NZ268592A - Mammamodulin, antibodies therefor, and pharmaceutical compositions, treatment of breast cancer - Google Patents

Description

New Zealand No. 268592 International No. PCT/EP94/01975 Priority Date(s): (4ll.9.w».

Complete Specification Class: Publication Data: P.O. Journal No: NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Novel organic compounds and uses therefor Name, address and nationality of applicant(s) as in international application form: F. HOFFMANN-LA ROCHE AG, of 124 Grenzacherstrasse, CH-4002 Basie, Switzerland A Sl^iSS Ccv-VAroO'-u_j I WO 95/02052 PCTYEP94/01975 268592 MAMMAMODULIN, A HORMONE-INDEPENDENT MAMMARY TUMOR CELLS SPECIFIC PROTEIN.

This invention relates to mammamodulin, which is a novel factor not known to exist so far in nature, produced by hormone-independent human mammary tumor cells affecting the morphology, growth and hormone 10 receptor expression of hormone-dependent tumor cells. More specifically the invention relates to mammamodulin in purified form, i.e. at least partially free from compounds with which it is associated in nature, up to high purity, e.g. enabling the determination of at least partial amino acid sequence(s). Means for the production and purification of mammamodulin are described, 15 as are related diagnostic and therapeutic strategies made possible by the discovery of this factor.

Growth of human mammary tumors is a complex process governed by 20 hormones and growth factors of endocrine, autocrine and paracrine origin. An important feature of mammary tumors is their dependency on hormones, particularly estrogen. This hormone dependency is often lost as the cancer develops, rendering endocrine treatment ineffective, and correlates closely with more aggressive behavior by the tumors. The prognosis for breast 25 cancer victims once the tumor enters the hormone-independent stage is extremely poor.

It has now been discovered that a previously unknown factor, which has 30 been named mammamodulin (MM), is expressed by hormone-independent mammary tumor cells. This factor stimulates less aggressive, hormone-dependent tumor cells to grow more rapidly while the expression of hormone receptors is down modulated. It is believed that, long term, MM plays a central role in the phenotypic switch from hormone-dependency to hormone-35 independency during tumor progression.

This factor has been, for the first time, identified, isolated and purified. Availability of substantially pure MM now enables various diagnostic and therapeutic strategies in the treatment of breast cancer. For example, MM levels may be assayed and used as a diagnostic indicator to determine the stage and seriousness of breast cancer. Substances which are observed to suppress MM*s cell activation properties, as well as monoclonal antibodies to MM or other MM binders, may be used as therapeutics to block MM*s stimulation of hormone-dependent cells, thereby stabilizing the cancer and preventing progression into the aggressive, hormone-independent stage of tumor growth. Finally, MM analogues may be used therapeutically to block MM receptors on hormone-dependent cells.

MM is a protein having an apparent molecular weight of approximately 52-55 kilodaltons, as measured by SDS polyacrylamide gel electrophoresis of highly purified preparations on 10-15% gradient gels. MM comprises partial amino acid sequences of Leu-Val-Leu-Arg-X-X-Glu-Thr (SEQ ID No: 1) Ser-Glu-Leu-Arg-He-Asn-Lys (SEQ ID No: 2), and X-Leu-X-Asn-Pro-X-X-Tyr-Leu (SEQ ID No: 3), wherein "X" represents an unidentified amino acid residue.

It is believed that the protein as a whole may contain allelic variations comprising additions, deletions, insertions or substitutions of residues comprising up to ten percent, but preferably no more than five percent, of the complete amino acid sequence, so long as the biological activity is retained. The protein may also contain various post-translational modifications such as gljcrsylation and formation of cysteine bridges.

MM is further characterized in that it is heat and acid labile, and trypsin- and mercaptoethanol-sensitive, and easily forms heterogeneous aggregates with other proteins.

MM can be extracted and purified to apparent homogeneity from cell cultures of fast-growing hormone-independent tumor cells, as described in the examples herein. MM may also be obtained by using amino acid sequence information to screen a cDNA library from a producer cell line to obtain the cDNA of the MM gene. The gene may then be cloned and introduced into an appropriate vector for expression in a suitable prokaryotic or eukaryotic system.

Purified MM may be used as an antigen to produce polyclonal or monoclonal antibodies by conventional processes. Antibodies, in particular monoclonal antibodies are useful (i) as therapeutics to block MM and prevent it from stimulating hormone-dependent cells; and (ii) in assays (including bioassays, radioimmunoassays (RIAs), fluoroimrminoassays (FIAs), Western blot assays, and ELISAs) to detect the presence of MM in biopsies of mammary tumors, thereby giving useful diagnostic information as to stage of development and prognosis of the cancer.

Purified MM may also be used in a binding assay to screen and identify compounds having affinity for MM and in competitive assays to screen and identify compounds having affinity for MM receptors on hormone-dependent cells. Such compounds are useful in inhibiting the biological activity of MM.

Additionally, compounds which inhibit the expression of MM in tumor cells, in particular mammary tumor cells, e.g. hormone-independent mammary tumor cells and/or have affinity for mammamodulin receptors on tumor cells, in particular on mammary tumor cells, e.g. hormone-independent mammary tumor cells are novel and useful. The only compound known to be a potent inhibitor of MM is heparin.

Esamntej, Production of MM-contnininpr nnnditinn medium from hormone independent cell cultures.

The human breast cancer cell line MDA-MB-231 was grown in HL medium supplemented with 5 % fetal calf serum. The optimized HL medium was developed for the human leukemia cell line HL60 as described in European Patent Application Publication No. 417563. The cells were grown in an adherent fashion with a doubling time of about 22 - 24 hours in T-flasks under 5 % C02-baIance air and 96 % water saturation. After achieving the confluent state, the growth medium was replaced by the serum-free HL medium. M«mmnmnri«1in (MM) is released into the culture medium during growth as well as in the non-growth confluent state.

A slightly modified 23 1 airlift fermentor (Chemap AG, Switzerland) was used for the scale-up production of MM . The outer volume of the draft tube was filled with 8.5 kg Baschig rings (8 z 8 x 5 mm, stone ware) and 10 Siraf 25 rings (25 z 25 z 2 mm, porous glass Baschig tings, Schott, Germany) giving about 20 m^ available surface area. The fermentor was inoculated with tryp-sinized MDA-MB-231 cells from 2 Cell Factories units (Nunc, 6000 cm^ per unit). Two medium ezchanges were performed during the growth phase of 15 days. The airlift fermentor was aerated with 1 normal liter /min air and the solved oxygen content (30 %), pH (7.2) and temperature (37°C) were continuously monitored.

MM-containing spent medium was collected by "hanging 17 - 181 serum-free fortified HL medium (glucose from 5 g to 7.5 g, glutamine from 5 mM to 6.5 mM, Primatone BL from 0.25 % to 0.3 %) daily. The metabolic state of the cells was monitored during the whole fermentation process by measuring glucose and glutamine consumption and lactate and ammonia formation as described (Schumpp B. and Schlaeger E.-J., J. Cell. Sci. 97: 639-647 [1990]).

The MM titre remained constant during the production campaign.

The MM containing cell-free feimentor volume was concentrated 20-fold (1001 to 5.51) by using an Ami con SF20 ultrafiltration unit (Amicon, Switzerland) equipped with two S10Y10 spiral-wound cartridges (1.8 m^ membrane area, MW cutoff 10 kDa). The MM-containing concentrates were frozen at -80°C.

Kwmnlft 2 Purification of MM from the supernatant of MDA-MB-231 cells to partial purity bv 4 chromatngranhic steps All operations were done at room temperature (RT) if not otherwise stated. Fractions of purification runs were characterized by determination of the absorbence at 280 nm, by SDS-PAGE reduced according to Laemmli, U.K. Nature 227,680-685 (1970) and by an assay for biological activity as described in Example 6. ,5 1 of frozen concentrated supernatant (prepared as described under Example 1, corresponding to 1001 of orignal supernatant) were thawn and centrifuged at 3000 g for 15 min (4°C).The absorbence of the supernatant at 280 nm at 1cm path length multiplied by the volume in ml (= OD28O) was 35000. The total activity in the assay was 5,8.10^ U (100%), the specific activity 165 U/OD28O.

The supernatant was then diluted by 121 of 8M urea 0,3 mM 3-{(3-Cholamidopropyl)- dimethylammonio}- <Buffer A> 1-propanesulfonate (CHAPS) 30mM Tris/HCl pH7,5 and 1,71 of dest. water. The solution was then applied to a Q-Sepharose FF column (Pharmacia, Diibendorf, Switzerland). The column (1,2 1, 4°-8°C) had been pre-equilibrated by 6M urea 042 mM CHAPS mM Tris/HQ <BufferB> pH7,5 The column was washed by 1 column volume of buffer A and was then eluted (10 ml/min, RT) by a linear gradient from 100 % buffer B to 40 % buffer B and 60 % of 6M urea 0,2 mM CHAPS mM Tris/HCl <Buffer C> 500 mM NaCl pH 7,5 within 7 h and 36 min. Fractions between 30 and 50 % of buffer C (1550 ml) contained 2630 OD280» 7,5 X10 (130%) and the specific activity was 2852 U/OD280- The chromatogram is shown in Figure 1. The fractions were pooled and concentrated (4°-8°C) to 210 ml by a Millipore Mini tan System equipped with 4 PTGC cassettes (Millipore, Volketswil, Switzerland).

The concentrate was 1: 15 diluted with 1 mM N-Dodecyl-N ,N-dimethyl- 3-ammonio- propansulfonate (Zwittergent 3-12) 50 pM Ethylene glycol-Hs(beta-aminoethyl ether) N.N.N'.N' - tetraacetic acid (EGTA) ^Buffer D> 50 mM Tris/HQ 150 mM NaCl 0,02% NaN3 pH7,6 to give a volume of 3150 ml. This volume was applied to a 80 ml - nnlnTmi of Heparin-Sepharose CL-6B (Pharmacia, 4°-8°C), which had been preequilibrated by buffer D. The column was washed by 24 column volumes of buffer D and then eluted (3 ml/min, RT) by a linear gradient from 100 % buffer D to 100 % of 1 mM Zwittergent 3-12 50 mM EGTA 50 mM Tris/HCl <Buffer E> 1350 mM NaCl 0,02 % NaN3 pH7,6 Fractions eluting between 39 and 64 % of buffer E (240 ml) contained 19,1 OD280» 7,7X10® U (134 %) and the specific activity was 403 000 U/OD28O (f°r chromatogram see Fig.2).The fractions were pooled and concentrated (4°-8°C) to 80 ml using a 400 ml Amicon stirring cell equipped with an YM10 membrane (Amicon, Ziirich, Switzerland). 80 ml of were added and the concentration/dilution procedure was 3 times repeated. The solution was finally concentrated to give 2,6 ml. This volume was applied to a Superdex 200 column (Pharmacia, 1,6 X 60, 120 ml, 1 ml/min). The column had been preequilibrated with buffer F. Fractions eluting between 47 and 63 % of the column volume (20 ml) contained 3,8 OD28O, 4X10 6 U (69 %) and the specific activity was 1.050.000 U/OD28O (for chromatogram see Fig. 3). The fractions were pooled and concentrated using a 50 ml Amicon stirring cell equipped with an YM10 membrane to give 5 ml, which were then diluted by 5 ml of Concentration/dilution was once repeated and the solution was concentrated to 1,6 ml and then 1:30 diluted to give a final volume of 48 ml. This volume was applied to a Mono S column (1 ml, Pharmacia) preequilibrated with buffer G. The column was washed with 12 column volumes of buffer G and then eluted by a linear gradient (0,5 ml/min) from 100 % buffer G to 100 % of 6 M Guanidinium hydrochloride 2,5 mM Zwittergent 3-12 50 uM EGTA 50 mM Tris/HCl pH7,5 <Buffer F> 6M urea 0,3 mM Zwittergent 3-12 20 mM Tris/HCl pH 7,5 <Bu£fer G> 6M b M urea 0,3 mM Zwittergent 3-12 mM Tris/HCl 500 mM NaCl pH7.5 <Buffer H> Fractions eluting between 36 and 56 % of buffer H contained 0,27 OD280» 4,4X10® U and the specific activity was 16.000.000 U/OD28O (for cbromatogram see Fig.4). The total yield based on activity was 76 % and the purification factor was 97000. Figure 5 demonstrates the protein composition of investigated fractions by SDS-PAGE. The biological activity correlates with the intensity of a protein band, which shows a molecular weight between 41 and 45 kD, when analyzed by SDS-PAGE (reduced) of 12,5 % T stained by Cocm&asie R 250. Part of the fractions was further purified (see Example 4).

A) Purification of MM out of the supernatant of MDA-MB-231 cells to high purity by five chromatographic steps In a similar experiment as described under Example 2 MM was purified from 1301 of supernatant. In difference to Example 2 more MM was found in the flow through of the Q-Sepharose and this chromatography was therefore repeated with the flow through. Also further chromatography via Heparin-Sepharose and Superdex 200 was done via 2 separate runs. The gradient of the Mono S chromatography was improved as follows: 0 % buffer H to 40 % buffer H in 48 min 40 % buffer H to 60 % buffer H in 120 min 60% buffer H to 100% buffer H in 46 min The eluted fractions between 42 and 45,5 % of buffer H contained 0,61 OD280» 4,1X10® U and the specific activity was 6,7X10® TJ/OD280- For further purification, these fractions were concentrated using an Amicon stirring cell of 10 ml equipped with an YM10 membrane. Volume was reduced from 11 ml to 0,7 ml. 2,5 ml of buffer F were added and the volume was reduced to 0,7 ml. Dilution and concentration were once repeated and the sample of 0,7 ml was applied to Superdex 200 column (1X 30 cm, 0,5 ml/min) preequlibrated in buffer F (for chromatogram see Fig. 6). 50 pi -aliquote of the resulting fractions were transfered into buffer G by the use of a Fast Desalting column (Pharmacia) in connection with the Pharmacia SMART system. The resulting samples were investigated by SDS-PAGE (reduced, 12,5 % T, Coomassie stain, see Fig. 7). The major fraction (m) exhibits only one protein band in the range from 41 to 45 kD. The yield in this fraction is 0,12 OD28O, the activity 4X10® U and the specific activity is 33X10® U/OD28O. The total yield based on activity is 36 % and the purification factor 170.000.

The fraction n eluting behind the major fraction (m) was further purified (see Example 4).

Purification of MM out of the supernatant of MDA-MB-231 cells to high rmritv hv maht. chromatographic steps In a similar experiment as described in Example 2, MM was purified from 1461 of supernatant with the following steps (omitting chromatography on Q-Sepharose): 6.61 of concentrate were diluted and loaded on a column of Heparin Sepharose (80 ml) using 1 mM CHAPS as detergent. The active fractions eluted were diluted and loaded on a column of Heparin Sepharose (50 ml) using 1 mM CHAPS as detergent and 2 mM spermidine as an additive in all buffers. The active fractions were pooled, diluted with a buffer containing 4 mM zwittergent 3-12 as detergent and loaded on a column of Heparin Sepharose (10 ml). The active fractions (8 ml) were pooled and, after supplementation with 6 M guanidinium hydrochloride, purified in 4 runs with 2 ml each on a Superdex 200 column (120 ml). The active fractions were pooled, diluted and concentrated in two steps using a 10 ml and a 2 ml column of Heparin Sepharose. The fraction with the highest activity (2 x 10-6 U/ml) of the final step was used for growth experiments as described in Example 5C). This fraction was run on a SDS acrylamid gradient gel (10-15%, Phast Gel, Pharmacia). After silver staining it showed a major band of approximately 52 kDa.

FfrffliiDlft 4 Purification of MM to apparent homogeneity The material from Example 2 ( fractions f to h) was purified according procedure A and fraction n from Example 3 was concentrated according procedure B, because of the high salt concentration (Buffer F) present in the sample.

Procedure A: The ionic strength of the material from Example 2 was reduced by fourfold dilution and then concentrated on a Mono S (PC 1.6/5) (Pharmacia, Uppsala, Sweden) column on a SMART chromatography system (Pharmacia).

The column was developed with a flow rate of 100 pi / min and a linear gradient (12.5%/min) from buffer G to H (for buffer composition see Example 2). 50 pi fractions were collected and active fractions (for activity test see Example 6) were pooled. A typical chromatogram of the concentration of MM is shown in Figure 8.

Procedure B: The material from Example 3u concentrated on a reversed phase column Poros R/H (Perseptive Biosystems, Cambridge, MA USA) (inner diameter 0.8mm, length 10 cm) packed by LC Packings, Amsterdam, Netherlands on the SMART chromatography system from Pharmacia using the following buffers: 0.1% TFA in water <Buffer J> 0.09% TFA in acetonitrile <Buffer K> The column was developed with a flow rate of 50 pi / min and a linear gradient (5%/min) from buffer J to K. 25 pi fractions were collected and active fractions were pooled.

The samples from procedures A and B were applied to agarose gels (Pro-Sieve Gel System from FMC Bioproducts, Rockland , ME, U.S.A) and run as described by the manufacturer under non-reducing conditions using high quality grade SDS.

After completion of the electrophoresis the agarose gel was cut into 2 mm slices from the anode to the cathode and the protein was eluted at ambient temperature for at least four hours in 6 M Urea mM Tris-HCl, pH 7.5 0.3 mM Zwittergent 3-12 <Buffer I> 0.3 M NaCl Activity could be measured in the gel eluates corresponding to the 45 to 55 kDa slices. Active eluates were pooled.

The pooled material was applied to a reversed phase column Poros R/H (Perseptive Bio systems, Cambridge, MA, U.S.A) (inner diameter 0.8mm, length 10 cm) packed by LC Packings, Amsterdam, Netherlands) on a SMART chromatography system (Pharmacia).

The column was developed with a flow rate of 50 pi / min and a linear gradient (2.5%/min) from buffer J to K 12 pi fractions were collected and active fractions (see Example 6) were pooled. A typical chromatogram for the separation of MM on a reversed phase column is shown in Figure 9.

Aliquots of the purified product were loaded on a SDS gel (Phast Gel 10-15%; Pharmacia) under nonreducing and reducing conditions together with low molecular weight protein standards (available from, e.g. Bio-Rad, Hercules, CA USA, 8ng/lane/protein, lane 1), and stained with silver (Fig. 10a). Scans of lanes 1-3 were also performed and are shown in Fig. 10b. 230 pmoles of the purified product were used for sequencing. MM was reduced, S-carboxy-amidomethylated and digested with endoproteinase Lys-C CWako, Neu8s, Germany) for 16 hours at 37°C. Enzyme to substrate ratio was 1:100. The digest was collected on a Mini S cation exchanger column (3x0.5 cm) equilibrated with 20 % acetonitrile in potassium phosphate, pH 2.5. The column was developed with a NaCl gradient and the peak-containing fractions were collected. Peaks were further purified on Vydac C4 (0.1x15cm) or Brownlee RP-300 (0.1x10cm) reversed phase columns, developed with a water/acetonitrile-0.1 % TFA gradient and sequenced on a 475A or 477A sequenator (Applied Biosystems, Foster City, CA). Sequences of three peptides were obtained having the amino acid sequences of Leu-Val-Leu-Arg-X-X-Glu-Thr (SEQ ID No: 1), Ser-Clu-Leu-Arg-Ile-Asn-Lys (SEQ ID No: 2), and X-Leu-X-Asn-Pro-X-X-Tyr-Leu (SEQ ID No: 3).

"X" represents a sequencing cycle in which the amino acid was not possible to identify. fiftflTTinlft fi Effects of MM on hormone-dependent cells A) Morphology: The morphological changes induced by exposing ZR-75-1 hormone-dependent cells to conditioned medium from hormone-independent human mammary tumor cells MDA-MB-231 are depicted in the photomicrographs of Fig. 11. 50,000 hormone-dependent cells (ZR-75-1, available, e.g., from American Type Culture Collection (ATCC), Rockville, Maryland, USA) per cm^ were seeded on cell culture dishes precoated with collagen type IV and grown for three days under serum-free conditions.

Then the culture medium was replaced by serum-free medium conditioned by hormone-independent MDA-MB-231 cells. Consecutive phase contrast photomicro-graphs were taken immediately before the medium exchange (Fig. 11a) and 15 minutes (Fig. lib), 35 minutes (Fig. 11c) and 3 hours (Fig. lid) after stimulation. The photomicrographs show that unstimulated hormone-dependent cells grow in culture as epithelial-like patches with tight intercellular contacts (Fig. 11a). The peripheral cells show ruffling membranes and start to produce lamellipodia (Fig. lib) within minutes of stimulation by MM-containing conditioned medium from the producer cells or by purified protein preparations of all purification stages. The colonies enlarge considerably due to cell flattening and cell- o-cell contacts get weaker in the following time (Figs. 11c and lld)„ Activation of the responding cells by highly purified MM lasted several hours. Then, the cells reassumed the original patch-like formation. The duration of the morphological changes was dependent on the concentration of MM and lasted longer at higher concentrations. Cell activation was observed in a wide variety of hormone-dependent cells as shown in Table I. All cell lines in Table I are publicly available from, e.g., American Type Culture Collection (ATCC) Rockville, Maryland, USA.

Hormone-independent human mammary tumor cells MDA-MB-453 did not respond with morphological changes upon stimulation by purified MM or by conditioned medium from MDA-MB-231 cells nor did they secrete a factor activating membrane ruffling of ZR-75-1 or T-47D cells.

B) Motility: The responding hormone-dependent cells showed a temporary activation of random motility associated with a fibroblast-like cell shape upon exposure to medium of fast growing hormone-independent cells or purified MM. Due to this induced motility, the cell patches formed after reassociation may not be composed by the same cells as the patches before stimulation with MM.

C) Proliferation: 1. Effects of MM on cell proliferation of estrogen receptor-positive human mammary tumor cells T47-D Hormone-dependent T47-D cells were seeded into 96 well cell culture plates previously coated with collagen type IV at a density of 10,000 cells per well and grown in serum- and phenol red-free media. Purified mammamodulin, prepared as described in Example 3B), was used at concentrations of 20 U/ml. For comparison, the effects of the mitogens insulin-like growth factor type I (IGF-I) and estradiol (E2) and the growth inhibitors tumor necrosis factor -alpha (TNF-alpha) and Interleukin-la (IL-1) in the absence as well as in the presence of mammnmnHnlin were determined. After 5 days, the cell numbers in the wells were determined as described by Kiing et al., Analyt. Biochem. 182,16-19,1989. The O.D. at 590 nm of the solubilized dye (crystal violet) which was taken up by the fixed cells during staining correlated linearly with cell numbers.

The purified TrmmmHTrinHiilin stimulated the proliferation of hormone-dependent cell line T47-D as shown in Fig. 12. In this figure, bars represent means of quadruplicates and standard deviations. IGF-I at the concentration used stimulated the cells maximally and mammamodulin had no additional effect on cell proliferation. Estradiol alone had little effect on cell growth but mammamodulin stimulated these cells to grow faster. The inhibitory effects of TNF-alpha and IL-1 on T47-D proliferation were, at least partially, reversed by simultaneous addition of mammamodulin. 2. Effects of MM on cell proliferation of estrogen receptor-positive human mammary tumor cells MCF-7 7,000 MCF-7 cells were seeded per well into cell culture microwell plates (Falcon) and grown under defined serum- and phenol red-free conditions [Kiing et al., Contr. Oncol. 22* 26-32 (1986)] without and with 25 U/ml mammamodulin or 1 x 10-10 M estradiol. The MM preparation used for this experiment was material of highest purity from reversed phase column as shown in Fig. 9, (pool material). Aliquots of this material were diluted immediately after elution from the column in serum-free cell culture medium. After 3 days, the cultures were refed. After 7 days, the cells were fixed and their numbers measured by die crystal violet assay as described by Kiing et al. (1989), supra. The O.D. at 590 nm of the solubilized dye (crystal violet) which was taken up by the fixed cells during staining correlated linearly with cell numbers.

The growth of MCF-7 was strongly stimulated by 25 U/ml MM as shown in Fig. 17. Cells were also stimulated by estradiol (E2) which was used as a control for normal function of the cells. In Fig. 17 the increases in cell numbers of stimulated cultures are expressed in percent of data obtained from the control cultures. The values represent means and standard deviations of quadruplicates.

D) Mitogenic activity: Cell cycle analysis by flow cytometry demonstrated that MM induced a marked increase in the proportion of hormone-dependent cells in the synthetic (S) phase. Cultures of hormone-dependent MCF7, ZR-75-1 and T47-D cells were washed twice with serum-and phenol red-free medium two and one day before starting the experiments for cell cycle analysis. Then the media were supplemented with 25 units of pure MM preparation (from pool of reversed phase column in Fig. 9). The cells were harvested 24 hours after stimulation and analyzed using a standard protocol for the FACScan analyzer (Becton Dickinson, CellFIT program). The results showed that treatment with pure MM considerably increases the percentage of S-phase cells of MCF-7, ZR-75-1 and T47-D cells. MCF-7 cells showed an increase of 186%, ZR-751 cells of 243% and T47-D cells of 161% above control cells. To provide a basis for comparison between MM and known mitogens for these cells, cells were treated with high concentrations of insulin-like growth factor (IGF-I, 10"8 M) or estradiol (3 xlO"9 M). The values for S-phase increase induced by IGF-I or estradiol were with MCF-7 cells 176% and 158%, with ZR-75-1 cells 280% and 180% and with T47-D cells 203% and 105%, respectively.

E) Conversion of hormone-dependent cells; MM-containing conditioned media from fast proliferating hormone-independent cell line MDA-MB-231 demonstrably suppressed estrogen receptor (ER) levels of hormone-dependent cells. In MCF-7 cells, exposure to MM-containing media (30 units/ml) for 2 days reduced the number of ERs to about 55% +/-10% (S.D. from 5 determinations), and in ZR-75-1 cells, the number of ERs was about 32% +/-20% in comparison to untreated cells, as measured by a binding assay with intact cells using tritiated estrogen (17-fi estradiol).

It was also shown that MM suppresses the ER mRNA expression in MCF-7 hormone-dependent cells. MCF-7 cells (2.5 x 106) in serum- and phenol red-free cultures were stimulated with 20 units/ml of highly purified MM (Example 2, MonoS fraction between g and h), or with 1 x 10"9 M estradiol in the absence or presence of MM. The cells were harvested 5 hours after treatment. RNA was then extracted from the cells and purified for mRNA on pre-packed spun columns (Pharmacia). The mRNA concentrations were measured and then 2.5 |ig of each extract was loaded and resolved on an agarose gel. After transfer to a membrane (Northern blot), the mRNA was probed for ER message by hybridization with a human ER probe (Fig. 13). ER mRNA was expressed in MCF-7 control cells (Fig. 13, lane 1). 10*9 M estradiol stimulated ER mRNA expression (Fig. 13, lane 2). MM reduces the mRNA expression considerably in the absence (Fig. 13, lane 3) as well as in the presence of 10"9 M estradiol (Fig. 13, lane 4).

In a separate experiment, 2.5 x 106 MCF-7 cells in serum and phenol red-free cultures were stimulated with highly purified MM (Example 3, fraction "m" of Fig. 6), IxlO*10 M estradiol or combination thereof for 24 hours. At this time point, mRNA was extracted from the cells and purified using a QuickPrep micro mRNA preparation kit from Pharmacia. The mRNA preparations were resolved on 1.1 % agarose gels and transferred to nitrocellulose membrane by Northern blotting. The blot was hybridized with a radioactive ER probe. The results (Fig. 14) demonstrate that estradiol inhibited the expression of mRNA for the ER. MM markedly reduced the mRNA level of ER. This effect was accentuated when estradiol was coincubated with MM.

ER and progesterone receptor (PR) protons were measured by extraction of stimulated MCF-7 cells and application of enzyme immuno assays (EIA). MCF-7 cells were seeded into 12-well cell culture plates (Costar) in the presence of 5 % FCS at a density of 150,000 cells per well. The next day, the medium was replaced by serum- and phenol red-free medium. The following day, cells in duplicate wells were stimulated with MM (200 U/ml) or estradiol (1 x 10-9).

After 6 and 48 h, the media were removed and the cells frozen at -70°C for at least 12 hours. After thawing, the cells were extracted according to the method described by Madeddu et al. [Eur. J. Cancer Clin. Oncol. 24,385-390 (1988)] by adding 225 pi of extraction buffer composed of 500 mM KC1,10 mM KH2PO4,1.5 mM EDTA and 5 mM Na-molybdate. The pH was adjusted to 7.4 with 1M KOH. Beta-mercaptoethanol was added freshly before use at a concentration of 0.01 %. The buffer was at 4° C. After 90 min, the extracts were centrifuged in 1 ml Eppendorf tubes for 5 min at 12,000 x g and 4°C The supernatants were collected and 100 pi of extract used for receptor determination. ER and PR levels were measured using EIA kits manufactured by Abbott Laboratories, North Chicago, IL, USA. The results (Fig. 15) showed that 6 h after stimulation with MM, the ER protein levels of MCF-7 cells were slightly and after 48 h strongly reduced in comparison to controls. PR levels were similar to controls at both time points when cells were stimulated with MM.

As a control for normal cell function of MCF-7 cells, ER and PR levels were also measured in cells treated with estradiol. Estradiol (E2) is known to inhibit the expression of both ER mRNA and ER protein and to stimulate PR expression of MCF-7 cells. Furthermore, PR expression is dependent on ER activation by estradiol [Ree et al., Endocrinology 124.2577-2583, (1989)]. The control experiments showed that estradiol reduced ER protein expression and stimulated PR protein expression. These results with MCF-7 cells are consistent with the data obtained by Ree et al., supra, and Read et al. [Molec. Endocrinol. 3, 295-304(1989)].

F) Tyrosine phosphorylation of cell membrane proteins of hormone-dependent cells: The effects of purified MM (fraction "m" of Fig. 6) on tyrosine phosphorylation of MCF-7 membrane proteins were tested.

MCF-7 cells were seeded into 24-well Falcon culture plates (2 cm2/well) at a density of 150,000 cells/well in cell culture medium containing 5 % fetal calf serum (FCS). After 24 hours, the medium was exchanged with medium containing 1 % FCS. The following day, the cells were incubated for two hours with serum-free medium before stimulation. Cells were treated with MM concentrations of 20 and 200 U/ml. An unstimulated control was included. After 30 min at 37°C in a cell culture incubator, the media were removed and the cells extracted by heating for 5 min in an oven at 100°C with 100 ill/well of reducing sample buffer for SDS polyacrylamide gel electrophoresis containing 4 % SDS and 2.5 % mercaptoethanol. 60 pi of each extract and an aliquot of EGFR reference sample were loaded and resolved on mini Tris-Tricine gels (6 %). The proteins were transferred to a FVDF membrane (Bio-Rad Laboratories, Hercules, CA) by Western blotting using 10 mM CAPS buffer, pH 11.0 with 10 % methanol and subsequently probed with an antibody raised against tyrosine phosphate. The detection of tyrosine phosphorylated proteins was performed with a second, alkaline phosphatase-labeled antibody obtained from Upstate Biotechnology Incorporated (UBI, Lake Placid, NY, product No. 17-105) and used as recommended by the manufacturer. The results showed that MM stimulated the tyrosine-specific phosphorylation of a membrane protein of MCF-7 cells with an apparent molecular mass of approximately 180-190 kDa when compared with molecular weight standards and with a phosphorylated EGF receptor reference from EGF-stimulatf 4 A431 (Fig. 16). The identity of the phosporylated protein(s) was not determined.

G) Elevated expression of cell surface EGF receptor and erbB2 levels after stimulation of hormone-dependent cells with MM: 12 x 106 MCF-7 cells were grown on culture plastic dishes wit}?. 10 cm diameter in serum- and phenol-free cell culture medium for 1 day and stimulated with 30 U/ml or 100 U/ml of highly purified MM (fraction "m" of Fig. 6) for 24 hours. The surface receptor numbers were determined with flow cytometry using a mouse monoclonal antibody directed against the cell surface domain of the human EGF receptor (Ab-1, Oncogene Science, Manhasset, NY) or the cell surface domain of erbB2 and a second, fluorescein isothiocyanate (FITC> labeled goat anti-mouse antibody (Becton Dickinson, San Jose, CA) was applied to stain the cell-bound mouse anti EGF receptor or and erbB2 antibodies. The cells were scraped from the dishes, washed in serum-free medium and counted using a micro cell counter (Sysmex F-300, TOA Medical Electronics, Kobe, Japan). To 106 cells in 80 pi sertun-free medium, 20 pi first antibody solution was added and incubated for 45 min on ice. Then the cells were washed 3 tunes in cold serum-free medium, resuspended in 100 pi and incubated with 4 pi FITC-labeled goat anti mouse antibody for 45 min on ice in the dark. Afterwards, the cells were washed twice with serum-free medium, resuspended in 250 pi serum-free medium and analyzed at room temperature in the flow cytometer (FACScan, Bee ton Dickinson, San Jose, CA). Histograms of the FTTC-fluorescence of the individual suspensions were acquired and data were expressed as the mean fluorescence channel numbers.

At least 90% of all cells were staining with the fluorescent antibody, as was confirmed by determining the number of cells with increased fluorescence in a gated dot plot on the flow cytometer. Bound fluorescence was calculated by subtracting the fluorescence of cells incubated only with the second (FITC-labeled) antibody from the fluorescence obtained from samples incubated with the first antibodies (the anti receptor antibodies) and the FITC-labeled antibodies.

The results of the experiment are shown in Figures 18 and 19. The values represent the means of 3 determinations. In hormone-dependent MCF-7 cells stimulated by MM, the EGF receptor levels were approximately 3-fold higher after 24 hours than the receptor level of control cells (Fig. 18). ErbB-2 levels were found to be elevated by 35 % after 24 h of stimulation by 100 U/ml of MM (Fig. 19).

Rrnmplft fi Determination of MM activity during isolation.

Determination of MM activity is based on the induction of fast morphological changes of the estrogen receptor-containing cell line T47-D. T47-D cells were grown in serum and phenol red-free cell culture medium supplemented with transferrin, low levels of insulin and 1 mg/ ml bovine serum albumin (Albumax I from Gibco). The seeding density was between ,000 and 35,000 cells per cm2. Two days after seeding, the cells were used for MM activity testing. T47-D cells reacted to MM similar to ZR-75-1 cells (compare Fig. 11a) but were slightly more sensitive and easier to handle because their attachment to the substrate is tighter. MM containing solutions were added in appropriate amounts to wells containing the test cells. The culture plates were then immediately returned to the cell culture incubator to maintain the normal 37°C temperature for cell cultures. After 10 to 15 minutes, and a second time after 30-45 minutes, the cells were inspected by phase contrast microscopy at a magnification of 200 x for induced membrane ruffling and lamellipodia formation. Five levels of activation were distinguished: a) no activation (-), b) weak and transitory, but unequivocally detectable formation of membrane ruffles in many cell patches (+), c) lamellipodia formation in many cell patches and membrane ruffles in all cell patches (++), d) formation of lammelipodia in all and of enlarged lamellipodia in many cell patches (+++) and e) intensive membrane ruffling and formation of large lamellipodia in all cell patches (++++). This gradation of test results corresponded roughly to the cell activation pattern of the 5 last steps in a 1 to 2 dilution of MM containing media or chromatographic fractions. The dilution with an assigned one plus (+) activity was defined as containing 1 unit MM per ml.

The results were subjected to Bome variability, depending mostly on the actual conditions of the cell cultures (cell density, age of cultures). Usually, differences between measurements of MM activity at other time points and other cultures of T47-D cells were not greater than 50% to 200%.

"Rrnmnlft 7 Antibody to MM MM is antigenic in nonhuman mammals, so that antibody is produced by the animals after they have been inoculated with the MM. Two doses of 5 \ig MM are sufficient to induce production of antibody in mice. Polyclonal anti sera can be isolated from the blood of the inoculated animals.

Monoclonal antibody to MM is produced by conventional KcShler-Milstein processes: immunization of a suitable animal species by injection with MM, recovery of antibody-producing cells (i.e., spleen cells) sensitized to MM, imxnortalization of the antibody-producing cells by fusion with a compatible myeloma cell line, and isolation of the monoclonal antibody from a selected immortalized cell line thus established. Preferably, female Balb/c mice are injected with MM in Titremax (CytRx, 150 -Technology Parkway, Technology Park Atlanta, Norcross, Georgia 30092), administered by i.p. injection. After two weeks, this iqjection is repeated. One week later, blood samples are collected, and tested, e.g., by western blot, for presence of antibody. The mice which exhibit the highest antibody levels are sacrificed, and their spleen cells are immortalized by fusion with mouse (Balb/c) myeloma cells, e.g., using PEG 4000, and distributed into 24 X 24 wells. The hybridoma lines thus produced are screened and selected for the production of antibody.

FiTFnmplft 8 filnnrng and mroi-ftHmnn of MM MM cDNA may be isolated from a random-primed cDNA library created using poly(A)+RNA from MDA-MB-231 cells. A vector, e.g., Lambda ZAP II vector, is utilized to form the MM-fusion protein, and the expressed protein is then screened using MM antibody. Alternatively, the cDNA library may be screened using MM amino acid sequence information, e.g., by colony hybridization techniques, for example expressing the library in an expression system, preferably E. coli, lysing the colonies, e.g., on nitrocellulose filters, denaturing their DNA in situ and fixing it on the filter, hybridizing with labeled, preferably radiolabeled, oligonucleotide probes of at least 24 base pairs having cDNA base sequences corresponding to all or a portion of the amino acid sequence of MM, identifying hybridized colonies, and retrieving the corresponding vectors from the library, using chromosome walking techniques if necessary to isolate and characterize the entire cDNA. Once the cDNA has been isolated, it may be expressed in a suitable expression system, e.g., a prokaryotic system such as E. coli. The MM may be isolated from the culture medium of the expression system, e.g., using the procedures outlined above.

Tframnlft ft Use of heparin as MM inhibitor Heparin is useful in the treatment or control of breast cancer due to its inhibitory effect on MM activity. Heparin for this use is preferably administered parenterally, most preferably by means of an implantable pump permitting long term, continuous intravenous infusion. Although heparin is relatively nontoxic, a trial dose of 1000 units should precede usual therapeutic dosages to confirm that the patient will not have an allergic reaction. Heparin preparations should be used which are designed for long-term administration, e.g., have low anticoagulant and angiogenic activity.

Fframirlfl 10 Therapeutic use of monoclonal antibody to MM Monoclonal antibodies to MM may be used therapeutically to block MM activity, thereby controlling or reducing the metastasis of breast cancer. Suitable dosages and forms of administration will be apparent to one skilled in the art.

Ffrflmplft 11 Diagnostic use of polyclonal or monoclonal antibody to MM Assay kits utilizing polyclonal or monoclonal antibodies (hereinafter collectively referred to as antibodies) to MM may be comprised of the following components: (i) antibody, preferably lyophilised; (ii) labeled (preferably radiolabeled) MM or fragment thereof; and (iii) MM standard containing a known amount of MM. If radiolabeled MM is used, it is preferably 125I-labeled MM, labeled to about 50-100 jiCi/fig.

The antibody is dissolved and incubated together with the labeled MM or fragment and either the sample to be assayed or the MM standard. Incubation takes place preferably at cool temperatures, e.g., about 4°C, and lasts for at least two hours, preferably 4-6 hours. The pH of the incubating mixture is preferably kept in the range of from 5 to 8, more preferably at 7 or 8, preferably with the aid of a buffering agent such as a citrate or trie buffer. After incubation, the fraction of labeled MM or fragment is separated from the unbound fragment, e.g., by the use of charcoal such as dextran-coated charcoal. The unbound fraction adsorbs to the charcoal and may then be separated by filtration or by centrifugation. The amount of radioactivity in one fraction is then measured by standard techniques, e.g., by liquid scintillation counting after the addition of a secondary solute. The proportion of labeled MM or fraction bound to the antibody is inversely proportional to the amount of MM in the unknown plasma sample. For quantitative analysis, a calibration curve may be prepared by analyzing solutions of MM of known concentration.

F/rnmplft 12 Animal models Nude mice inoculated with human mammary tumor cells are a preferred animal model for study of MM action and effects of blocking MM action by antagonists. For example, nude mice are inoculated with hormone-dependent mammary tumor cells, e.g., MCF-7 cells, and the developing tumors are treated with MM (infusion) in the absence and presence of MM blockers such as heparin, neutralizing MAbs, and MM receptor blockers, to evaluate the actions of MM in vivo on responder cells. Alternatively, nude mice are inoculated with hormone-independent mammary tumor cells, e.g., MDA-MB-231 cells, and the developing tumors are treated with heparin, blocking MAbs for MM, and MM receptor blockers to evaluate the role of MM in producer cell proliferation in vivo.

Table L Human tumor and normal mammary cells producing or responding to a factor (mammamodulin, MM) inducing membrane ruffling and cell motility. cells and expression of secretion of reaction to reaction to cell lines estrogen cell activating CM from purified receptors factor (^) MDA-MB-231 MM (2) MDA-MB-231 ATCCHTB 26 - + - - HBL-100 ATCC HTB 124 - + - - Hs578T(3) ATCCHTB 126 - + - - Hs578Bst (4) ATCC HTB 125 - (+) - - SK-BR-2 HI ATCCHTB 29 - + - - MDA-MB-453 ATCC HTB 131 - - - - BT-20 ATCCHTB 19 - - - - MCF-7 ATCC HTB 22 + + + ZR-75-1 ATCC CRL1500 + - + + ZR-75-30 ATCC CRL1504 + - + + T-47D ATCC HTB 133 + - + + MDA-MB-361 ATCCHTB 27 + - + + BT-474 (5) ATCC HTB 20 (+) - (+) + normal epithelial cells/ - (+) organoids normal stroma cells (*>) + (*) Results are based on the observation of morphological changes after stimulation of the mammamodulin responder cells ZR-75-1 and T-47D by conditioned media (CM) of the cells listed. Fresh serum-free medium was added to confluent cell cultures of each cell (grown serum-free) and collected 3 days later for the testing. The tests were performed as described in Example 6. "+" stands for unequivocally detectable cell activation which is inhibited by 100 H-g/ml heparin; (+) stands for marginal cell activation. (2) highly purified factor (fraction "m" of Fig. 6 ). (3) tumor and (4) myoepithelial cell lines of the same tumor specimen. (5) BT-474 cells express very low levels of ER and PR. (6) from dysplastic mammary tissues.

Hs578Bst, MDA-MB-453 and BT-20 are estrogen receptor-negative cells that have low growth rates in cell culture and produce only very low or undetectable amounts of mammamodulin activity.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: F.HOFFMANN-LA ROCHE AG (B) STREET: Grenzacherstrasse 124 (C) CITY: Baale (D) STATE: BS (E) COUNTRY: Switzerland (F) POSTAL CODE (ZIP): CH-4002 (G) TELEPHONE: 061 - €88 42 56 (H) TELEFAX: 061 - 688 13 95 (I) TELEX: 962292/965542 hlr ch (ii) TITLE OF INVENTION: NOVEL ORGANIC COMPOUNDS AND USES THEREFOR (ill) NUMBER OF SEQUENCES: 3 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: Apple Macintosh (C) OPERATING SYSTEM: System 7.1 (Macintosh) (D) SOFTWARE: Word 5.0 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Leu Val Leu Arg Xaa Xaa Glu Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (V) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Ser Glu Leu Arg He Asn Lys 1 5 INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO Xaa Leu Xaa Asn Pro Xaa Xaa Tyr Leu 1 5

Claims (5)

WHAT WE CLAIM IS: -26- 26 8 5 92

1. Mammamodulin in purified form which a) is a protein with a molecular weight of approximately 52 to 55 kilodaltons on 10-15% gradient SDS polyacrylamide gels; b) comprises partial amino acid sequences of Leu-Val-Leu-Arg-X-X-Glu-Thr (SEQ ID No: 1) Ser-Glu-Leu-Arg-Ile-Asn-Lys (SEQ ID No: 2), and X-Leu-X-Asn-Pro-X-X-Tyr-Leu (SEQ ID No: 3), wherein "X" represents an unidentified amino acid residue; c) is heat and acid labile and trypsin- and mercaptoethanol-sensitive; and d) is obtained from hormone-independent mammary tumor cells.

2. A DNA or cDNA sequence encoding mammamodulin according to claim 1.

3. Mammamodulin according to claim 1 for the production of polyclonal or monoclonal antibodies.

4. Mammamodulin according to claim 1 for identification of compounds which inhibit its activity.

5. A process for the production of mammamodulin according to claim 1 comprising the steps of a) culturing hormone-independent mammary tumor cells, and b) isolating the mammamodulin from the culture medium. 268592 -27- A process for the production of mammamodulin according to claim 1 comprising the steps of a) inserting DNA or cDNA according to claim 2 into a vector, b) transfecting a prokaryotic or eukaryotic cell line with the vector, c) selecting from the transfected cell line cells which express mammamodulin, d) culturing the selected cells in a suitable medium, and e) extracting the mammamodulin from the biomass. Compounds other than heparin, which inhibit the biological activity of mammamodulin according to claim 1. Compounds according to claim 7 which have at least one of the following activities: a) inhibition of mammamodulin expression in mammary tumor cells; b) affinity for mammamodulin; or c) affinity for mammamodulin receptors on mammary tumor cells. Polyclonal or monoclonal antibody which is able to recognize at least one epitope on mammamodulin according to claim 1. Antibody according to claim 9 which is monoclonal. A pharmaceutical composition for treatment and control of breast cancer comprising one or more of the following inhibitors of mammamodulin activity: a) a compound according to claim 7 or claim 8; and/or 268592 -28- b) antibody to mammamodulin according to claim 9 or claim 10; together with a pharmaceutically acceptable carrier or diluent. A kit for assaying mammamodulin levels comprising a polyclonal or a monoclonal antibody as claimed in claim 9 or 10. A method of identifying compounds which inhibit mammamodulin activity comprising the step of measuring the effect of the compounds to be tested on any one or more of the following parameters in test systems comprising mammamodulin and hormone-dependent mammary tumor cells: a) morphology of hormone-dependent cells; b) motility of hormone-dependent cells; c) proliferation of hormone-dependent cells; d) mitogenic activity in hormone-dependent cells; or e) conversion of hormone-dependent cells to hormone-independent cells. The use of mammamodulin according to claim 1 for the production of polyclonal or monoclonal antibodies. The use of mammamodulin according to claim 1 for identifying compounds which inhibit mammamodulin activity. Mammamodulin according to claim 1 whenever prepared by a process as claimed in claims 5 and 6. Mammamodulin according to claim 1, substantially as hereinbefore described with particular reference to any one of the foregoing Ex, 1_~ ° to 4 and 8. 268 5 92 -29- A process according to claim 5, substantially as hereinbefore described with particular reference to the foregoing Example 1 and any one of the foregoing Examples 2 to 4. A process according to claim 6, substantially as hereinbefore described with particular reference to the foregoing Example 8. DATED THIS A. J. PARK & SON PER AGENTS FOB TWF APPt.!nAMT$