enhanced compared with surface binding at either 37 ^* C or 4 ^* C (unpublished observations).

Analysis by SDS-PAGE of cells which were incubated with StxM ^{1 5} I-GR? at ,15 ^* C for 2.5 h, and treated with the homobifunctional cross-linking agents EGS, DSS, DSP or BSCOES revealed the presence of a single major band of Mr 75000-85000 (Fig. 1B). Identical results were obtained

125 using either commercially available . I-GRP or GRP radiolabelled with

125 I in our laboratory. In the presence of 500-fold excess unlabelled

GRP(+) this band was completely abolished. EGS displayed the greatest efficiency of CΓOBB-linking and this agent was therefore used in subsequent experiments. The effect of EGS on the level of the Mr

75000-85000 protein was concentration-dependent; the half-βaximal effect of EGS was obtained at a concentration of 2 mM (Fig. 1C). The rank order of cross-linking efficiency (EGS>DSS>DSP>BSCOES) may be related to the chain length of the anas of these bifunctional molecules. Thus BSCOES which has the shortest chain length was

virtually ineffective (Fig. 1B). Die Mr 75000-85000 protein appeared as a fflulticomponent spot migrating with an iaoeleetic point of 6.4 to 6.9 when cross-linked cultures of Swiss 3T3 cells were analysed by two-dimensional gel electrophoresis using isoelectric focusing in the first dimension and SDS-PAGE in the second dimension (rββultβ not shown).

The Mr 75000-85000 band was not obtained when the cross-linking reaction waβ earried out either in the absence of the cross-linking - agent, in plastic dishes without cells, or using other cell lines including Rat-1, 'whole mouse embryo fibroblastβ and Balbc/3T3, which

125 neither exhibit significant specific I-GRP binding, nor respond mitogenically to bombβBin-related peptides (13). The possibility that the affinity labelled band was a degradation product of a higher molecular weight protein was tested by extracting cultures after the cross-linking reaction in the presence of the protease inhibitors aprotinin (100 μg/ml), pepstatin (4μg/ml), phenylmethyl- sulphonylfluoridθ (2 mM) and ethy eneglycol-bis-fB-aninoethyl ether)N,H«-»tetra-acetic acid (4-mM). These treatments had no significant effect on the level of the Mr 75000-85000 protein and did not result in the appearance of any higher molecular weight proteins. In another experiment performed at 4 ^* C to prevent ligand internalization and degradation identical results were obtained to those shown in fig. 1 (results sot shown). Thus, the Mr 75000-85000 protein was neither sn intraeellular component associated with

125 internalized I-GRP or a product of peptide degradation, nor a fragment arising from protβolysis of a larger molecule. In addition, treatment with: 0.6 M 2-αercaptoethanol did not result in the appearance of additional bands of lower molecular weight suggesting that the Mr 75000-85000 protein consists of a single polypeptide

chain. These important controls strongly suggested that the Mr 75000-85000 band was a surface component of Swiss 5T3 cells closely related to the receptor for peptides of the bombesin family.

The above conclusion was further substantiated by cross-linking

125

5 I-GRP to cultures incubated in the presence of different concentrations of unlabβllβd peptide. The decrease in the level of the Mr 75000-85000 band with increasing concentrations of unlabβlled

(HIP (Fig. 2A, open symbols) closely paralleled the ability of GRP to

125 inhibit the binding of an identical concentration of I-GRP in a

10 parallel set of cultures (Fig. 2A, closed symbols). The cross-linking

1 5 of I-GRP to Swisβ 3T3 cells was also markedly inhibited by other peptides structurally related to GRP including bombesin, neuromedin B,

1 2 7 9 11 litorin and the bombesin antagonist [D-Arg ,D-Pro ,D-Trp ' ,Leu ] substance P (13,17,35,36). The amino-terminal fragment of GRP 15 (GRP(1-16)) which neither inhibits ¹²⁵ I-GRP binding, nor stimulates DNA synthesis (13) caused no reduction in the level of the Mr

75000-85000 protein (Table 1). To ascertain the specificity with

1 5 which I-GRP recognizes the Mr 75000-85000 protein, Swiss 3T3 cells

125 were incubated with I-GRP in the presence of a variety of other

2.0 mitogens for these cells. As shown in Table 1 , the level of the protein obtained by treatment with EGS was not substantially affected by saturating concentrations of PDGP, epidermal growth factor (EGF), wasopressin, insulin, and phorbol 12,13-dibuty ate (1,37,58). In addition the neuropeptides substance P, substance X and somatostatin 5 also had no effect on affinity labelling of the Mr 75000-85000 band

(Table 1). This result is in accord with the finding that the binding

125 of I-GRP to intact STS cells is also not inhibited by these mitogens (13).

The conclusion that the Mr 75000-85000 protein is a major component of the receptor for peptides of the bombesin family in Swiss ST3 cells was further strengthened by measuring the level of the protein as a function of the concentration of the radioiodinated ligand. Fig. 2B showβ that the cross-linking of ¹²⁵ I-GRP to the Mr

75000-85000 band increased in a saturable manner with increasing- - ~÷— concentration of the labelled peptide. A double-reciprocal plot ef ^" ~- ^~ these data (not shown) produced a straight line and gave a value for the Ed of 1 nM which compares very favourably with the Ed (0.5 x 10 M) obtained from Scatchard analysis of binding curves (13).

Furthermore, the dependence of affinity labelling of the Mr

125 75000-85000 protein on I-GRP concentration closely parallels the ability of the peptide to stimulate a variety of early biological responses (16,17) and DNA synthesis (13) in quiescent Swisβ 3T3 cells.

125 At high concentrations of I-GRP a higher molecular weight band of approximately Mr 160,000 was observed (Fig. 2B). This band represented 4% of the total cross-linked material and was almost undetectable either at lower levels of ¹²⁵ I-GRF or at 4 ^* C.

Footnote The abbreviations used are: GRP, gaβtrin releasing peptide; BSA, bovine serum albumin; PBS, phosphate-buffered saline; IGF, epidermal growth factor; PDGF, platelet-derived growth factor; ΪGS, ^β thyl ^β n ^β glycelbi ^β _^ (succinimidyl ^β ucciaate); DSS, diβuccinimidyl ^β ub ^β rat ^β ; DSP, dithio-bi ^β -(succinimidylprepio»atβ}; BSCOES, fei [2-( ^β uccimidooxycarbo__yloxy)ethyl]sulphone; SDS-PAGE, sodium dodecyl sulphate-polyacrylamidβ gel elβctrophoreβia.

EXAMPLE 2 This illustrates the more than Fivefold potency of Antagonist D over Antagonist A.

Substance P has a slight amino acid sequence homology with bombesin (Table 2) and neither inhibits the binding of GRP to Swiss 3T3 cells nor stimulates DNA

2 synthesis. However, [DPro ] spantide (Antagonist A,

Table 2) which was synthesized as a substance P antagonist was found to be a bombesin antagonist in pancreatic acinar cells and to block the growth-promoting effects of bombesin in Swiss 3T3 cells. In order to identify a more potent antagonist of bombesin-like peptides, we have tested ten substance P antagonists at 50 iiM (Table 2) for their ability to inhibit mitogenesis stimulated by GRP (the mammalian ho ologue of bombesin in Swiss 3T3 cells.

[DPhe ]spantide (Antagonist D) was clearly the most potent GRP antagonist. In contrast, peptides, B, C, E, F, G, H, J and K were less potent than either A or D. Spantide (B) had no antagonist activity even at 100 uM. None of the peptides stimulated DNA synthesis when tested at 20 uM with insulin at 1 eg/ml i.e. none exhibited any agonist activity.

Following the identification of [DPhe ]spantide as the most promising GRP antagonist, we compared the potency of [DPhe 5]spantide with that of [DPro2]spantide.

Figure 3 shows that [DPhe ]spantide at 20 ^ιH markedly increased the concentration of GRP required to produce half-maximal stimulation of DNA synthesis whereas addition of [DPro 2]spantide also at 20 πK had only ^" a'slight effect. ς Inhibition of DNA synthesis by [DPhe ]spantide was completely reversed by high concentrations of GRP, indicating that its inhibitory effect was competitive and reversible. The dose response curves for the two antagonists in the presence of GRP 3.6 nM are shown in

Figure 3 (right) . Half-maximal inhibition of DNA synthesis was obtained with 22 uM [DPhe ] spantide and 118 μM [DPro 2]spantide. Thus, [DPhe5]spantide is 5.4-fold more potent than [DPro 2] spantide in inhibiting DNA

^• if synthesis ^* induced by GRP.

The following Examples demonstrate that Antagonist D inhibits all of the events initiated by Bombesin/GRP in a specific manner.

EXAMPLE 3 [DPhe 3 spantide binds competitively to the GRP receptor

The preceding results demonstrate that [DPhe ]spantide is a potent inhibitor of GRP-induced DNA

synthesis which exhibits specificity against other mitogens. To elucidate its mechanism of action, we

5 examined the effect of [DPhe 3 spantide on the specific binding of [ ¹²⁵ I]GRP to Swiss 3T3 cells. Figure 4 (left) shows that both [DPro 23 spantide and [DPhe5] spantide caused a concentration-dependent inhibition in the specific binding of [ 125I]GRP (1 nM) . Half-maximal inhibition of

5 binding was achieved with 2.3 _M of [DPhe 3 spantide and

2

14 uM of [DPro ] spantide, a 6.1-fold difference in potency. This is consistent with the relative potencies of the two antagonists in inhibiting DNA synthesis induced by GRP.

The binding of different concentrations of [ 12513GRP was measured in the absence and presence of

10 uM [DPhe 3 spantide. A double reciprocal plot of these

5 data (Figure 4, center) shows that [DPhe 3 spantide markedly reduces the affinity of the receptors of [ 125I3GRP, although the number of binding sites is unchanged. This is consistent with results previously

2 obtained with [DPro [spantide and strongly suggests that these peptides bind competitively to the GRP receptor.

To further substantiate these findings, we investigated the effects of the two antagonists on the affinity-labelling of the recently described Mr

75000-85000 protein which is a putative bombesin receptor

(Figure 4, right) . They were both able to differentially inhibit the Mr 75000-85000 protein obtained by cross-linking [ ¹²⁵ I}GRP to Swiss 3T3 cells with EGS. Half-maximal inhibition (obtained by scanning densitometry of the autoradiographs) was achieved with [DPhe 3spantide

2 at 5.5 μH and [DPro 3spantide at 20 ^oM, again demonstrating the superiority.of. [DPhe-3-spantide.

EXAMPLE 4

5 [DPhe 3spantide inhibits the early events elicited by GRP

One of the earliest events stimulated by addition of bombesin or GRP to quiescent Swiss 3T3 cells is an increase in cytosolic Ca 2+ concentration ( [Ca2+].)•

--" ^~ 2+ Figure 5 (left) shows that the rise in [Ca ] . caused by the addition of GRP (1 nM) to quiescent Swiss 3T3 cells was prevented by the addition of [DPro 23spantide at 20 uM but not at 5 uM. In contrast [DPhe 3spantide was effective at 5 iM, demonstrating that [DPhe 3 spantide is

2 at least 4-fold more potent than [DPro 3 spantide in this assay. These effects were specific and reversible since

5 [DPhe 3 spantide at 5 μΑ did not prevent a response to PDGF and because the effects of the antagonist in preventing Ca 2+ mobilization in response to 5 nM GRP was reversed by addition of GRP at 50 nM.

Inhibition of [125I]EGF binding by GRP, which is mediated by the protein kinase C pathway, was reversed in

2 a concentration-dependent fashion by [DPro 3spantide and

[DPhe 3 spantide (Figure 5, right). Half-maximal reversal of inhibition was obtained with [DPhe 3spantide at 8.7 uM

2 and [DPro 3spantide at 30 ^αM. These findings further substantiate the conclusion that [DPhe ]spantide is a potent GRP antagonist.

EXAMPLE 5

In addition to the demonstration of inhibition of the cellular effects of Bombesin/GRP in mouse 3T3 cells we have now shown that Antagonists A and C can inhibit the growth of SCLC cells in a specific and reversible fashion.

SCLC is known to secrete bombesin-like peptides which have been suggested to act as autocrine growth factors. _^ ,ι Thus it is plausible but as yet unproven thatan antagonist to bombesin/GRP will inhibit SCLC growth, so we

2 have now tested the effects of [DPro 3 spantide and

[DPhe ] spantide on SCLC _in vitro.

Figure 6 shows that the rate of growth of the

SCLC lines H69, H128 and H417 in serum-free medium was

2 abolished by addition of [DPro 3spantide at 150 ^ιM, a concentration that reversibly inhibits GRP-induced mitogenesis in Swiss 3T3 cells. The inhibition of growth by the antagonist in SCLC cell lines was reversed by

washing the cells and resuspending them in serum-free medium.

2

The effects of [DPro ]spantide and ς [DPhe ]spantide on H69 cells are compared in Figure 7.

The cells achieve 10-fold increase in number in about 12 days in serum-free medium (inset) . Both antagonists inhibited growth in a dose-dependent-manner; half-maximal effect was seen at 24 pΑ and [DPhe 3spantide and at 82 μH with [DPro 23spantide. The difference in potency is of the same magnitude as that demonstrated in Swiss 3T3 cells and supports the contention that bombesin-like peptides (or vasopressin) may be important growth factors for SCLC.

Figure Legends

125 Figure 1; A. Time-dependence of I-GRP binding to Swiss 3T3 cells at 15 ^* C. Cultures were washed and then incubated at 15*C in 1

125 ml binding medium containing '""I-GRP (2 nM). After various times the cells were rapidly washed four times with cold (4 ^* C) PBS supplemented with 1 eg/ml bovine serum albumin (BSA) and then incubated with 1 ml

0.2 M acetic acid, 0.5 M Had at 4 ^* C for 6 min to remove cell-surface associated ligand. This medium was then removed for counting and the remaining intracellular cell-associated radioactivity extracted with 1 ml 0.1 M NaOH containing 2% Na_C0_ and 1% SDS. Open squares denote— surface bound ligand and closed βquares intracellular radioactivity.

B. ²⁵ I-GRP affinity labelling of an Mr 75000-85000 cellular protein using diβsuccinimidyl cross-linking agents. Confluent cultures of

125 Swiss 3T3 cells were washed and then incubated with I-GRP (5 nM) in the presence (f) or absence (-) of 0.9 μM unlabelled GRP for 2.5 h at 15 ^* C. The cells were then rinsed to remove free ligand and treated with either EGS, DSS, DSP or BSCOES at concentrations of 6 mM, 2 mM, 2 mM and 4 mM respectively. The cells were solubilized in SDS sample buffer and electrophoresed on a 7.5% polyacrylamidβ gel. The arrow indicates the position of the Mr 75000-85000 protein. All other experimental procedures used in this and similar experiments were as described in Materials and Methods. A broad and intensely radioactive band migrating at low molecular weight was observed is all our gels. This material was not obtained in the presence of excess unlabelled GRP and therefore is most likely to represent the unreacted peptide.

C. Concentration-dependence of the effect of EGS on the affinity-labelling of the Mr 75000-85000 protein. Confluent cultures of Swiss 5T3 cells were incubated with 1 nM ^1iJ5 I-GRP at 15 ^* C and then treated with various concentrations of EGS as indicated. Samples were prepared for SDS PAGE as described in Materials and Methods. The arrow indicates the position of the Mr 75000-65000 protein. In this and other experiments the efficienc -of cross-linking using EGS ranged ^' from 5 to 10% of cell-surface associated radioactivity.

Figure 2: A. Effect of varying concentrations of unlabelled GRP on the ²⁵ I-GRP binding to intact Swiss 5T3 cells (#)) and the affinity labelling of the Mr 75000-85000 protein (£))• Confluent cultures of 3T3

125 cells were incubated with 5 nM I-GRP in the presence of varying concentrations of so -radioactive ligand at 15 ^* C for 2.5 h. After this time some cells were washed four times with cold (4 ^* C) PBS supplemented with 1 mg/ml BSA, solubilized and total cell associated radioactivity was then determined in a gamma counter. Parallel cultures were rinsed with PBS at 15 ^* C and then treated with EGS (6m!-)

125 to cross-link bound I-GRP. The cells were then solubilized and samples were analyzed by SDS-PAGE. After electrophoresis, gels were dried, exposed to film and scanned as described is Materials and

Methods. The areas under the individual peaks for the Mr 75000-85000 protein are shown as a function of the concentration of the unlabelled peptide and are expressed as a percentage of the control. The inset shows the region of the autoradiogram used for the scanning. B.

1 5 Concentration-dependence cf I-GRP affinity labelling of the Mr

75000-85000 protein. Confluent cultures of Swiss STS cells were

125 incubated with different concentrations of I-GRP at 15 ^* C for 2.5h, and treated with EGS (6 mM)- The level of the Mr 75000-85000 protein expressed is arbitrary units is shown as a function of the concentration of the labelled ligand. .Inset: autoradiogram of the gel used for scanning; the Mr 75000-85000 and 160,000 bands are indicated by arrows. All other experimental details were as described is Materials and Methods.

Figure 3. Inhibition of GRP-induced DNA synthesis by

2 5

IDPro Ispantide (A) and [DPhe Jspantide (D). Left: Confluent and quiescent cultures of Sviss 3T3 cells in 35 am plastic dishes vere washed twice vith DHEM then incubated at 37°C in 2 ml of a 1:1 mixture

3 of DMEH/Vaymouth medium containing [ HJthymidine at 1 wCi/ml (1 uM), insulin at 1 vg/ml and increasing concentrations of GRP in the absence

(■) or presence of antagonist A at 20 vM (o) or antagonist D at 20 yK

3 (Δ ). After 40 hours DNA synthesis was estimated by [ HJthymidine incorporation into acid-precipitable material. Values are expressed as a percentage of [ BJthymidine^incorporation obtained vith a saturating level of GRP (36 nM) in the absence of antagonist (100% -

8.4 x 10 cpm per dish). Each point represents the mean of duplicate determinations. Right: Cultures of Sviss 3T3 cells vere vashed and incubated as above, except that the concentration of GRP vas fixed at

3.6 nM vith various concentrations of antagonists A (o) and D ( Δ ).

3 Values are expressed as a percentage of [ HJthymidine incorporation obtained in the absence of antagonists (100% - 8.6 x 10 cpm per dish). Each point represents the mean of 4 determinations.

2 5

Figure 4. Effects of [DPro Jspantide (A) and [DPhe Jspantide (D) on the binding of ¹²⁵ I-labelled GRP ([ ¹²⁵ IJGRP) to Sviss 3T3 cells.

125 Left: Inhibition of specific [ IJGRP binding by antagonists A and

D. Confluent and quiescent cells vere vashed tvice vith DHEM then

125 incubated at 37 ^# C in 1 ml of binding medium (3) containing _ ^A< "ljGRP at 1 nM and various concentrations of antagonist A (o) and D (Δ ).

125 Cell-associated { IJGRP binding vas measured after 30 minutes.

Values are expressed as percentage of the-specific binding obtained in the absence of antagonists. Non-specific binding vas determined by the addition of 300-fold excess of unlabelled GRP. Each point represents the mean of 3 determinations. Center: Effect of antagonist D on the affinity of binding sites in Sviss 3T3 cells for

125 { IJGRP. Confluent and quiescent cells vere vashed tvice vith DMEH then incubated for 30 minutes at 37°C in 1 ml of binding medium

125 containing various concentrations of [ IJGRP in the absence (e) or presence (Δ) of D at 10 yM. Specific binding (B) is expressed in pmol/10 cells and is shovn in a double-reciprocal plot. Each point represents the mean of duplicate determinations. Right: Effects of antagonists A and D on the affinity labelling of the bombesin receptor-associated Mr 75000-85000 protein. Confluent cultures of Sviss 3T3 cells vere vashed tvice vith DMEH and incubated at 15°C in 1 ml of binding medium (pH 7.0) (15) containing 0.5 nM [ ¹²⁵ IJGRP and various concentrations of the antagonists. After 2 hours, the cultures vere vashed tvice vith binding medium then incubated in 1 ml containing 6 mM ethylene gl col bis (succinimidylsuccinate) (EGS) at pH 7.4 for 15 minutes at 15 ^β C. The cultures vere then vashed tvice vith cold phosphate-buffered saline and solubilised in 0.1 ml of 2x sample buffer, then immediately heated at 100°C for 5 minutes and electrophoresed on a 10% polyacrylamide gel.

2 5

Figure 5. Effects of [DPro Jspantide (A) and [DPhe Jspantide (D) on the early cellular responses stimulated by GRP. Left: Effects of antagonists on [Ca ⁺ J.. Quiescent Sviss 3T3 cells grovn on Cytodex 2 beads vere vashed tvice vith DHEM and incubated for 10 minutes vith fura-2 tetracetoxymethy1 ester at 1 yM, then vashed three times and suspended in 2 ml of electrolyte solution (6) in the fluori eter at

37°C and stirred. Fluorescence vas recorded continuously in a

Perkins-Elmer LS5 luminescence spectrometer vith an excitation

vavelength of 335 nM and emission vavelength of 510 nM. After a period of equilibration, the following additions vere made: solvent, S; antagonist A at 5 yM, A, 5 and 20 yM, A, 20; antagonist D at 5yH, D, 5. After 3 minutes in each case, GRP vas added at 1 nM, G or at 50 nM, G, 50; PDGF at 1 nM, P. Right: Antagonists A and D reverse the

125 inhibition of [ IJEGF binding induced by GRP. Confluent and quiescent cultures of Sviss 3T3 cells vere vashed tvice vith DMEH then incubated for 1 hour at 37°C in 1 ml of binding medium (9) containing

175 ' ^• "

I IJEGF at 0.2 ^' nH and GRP at 3.6-nM alone ( ■ ) or in the presence of various concentrations of A (o) or D (Δ ). Values are expressed as

125 percentages of the specific binding obtained vith [ IJEGF alone at

0.2 nM. The non-specific binding vas obtained by the addition of

500-fold excess of unlabelled EGF. Each point represents the mean of

6 determinations.

2 Figure 6. [DPro Jspantide reversibly inhibits the growth of SCLC cell lines. Stock cultures of cell lines H69, H128 and H417 vere maintained in RPMI 1640 medium vith 10% fetal bovine serum (heat inactivated) in a humidified atmosphere of 10% C0-:90% air at 37°C.

88/07551

- 26 -

They vere passaged every 7 days. Identical grovth vas obtained in the serum-free medium of RPMI 1640 supplemented vith EITES (29) (hydrocortisone, 10 nM; insulin, 5 μg/ml; transferrin, 100 yg/ml; 17β-estradiol, 10 nM; sodium selenite, 30 nM) and 0.25% bovine serum albumin. Cells vere vashed tvice vith RPMI 1640 medium then incubated in the serum-free medium in the absence (•) or presence (o,a) of [DPro Jspantide at 150 yM. After 4 days they vere again vashed tvice vith RPMI 1640 medium then resuspended at a density of 5 x 10 cells

2 per ml in the absence (e,α) or presence (■ ) of [DPro Jspantide at 150 yM (Day 0). Cell number vas determined at intervals over 14 days in a

Coulter Counter after disaggregation of cell clumps by syringing through 19G and 21G needles. Each point represents the mean of 3 deter inations.

Figure 7. Inhibition of SCLC grovth in vitro by [DPro Jspantide and [DPhe Jspantide is concentration-dependent. Cultures of H69 cells vere vashed tvice, vith RPMI 1640 medium then incubated in serum-free medium (as in Figure 6) in the absence (*) or presence of various

2 5 concentrations of [DPro Jspantide (o,A) and [DPhe Jspantide (Δ,D).

Cell number vas determined in a Coulter Counter after disaggregation of cell clumps by syringing through 19G and 21 G needles. Samples vere incubated for 13 days, vhen the controls (inset) had achieved

10-fold increase in number, indicated by the arrov. Each point represents the mean (± standard deviation) of 5 determinations.

Table 1

Specificity of ¹²⁵ I-GRP affinity labelling of the Mr 75000-85000 φrotein

Addition M 75000-85000 protein of control

100

GRP 0.8

GRP (14-27) 0.4

Bombesin 0.5

Litorin 2

Neuromedin B 10

[D-Arg ¹ ,D-Pro ² ,D-Trp ⁷ ' ⁹ ,Leu ¹¹ J 9.7

Substance P GRP (1-16) 93

PDGF 99

EGF 94

Tasopreβsis 94

Insulin 90 ϊfcorbol 12,15-dibutyrate 99

Substance P 91

Substance X 94

Somatostatin 97

Keurotensin 93

125 Confluent cultures of Swiss 3TS cells were incubated with I-GRP

(0.5 nM) at 15 ^* C for 2.5 h in either the absence (-) or"presence of the following GRP-related peptides at the concentrations indicated: GRP (360 nM), the 14-27 amino acid fragment of GRP (GRP (14-127)) (30 sM), bombesin (50 nM), litorin (92 sM), seuromedis B (220 sM), the

1 2 7 θ 11 bombesin antagonist [D-Arg ,D-Pro ,D-Trp » ,Lβu J substance P (100 pM) and the 1-16 amino acid fragment of GRP (GRP (1-16)) (5230 nM).

Parallel cultures were-incubated with the -same concentration of

125

I-GRP in the presence of the following unrelated factors: PDGF (5 sM), EGF (83 nM), vasopreβsin (1000 sM), insulin (1 μg/ml), phorbol 12,13 dibutyrate (2000 nM), substance P (740 nM), substance Σ (880 sM), somatostatis (610 nM) and seurotensin (600 nM). After the incubation period cultures were treated with 6 mM EGS as described in

Materials and Methods. Each value is expressed as a percentage of the level of the Mr 75000-85000 protein obtained with so additons (-). Other experimental details are as described in Materials and Methods.

TABLE 2

Bombesin PGlu - Gin - Arg - Leu - Gly - Asn - Gin - Trp - Ala - val - Gly - His -Leu -Met -NH ₂

1 2 3 1 5 6 7 8 9 10 11

Substance P Arg - Pro - Lys - Pro - Gin - Gin - Phe - Phe Gly -Leu -Met -HH ₂ Antagonist TT DArg - DPro - Lys - Pro - Gin - Gin - DTrp- Phe - DTrp -Leu -Leu -NH

B DArg - Pro - Lys - Pro - Gin - Gin - DTrp- Phe DTrp -Leu -Leu -NH- C Arg - DPro - Lys - Pro - Gin - Gin - DPhe- Phe DTrp -Leu -Met -m_

F DPro - Gin Gin - DTrp- Phe - DTrp -DTrp -Met -HH ₂ G Arg - DTrp- MePhe- DTrp -Leu -Met -HH ₂ H DArg - DPro - Lys - Pro - Gin Gln - DPhe- Phe ^■ DHls -Leu -Met -NH ₂ J HArg - Gly Gin - DTrp- Phe ^■ Gly -Asp -(OtBu) ₂ K DPro - Gin Gin - DTrp- Phe • DTrp -Leu -Met -NH,

o

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