CN1111910A - Mammamodulin, a hormone-independent mammary tumor cells specific protein - Google Patents

Abstract

Mammamodulin (MM), a novel 52-55 kDa protein expressed by hormone-independent mammary tumor cells, has been found to affect the morphology, motility, growth and hormone receptor expression of hormone-dependent mammary tumor cells. The presence of MM, e.g., in tumor biopsies, is an important diagnostic indicator. Inhibitors of MM activity, such as MAbs to MM and MM antagonists, are useful in the treatment and control of breast cancer.

Description

Mammamodulin, a hormone-independent mammary tumor cells specific protein

The invention provides the MM that produces by the non-hormone dependence mankind mastopathy cell, it can influence the expression of form, growth and the hormone receptor of hormone-dependent tumor cell, this is a kind of still undiscovered so far new factor that exists naturally, especially the invention provides the MM of purified form, promptly to the compound of small part removing, until the high-purity forms that can measure the part amino-acid sequence at least with its natural combination.Here introduced the production of MM and the method for purifying, and the discovery of having set forth this factor makes diagnosis and methods of treatment become possibility.

The growth of human breast cancer is the process of a complexity, is controlled by internal secretion, hormone and somatomedin that autocrine and paracrine produce.A key character of mammary cancer is exactly for hormone, especially estrogenic dependency.This hormonal dependent disappears along with the development of tumour usually, makes hormonotherapy invalid, and closely related with the neoplasm invasiveness enhancing.In a single day tumour enters non-hormone and relies on stage, mammary cancer patient's prognosis extreme difference.

Have now found that, called after MM (MM), the factor of not recognizing is in the past expressed by the non-hormone dependence breast cancer cell.When the expression of hormone receptor was suppressed, this factor stimulated the more weak hormone-dependent tumor cell growth of wettability to accelerate.Think at present that in the growth of tumor process in a very long time, MM plays an important role for the phenotype conversion that relies on non-hormone dependence from hormone.

This factor is differentiated, is separated and purifying first.Obtain various diagnosis and treatment means that the MM of purifying basically can be used as mammary cancer at present.For example, can measure the MM level and be used as diagnostic markers, to determine residing stage of mammary cancer and severity.The material that can suppress MM cellular activity characteristic, MM monoclonal antibody or other MM binding substances all can be blocked the hormesis of MM to the hormone dependent cells as medicine, can make cancer stable thus, the non-hormone of avoiding developing into tumor growth of wettability relies on the stage.In addition, the MM analogue can be used to seal the MM acceptor of hormone dependent cells aspect treatment.

MM is a protein, on the 10-15% gradient gel highly purified preparation is carried out sds polyacrylamide gel electrophoresis and records its apparent molecular weight and be about the 52-55 kilodalton.The partial amino-acid that MM contains is in proper order

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 amino-acid residue is not identified in " X " representative.

Think that at present as long as keep biologic activity, this protein can contain allelic variation on the whole, comprises interpolation, disappearance, insertion or the replacement of residue, variation can reach 10% of whole amino-acid sequences, but preferably is no more than 5%.This protein can also contain various posttranslational modifications, and for example, glucosides turns the formation of usefulness and halfcystine bridge into.

Another characteristic of MM is unstable to heat and acid, to trypsinase and mercaptoethanol sensitivity, and easily and other protein form inhomogenous polymer.

According to the method for being introduced in the example of the present invention, the cell culture extraction MM and the purifying that can rely on tumour cell from the non-hormone of quick growth become apparent uniform substance.The other method that obtains MM is the cDNA library that utilizes the information of aminoacid sequence to screen from production clone, obtains the cDNA of MM gene, can and import appropriate carriers with gene clone then, expresses in suitable prokaryotic cell prokaryocyte or eukaryotic cell system.

The MM of purifying can be used as antigen, produces polyclone or monoclonal antibody with conventional method.Antibody, especially monoclonal antibody can (ⅰ) be blocked MM as medicine in order to avoid stimulate the hormone dependent cells; (ⅱ) be used for detection method (comprising biological assay, radioimmunoassay (RIAs), fluorescence immunoassay (FIAs), western blotting test and ELISAs), can obtain valuable diagnostic message thus about cancer development stage and prognosis aspect to detect existing of MM in the mammary tumor living tissue.

The MM of purifying also can be used for screening and identify the compound that MM is had affinity in conjunction with test, can also be used for competitive trials and screen and identify the compound that has affinity for the MM acceptor of hormone dependent cells.These compounds can be used for suppressing the biologic activity of MM.

In addition, can suppress tumour cell, especially breast cancer cell, the compound that MM expresses in the non-hormone dependence breast cancer cell and/or for example for tumour cell, especially breast cancer cell, for example to have the compound of affinity all be new drug to the MM acceptor of non-hormone dependence breast tumor cell and have using value.Unique compound of the effective inhibitor of the known MM of can be used as is exactly a heparin at present.

Example 1

From the non-hormone dependence cell culture, produce and contain the MM conditioned medium

Human breast cancer cell line MDA-MB-231 grows in the HL substratum that has replenished 5% calf embryo serum.Press the same procedure that the open No.417563 of european patent application introduces, the preparation human leukaemia cell is the optimal medium of HL60.Cell in T shape culturing bottle at 5%CO ₂Grow in the adhesion mode under the condition of balanced gas and 96% water saturation degree, the doubling time is about 22-24 hour.Replace growth medium with the HL substratum that does not contain serum after being the fusion state, MM (MM) is discharged in the substratum in process of growth and when state is merged in non-growth.

(Chemap AG is Switzerland) with scale operation MM to adopt the 23l ventilation fermentation tank of improveing a little.The external volume of vent-pipe can obtain about 20m with Raschig ring (8 * 8 * 5mm, made of stones) and 10 Siraf25 rings (25 * 25 * 2mm, sintered glass Raschig ring, the Schott Germany) filling of 8.5kg ²Surface-area.Fermentor tank inoculation 2 Cell Factories units (Nunc, per unit 6000cm ²) through the MDA-MB-231 of trypsin treatment cell.Ventilation fermentation tank is with 1 standard liter/min air draft, and dissolved oxygen content (30%) is controlled in continuous monitoring, pH(7.2), and temperature (37 ℃).

Collect the substratum that exhausts that contains MM by changing the reinforcement HL substratum (5-7.5g glucose, 5-6.5mM glutamine, 0.25-0.3%Primatone RL) that 17-18 1 do not contain serum every day.In whole glycolysis process according to Schumpp B.and Schlaeger E.J., J.Cell.Sci.97: 639-647[1990] method the introduced growing amount of measuring glucose and glutamine consumption and lactic acid salt and ammonia monitors the metabolism state of cell.

The MM titre remains unchanged in the production process.

2 S10 Y10 volution filter core (1.8m are equipped with in employing ²Membrane area is 10KDa by molecular weight) Amicon SP20 ultra-filtration equipment (Amicon Switzerland) includes MM with the glycolysis jar but not celliferous nutrient solution concentrates 20 times (100 liters are concentrated into 5.5 liters).The concentrated solution that contains MM put-80 ℃ freezing.

Example 2

With the four one-step chromatographies MM that from partially purified MDA-MB-231 cell conditioned medium liquid, purifies

If there is not other explanation, all operations all carries out under room temperature (RT).Component to each flow process purifying is all carried out specificity analysis, and method is to measure the light absorption ratio at 280nm place, adopts Laemmli, and U.K.Nature 227,680-685(1970) Determination of biological activity of introducing in reduction SDS-PAGE method of Jie Shaoing and the example 6.

5.5l refrigerated concentrated supernatant (press the method preparation of introducing in the example 1, be equivalent to the 100l original supernatant) is dissolved centrifugal 15 minutes of 3000g (4 ℃).280nm, the light absorption ratio of supernatant liquor multiply by volume milliliter number (=OD when path length was 1cm ₂₈₀) equal 35000.The gross activity of measuring is 5.8 * 10 ⁶U(100%), specific activity is 165U/OD ₂₈₀

Then with the supernatant liquor following material of 12l

The 8M urea

0.3mM 3-{ (3-courage amido propyl)-dimethylamino }-1-propanesulfonic acid salt (CHAPS)

30mM Tris/HCl＜buffer A＞

pH 7.5

With 1.7 1 distilled water dilutings.(Switzerland), this chromatography column (1.2 l, 4-8 ℃) is used for Pharmacia, Dubendorf this solution to be added Q-sepharose FF chromatography column again

The 6M urea

0.2mM CHAPS

20mM Tris/HCl＜buffer B＞

pH?7.5

Pre-equilibration.With the buffer A of 1 column volume washing chromatography column, then in 7 hours 36 minutes with the following substance solution of 100% buffer B to 40% buffer B and 60%:

The 6M urea

0.2mM CHAPS

20mM Tris/HCl＜damping fluid C＞

500mM NaCl

pH 7.5

Carry out linear gradient elution (10ml/ minute, RT).Damping fluid C concentration is that the outflow component (1550ml) between the 30-50% time contains 2630 OD ₂₈₀, 7.5 * 10 ⁶U(130%), specific activity is 2852U/OD ₂₈₀Chromatogram is seen Fig. 1.Component is merged, and with being furnished with the Millipore Minitan System(Millipore of 4 PTGC chambers, Volketswil Switzerland) is concentrated into it 210ml(4-8 ℃).

Concentrated solution is used the solution of forming by following material

1mM N-dodecyl-N, N-dimethyl-3-aminopropanesulfonic acid salt (Zwittergent 3-12)

50 μ M ethylene glycol-two (β-aminoethyl ether) N, N, N ', N '-four acetic acid (EGTA)

50mM Tris/HCl

150mM NaCl＜damping fluid D＞

0.02% NaN ₃

pH 7.6

Carry out dilution in 1: 15, final volume is 3150ml.With the Heparin-agarose gel cl-6b chromatography column (Pharmacia, 4-8 ℃) of a 80ml of this diluent adding, this post has been used damping fluid D pre-equilibration.With the damping fluid D of 24 column volumes washing chromatography column, carry out then wash-out (3ml/ minute, RT) wash-out is to be changed to 100% the solution of being made up of following material with 100% damping fluid D:

1mM Zwittergent?3-12

50 μ m EGTA＜damping fluid E＞

50mM Tris/HCl

1350mM?NaCl

0.02% NaN ₃

pH 7.6

Carry out linear gradient elution.Damping fluid E be 39% between 64% the elution fraction (240ml) during concentration contain 19.1 OD ₂₈₀, 7.7 * 10 ⁶U(134%), specific activity is 403000U/OD ₂₈₀(chromatogram is seen Fig. 2).Component is merged, and (Amicon, Zurich Switzerland) are concentrated into 80ml with it with the 400ml Amicon agitated pool that the YM10 filter membrane is housed again.To wherein adding the solution that 80ml is made up of following material:

The 6M Guanidinium hydrochloride

2.5mM Zwittergent?3-12

50 μ m EGTA＜damping fluid F＞

50mM?Tris/HCl

pH 7.5。

Concentrated/dilution program repeats 3 times.With solution concentration to final volume 2.6ml.Concentrated solution is added Superdex 200 chromatography columns (Pharmacia 1.6 * 60,120ml, 1ml/ minute).Chromatography column has been used damping fluid F pre-equilibration.Column volume (20ml) 47% to 63% between elution fraction contain 3.8 OD ₂₈₀, 4 * 10 ⁶U(69%), specific activity is 1,050,000 U/OD ₂₈₀(chromatogram is seen Fig. 3).Component is merged, with the 50ml Amicon agitated pool that the YM10 filter membrane is housed it is concentrated into 5ml again, contain the solution of following material again with 5ml:

The 6M urea

0.3mM Zwittergent?3-12

20mM Tris/HCl＜damping fluid G＞

pH 7.5

With its dilution, concentrate/dilute and repeat once, to 1.6ml, doing then and being diluted to final volume at 1: 30 is 48ml with solution concentration.This solution is added the Mono S chromatography column (1ml Pharmacia) of using damping fluid G pre-equilibration, damping fluid G washing chromatography column with 12 column volumes, wash-out (0.5ml/ minute) then, the used linear gradient of wash-out are changed to 100% the solution of being made up of following substances by 100% damping fluid G:

The 6M urea

0.3mM?Zwittergent?3-12

20mM Tris/HCl＜damping fluid H＞

500mM?NaCl

pH 7.5

Damping fluid H is that 36% elution fraction between 56% time contains 0.27 OD ₂₈₀, 4.4 * 10 ⁶U, specific activity are 16,000,000U/OD ₂₈₀(chromatogram is seen Fig. 4).By the activity overall yield is 76%, and purification factor is 97000.Fig. 5 has shown the protein composition of the component of analyzing with SDS-PAGE of studying.The SDS-PAGE(reduction of employing 12.5%T) analyzes and uses Coomassie R ₂₅₀During dyeing, biologic activity is relevant with the intensity of protein band, and protein band intensity illustrates that its molecular weight is between the 41-45KD.To be further purified (seeing example 4) to the part component.

Example 3

A) with five one-step chromatographies from MDA-MB-231 cell conditioned medium liquid purifying MM to high purity

With with example 2 described similar experiment method purifying MM from the 130l supernatant liquor.Different with example 2 is to contain more MM in the omnidistance effluent liquid of Q-sepharose chromatography column, therefore, need carry out this chromatography repeatedly with omnidistance effluent liquid, also will carry out the further chromatography of 2 flow processs respectively with Heparin-sepharose and Superdex 200 chromatography columns.The gradient of Mono S column chromatography is improved as follows:

Be changed to 40% damping fluid H by 0% damping fluid H in 48 minutes

Be changed to 60% damping fluid H by 40% damping fluid H in 120 minutes

Be changed to 100% damping fluid H by 60% damping fluid H in 46 minutes

Damping fluid H is that 42% elution fraction between 45.5% time contains 6.1 OD ₂₈₀4.1 * 10 ⁶U, specific activity are 6.7 * 10 ⁶U/OD ₂₈₀

For being further purified, adopt the 10ml Amicon agitated pool that the YM10 filter membrane is housed that these components are concentrated, volume is reduced to 0.7ml from 11ml.Add 2.5ml damping fluid F, again volume is concentrated into 0.7ml.Dilution and concentrated the repetition once.The sample of 0.7ml is added Superdex 200 chromatography columns (1 * 30cm, 0.5ml/ minute) (chromatogram is seen Fig. 6) of using damping fluid F pre-equilibration.Adopt the Fast Desalting chromatography column (Pharmacia) that is connected with the Pharmacia SMART System that 50 μ l aliquots containigs of obtained component are transferred among the damping fluid G.The gained sample reduces with SDS-PAGE(, 12.5%T, Fig. 7 is seen in Coomassie dyeing) analyze.Main ingredient (m) only shows a protein band in the scope of 41-45KD.Yield is 0.12 OD in this component ₂₈₀, activity is 4 * 10 ⁶U, specific activity are 33 * 10 ⁶U/OD ₂₈₀By the activity total recovery is 36%, and purification factor is 170,000.

Will main ingredient (m) afterwards the component n of wash-out be further purified (seeing example 4).

B) with eight one-step chromatographies from MDA-MB-231 cell conditioned medium liquid purifying MM to high purity

With with example 2 described similar experiment methods, with the following step (omit Q-sepharose column chromatography) purifying MM from the 146l supernatant liquor: with the dilution of 6.6l concentrated solution and add Heparin sepharose chromatography column (80ml), with 1mM CHAPS as washing agent.With the active ingredient dilution and the adding Heparin sepharose chromatography column (50ml) of wash-out, use 1mM CHAPS as washing agent.With the additive of 2mM spermidine as all damping fluids active ingredient is merged, the damping fluid dilution with containing 4mM.Zwittergent 3-12 washing agent adds Heparin sepharose chromatography column (10ml) again.Active ingredient (8ml) is merged, behind additional 6M Guanidinium hydrochloride, divide 4 flow processs Superdex 200 chromatography columns (120ml) purifying, every flow process 2ml.Heparin sepharose chromatography column with 10ml and 2ml merges active ingredient in two steps, dilution and concentrated.Activity is the highest by (2 * 10 in the final step ⁶U/ml) component is used for the described growth experiment of example 5C.(10-15%, Phast Gel Pharmacia) analyze this component, occur the master tape of a treaty 52KDa after silver dyeing with SDS acrylamide gradient gel.

Example 4

With the MM purifying is apparent even thing

The material (component f to h) that obtains according to method A purifying example 2, the component n that obtains according to method B purifying example 3 is because salt concn is very high in the sample (damping fluid F).

Method A: do 4 times of dilutions to reduce the ionic strength of example 2 gained materials, use SMART chromatographic system (Pharmacia) again at Mono S(PC 1.6/5) (Pharmacia, Uppsala Sweden) on the chromatography column concentrate it.

Chromatography column with 100 μ l/ minutes flow velocity and by damping fluid G be changed to damping fluid H(damping fluid composition see example 2 ') linear gradient (12.5%/minute) launch.Component is collected by 50 μ l are a, and active ingredient (example 6 is seen in determination of activity) is merged.The typical chromatography spectrum signature of MM concentration is seen Fig. 8.

Method B: the following damping fluid of example 3 gained materials:

The 0.1% TFA aqueous solution＜damping fluid J＞

0.09% TFA acetonitrile solution＜damping fluid K＞

At reversed-phase partition chromatography post PorosR/H(Perseptive Biosystems, Cambridge, MA USA) (internal diameter 0.8mm, length 10cm) upward concentrates, this chromatography column LC Packings, Amsterdam, Netherlands filling and the SMART chromatographic system of producing with Parmacia.

Chromatography column is with 50 μ l/ minutes flow velocity, and launched by the linear gradient (5%/minute) that damping fluid J is changed to K.Component is collected by 25 μ l are a, and active ingredient is merged.

With the sepharose method (ProSieve Gel System from FMC Bioproducts, Rockland, ME, U.S.A) separation method A and B gained sample adopt high-quality magnitude SDS according to manufacturers explanation electrophoretic analysis under non-reduced condition.

After finishing, electrophoretic analysis from the positive pole to the negative pole, sepharose is cut into the thick thin slice of 2mm, with the solution that contains following material

The 6M urea

20mM?Tris/HCl，pH?7.5

0.3mM Zwittergent 3-12＜damping fluid I＞

0.3mM?NaCl

At room temperature elute protein is at least 4 hours.

The gel elutriant of available thin slice corresponding to 45-55KDa carries out determination of activity, and active eluant is merged.

The material that merges is added reversed phase chromatography post Poros R/H(Perseptive Biosystems, Cambridge, MA U.S.A) (footpath 0.8mm, length 10cm) this chromatography column is by LC Packings, Amsterdam, Netherlands preparation and with SMART chromatographic assay system (Pharmacia).

Chromatography column is with 50 μ l/ minutes flow velocity, and launched by the linear gradient (2.5%/minute) that damping fluid J is changed to damping fluid K.Component is collected by 12 μ l are a, merges active ingredient (seeing example 6).Separate the chromatogram feature of MM with the reversed-phase partition chromatography post and see Fig. 9.

Under non-reduced and reductive condition with the aliquots containig of purified product and lower molecular weight standard protein (for example, can be from Bio-Rad, Hercules, CA USA, 8ng/ swimming lane/protein, swimming lane 1) application of sample is in sds gel (Phast Gel 10-15%:Pharmacia) together, and (Figure 10 a) with silver dyeing.1-3 scans to swimming lane, and it the results are shown in Figure 10b.The purified product of 230 PM is as sequence analysis.With the MM reduction, S-carboxyl-amino methylization is used protein incision enzyme Lys-C(Wako again, and Neuss is Germany) 37 ℃ of digestion 16 hours.The ratio of enzyme-to-substrate is 1: 100.(3 * 0.5cm) collect Digestive system, and this post has been used the 20% acetonitrile potassiumphosphate balance of pH2.5 with Mini S cationic exchange coloum.Post is launched with gradient NaCl, collect each chromatographic peak component.Use Vydac C ₄(0.1 * 15cm) or Brownlee RP-300(0.1 * 10cm) the reversed-phase partition chromatography post is further purified the peak component, water/acetonitrile-0.1%TFA gradient development, (Applied Biosystems, Foster City CA) carry out sequence analysis with 475A or 477A sequence analyser again.The amino-acid sequence of three kinds of peptides is as follows:

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），

" X " represents an order-checking cycle, and wherein this amino acid is not identified.

Example 5

MM is to the influence of hormone dependent cells

A) morphology: the restricted substratum of ZR-75-1 hormone dependent cells with non-hormone dependence mankind mastopathy cell MDA-MB-231 acted on mutually, and the inductive morphological change is seen the Photomicrograph of Figure 11.Tissue Culture Dish with type overlay by, inoculate therein the hormone dependent cells (ZR-75-1, for example, can be from U.S. typical case culture collection center (ATCC), Rockville, Maryland, USA), 50,000/cm ², growth is 3 days under the condition that does not contain serum.Replace former substratum with the blood serum medium that do not contain of non-hormone dependence MDA-MB-231 cell restriction then.(Figure 11 a) stimulates back 15 minutes (Figure 11 b), 35 minutes (Figure 11 c) to carry out position photomicrography mutually continuously at once with 3 hours (Figure 11 d) before changing substratum.Photomicrography shows that unprovoked hormone dependent cells is the growth of epithelial cell sample patch in cultivation, and iuntercellular closely links to each other, and (Figure 11 a).Stimulate with the restricted substratum that contains MM of producing cell or with the protein purification thing of each purification phase that peripheral cells presents the film shrinkage and begins to produce [(Figure 11 b) in afterwards several minutes.Because cell becomes flatly, and cell colony obviously increases, iuntercellular connects die down (Figure 11 c and Figure 11 d) after this.The activity of the reacting cells that is stimulated by highly purified MM can last for hours, and cell presents original patch shape again then.The time that morphological change continues is depended on the concentration of MM, continues more of a specified duration during high density.As show shown in the I, all can be observed cellular activity at a variety of hormone dependent cells.All there is disclosed source in all cells system of table in the I, U.S. typical case culture collection center (ATCC) for example, Rockville, Maryland, USA.

For the stimulation of purifying MM or MDA-MB-231 cell restriction substratum, non-hormone relies on not genetic morphology change of mankind mastopathy cell MDA-MB-453, does not also secrete the factor that causes ZR-75-1 or the shrinkage of T-47D cytolemma.

B) mobility: present the of short duration random motion activity relevant after the non-hormone dependent cells substratum of hormone dependent reaction cell and fast growth or purifying MM act on mutually with fibrocyte like cell form.Because this inductive mobility, the cell spot that forms after reconnecting may be by stimulating the identical cell of patch before to form with MM.

C) propagation:

1.MM influence to the cell proliferation of estrogen receptor positive mankind mastopathy cell T47-D.

In spreading in the 96 porocyte culture dish of quilt with type in advance, density is 10,000 cells in every hole, does not have in the phenol red medium at serum-free and grows with hormonal dependent T47-D cell inoculation.Adopt the MM (pressing the preparation of example 3B method) of purifying, concentration is 20U/ml.In contrast, measured mitogen under the condition that MM does not exist and exists, be I type rhIGF-1 (IGF-I) and estradiol (E2) and growth inhibiting factor, i.e. the effect of tumor necrosis factor-alpha (TNF-α) and interleukin 1 α (IL-1).After 5 days according to Kung et al., Analyt.Biochem.182, the method that 16-19 1989 introduces is measured the cell quantity in the hole.Soluble dye (Viola crystallina) is in the optical density(OD) and the cell quantity linear dependence at 590nm place, and this dyestuff absorbs for the fixed cell when dyeing.

As shown in figure 12, to stimulate the hormone dependent cells be the propagation of T47-D to the MM of purifying.Bar is represented the mean and the standard deviation of four replications in the figure.The IGF-1 of used concentration is irritation cell to greatest extent, and MM does not have additional effect for cell proliferation.The effect of simple estradiol cell growth is little, and MM is accelerated these cell growths.Add MM simultaneously TNF-α and IL-1 are reversed for T47-D inhibition of proliferation effect, can partly reverse at least.

2.MM influence for the cell proliferation of estrogen receptor positive mankind mastopathy cell MCF-7

With the MCF-7 cell inoculation in micropore Tissue Culture Dish (Falcon), 7,000 cells in every hole do not have phenol red condition [Kung et al. at the serum-free of stipulating, Contr.Oncol.23,26-32(1986)] and do not have and have 25U/ml MM or 1 * 10 ^-10Grow under the M estradiol condition.The used MM preparation of this experiment be as shown in Figure 9 by the isolating pure substance (merging material) of reversed-phase partition chromatography post.The aliquots containig of this material is that wash-out goes out afterwards at once with not containing the blood serum medium dilution from chromatography column.Give the nutrition of culture restock after 3 days.With cell fixation, with above-mentioned Kung et al., the Viola crystallina method that (1989) are introduced is measured its quantity after 7 days.Soluble dye (Viola crystallina) is in the optical density(OD) and the cell quantity linear dependence at 590nm place, because this dyestuff is that fixed cell absorbs when dyeing.

As shown in figure 17, the growth of 25U/ml MM intense stimulus MCF-7.Also use estradiol (E2) irritation cell, as the contrast of cell normal function.The increase of irriate cells in culture quantity is to use the percentage ratio of the data that obtain from control cultures to represent in Figure 17.Numerical value is represented the mean and the standard deviation of four replications.

D) mitogen activity:

Carry out cell cycle analysis with the flow cytometry method, the result shows that MM can induce synthesis phase the ratio of (S) hormone dependent cells obviously to increase, experiment at cell cycle analysis began preceding 2 days and preceding 1 day, hormone relies on MCF-7, and the culture of ZR-75-1 and T47-D cell does not have the phenol red medium washed twice with serum-free.In substratum, replenish the purifying MM preparation (taking from the merging component of reversed phase chromatography post among Fig. 9) of 25 units then.Stimulate back 24 hours harvested cells, adopt the standard scheme that is fit to FACScan analyser (Becton Dickinson, CellFIT program) to analyze.The result shows the per-cent with purifying MM processing can significantly increasing S-phase cell in MCF-7, ZR-75-1 and the T47-D cell, compares with control cells, and the MCF-7 cell increases by 186%, and the ZR-75-1 cell increases by 243%, and T47-1) cell increases by 161%.For the basis of comparing between MM and the known mitogen of these cells is provided, cell is used rhIGF-1 (IGF-I, 10 of high density ^-8M) or estradiol (3 * 10 ^-9M) handle.The numerical value that is increased by IGF-I or estradiol inductive S-phase cell is: the MCF-7 cell is respectively 176% and 158%, and the ZR-75-1 cell is respectively 280% and 180%, and the T47-D cell is respectively 203% and 105%.

E) conversion of hormone dependent cells: the non-hormone dependent cells of fast breeding is the restricted substratum that the contains MM level of the estrogen receptor of inhibitory hormone dependent cell (ER) significantly of MDA-MB-231.MCF-7 cell and the substratum that contains MM (30 units/ml) act on 2 days, its estrogen receptor quantity reduces to the standard deviation of about 55% ± 10%(5 time measured value), handle in the same manner, the estrogen receptor quantity of ZR-75-1 cell is about 32% ± 20%, above result relatively obtains with undressed cell, and its measuring method is the test that combines of adopting tritiate oestrogenic hormon (17-β estradiol) and intact cell.

Simultaneously proved that also MM can suppress the expression of estrogen receptor mRNA in the MCF-7 hormone dependent cells.In serum-free and no phenol red culture with the highly purified MM(example 2 of 20 units/ml, Mono S component g to h) stimulate MCF-7 cell (2.5 * 10 ⁶), perhaps MM exist or non-existent condition under with 1 * 10 ^-9The M estradiol stimulates.Handle back 5 hours harvested cells, extract the RNA in the cell then, with glass yarn chromatography column (Pharmacia) purified mRNA of pre-preparation.Measure mRNA concentration, then each extract 2.5 μ g application of sample is also separated in sepharose.Transfer on the film (RNA blotting) and hybridize the mRNA combination that to carry ER information with human ER probe (Figure 13) afterwards.ERmRNA expresses (Figure 13, swimming lane 1) in the MCF-7 control cells.10 ^-9The M estradiol can stimulate the expression (Figure 13, swimming lane 2) of ERmRNA.MM is 10 ^-9The M estradiol not under the existence condition under (Figure 13, swimming lane 3) and the existence condition (Figure 13, swimming lane 4) all can significantly reduce the expression of mRNA.

In another one experiment, in serum-free and the no phenol red culture 2.5 * 10 ⁶Individual cell is with highly purified MM(example 3, the component among Fig. 6 " m "), 1 * 10 ^-10M estradiol or both merge thing to be stimulated 24 hours, at this moment extracted mRNA in the cell and the QuickPrep trace mrna test kit produced with Pharmacia comes purifying.The mRNA preparation separates with 1.1% sepharose, adopts the RNA blotting that it is transferred on the nitrocellulose membrane, with ER probe and blot hybridization with radioactivity.Result (Figure 14) shows that estradiol can suppress the expression of mRNA for ER, and MM can obviously reduce the mRNA level of ER.This effect strengthens when estradiol and MM are incubated simultaneously.

MCF-7 cell extract and enzyme immunity test (EIA) with irriate are measured ER and PgR (PR) albumen.Under the 5%FCS existence condition with the MCF-7 cell inoculation in 12 porocyte culture dish (Costar), density is 150,000 cells in every hole.Replaced former substratum with serum-free and no phenol red medium in second day.Used MM(200U/ml after one day) or estradiol (1 * 10 ^-9M) stimulate the interior cell of parallel hole.Remove substratum after 6 and 48 hours, cell is freezing at least 12 hours in-70 ℃.Dissolve the back adding and contain 500mM KCl, 10mM KH ₂PO ₄, the 225 μ l extraction damping fluid of 1.5mM EDTA and 5mM Sodium orthomolybdate, according to Madedu et al., the method for introduction [Eur.J.Cancer Clin.Oncol.24,385-390(1988)] extraction cell is adjusted to 7.4 with 1MKOH with pH.Facing with the preceding concentration that now adds is 0.01% beta-mercaptoethanol.4 ℃ of placements of damping fluid.After 90 minutes, with 1ml Eppendorf pipe with extraction liquid with 12,000xg, 4 ℃ centrifugal 5 minutes.Collect supernatant liquor, get 100 μ l extraction liquids and be used for receptor determination.Use Abbott Laboratories, North Chicago, IL, the EIA test kit that USA produces is measured the level of ER and PR.Result (Figure 15) shows, stimulates back 6 hours with MM, and compares, and the ER protein level of MCF-7 cell has decline slightly, sharply descends after 48 hours.When cell stimulated with MM, the PR level was all similar to contrast on above-mentioned two time points.

As the contrast of MCF-7 cell normal cell function, also measured ER and PR level in the cell of handling with estradiol.Present known estradiol (E2) can suppress the ERmRNA and the proteic expression of ER of MCF-7 cell, but the expression that can stimulate PR.And the activation [Ree et al., Endocrinology 124,2577-2583, (1989)] of estradiol to ER depended in the expression of PR.Control experiment shows that estradiol reduces the proteic expression of ER, but can stimulate the proteic expression of PR.These results of MCF-7 cell and above-mentioned Ree et al. and Read et al.[Molec Endocrinol.3, the 295-304(1989)] data consistent that obtains.

F) tyrosine phosphorylation effect of the epicyte protein of hormone dependent cells: the component " m " of measuring purifying MM(Fig. 6) to the effect of the tyrosine phosphorylation effect of MCF-7 membranin.

The MCF-7 cell inoculation is in 24 hole Falcon Tissue Culture Dish (2cm ²/ hole) in to contain 5% calf embryo serum (FCS) density be in the cell culture medium of 150,000 cells/well.Replace former substratum with containing the 1%FCS substratum after 24 hours.After one day, before stimulating with serum free medium with cell cultures 2 hours.With concentration be 20 and the MM of 200U/ml handle cell, comprise unprovoked contrast.Put in 37 ℃ of cell cultures incubators after 30 minutes, remove substratum, add the reduction sample buffer in 100 μ l/ holes, put in the baking oven 100 ℃ of heating extraction in 5 minutes cell for the sds polyacrylamide gel electrophoresis analysis that contains 4%SDS and 2.5% mercaptoethanol.The EGFR sample for reference of getting each extraction liquid 60 μ l and aliquot adds Tris-Tricine microgel (6%) and also separates.In Western blotting with the 10mM CAPS damping fluid that contains 10% methyl alcohol pH11.0 with protein transduction move on to pvdf membrane (Bio-Rad Laboratories, Hercules, CA) on, use anti-tyrosine phosphatase salt antibody detection protein matter then.Second antibody with alkali phosphatase enzyme mark detects tyrosine-phosphorylated protein, this antibody is from Upstate Biotechnology Incorporated(UBI, Lake Placid, NY, product No.17-105), and according to the method that the manufacturer recommends use.The result shows, stimulates phosphorylation EGF acceptor object of reference that A431 obtains relatively with standard molecular weight with EGF, and MM can stimulate the special phosphorylation of the tyrosine of MCF-7 cell membrane protein, and the apparent molecular weight of membrane protein is about 180-190KDa(Figure 16).The characteristic of phosphorylated protein is not analyzed.

G) improve with cell surface EGF expression of receptor and erbB2 level behind the MM stimulation hormone dependent cells: 12 * 10 ⁶The MCF-7 cell is growth 1 day in serum-free in the plastic culture dish of 10cm and the no phenol red cell culture medium at diameter, again with component " m " among highly purified MM(Fig. 6 of 30U/ml or 100U/ml) stimulation 24 hours.Mouse monoclonal antibody (Ab-1 with flow cytometry method and territory, anti-human EGF recipient cell surface, Oncogene Science, Manhasset, NY) or the mouse monoclonal antibody in anti-erbB 2 cell surface territory measure the quantity of surface receptor, and with goat-anti mouse second antibody (the Becton Dickinson of fluorescein isothiocyanate (FITC) mark, San Jose, CA) pair cell bonded mouse anti EGF receptor antibody or anti-erbB 2 antibody dye.Scrape cell from culture dish,, use micro-cell counter (Sysmex F-300, TOA Medical Electronics, Kobe, Japan) counting again with the serum free medium washing.To containing 10 ⁶Add 20 μ l first antibody solution in the 80 μ l serum free mediums of individual cell, ice is deposited and is hatched 45 minutes, uses the cold substratum of serum-free with cell washing 3 times, with 100 μ l resuspending then, the goat anti-mouse antibody that adds 4 μ l FITC marks is put dark place ice and is deposited and hatch 45 minutes.Then use the serum free medium washed cell 2 times,, analyze with flow cytometry (FACScan, Becton Dickinson, San Jose CA) under the room temperature with 250 μ l serum free medium resuspending.Can obtain the column diagram of the FITC-fluorescence of each suspension, data are expressed as the mean fluorecence port number.

Have to have 90% in all cells at least by fluorescent antibody staining, this can measure by cell quantity and confirm, because fluorescence intensity increases in flow cytometry gate pulse point diagram.Combined with fluorescent is calculated as follows, and the fluorescence intensity of the sample of hatching with first antibody (antireceptor antibody) and FITC traget antibody deducts the fluorescence intensity of the cell of simple usefulness second (FITC mark) antibody incubation.

Figure 18 and Figure 19 have provided experimental result.The mean that numeric representation is measured for 3 times.EGF receptor level receptor level than control cells after 24 hours exceeds about 3 times (Figure 18) in the hormone dependence MCF-7 cell that stimulates with MM.Stimulate ErbB2 level rising 35%(Figure 19 after 24 hours with the MM of 100U/ml).

Example 6

MM determination of activity in the sepn process

The MM determination of activity is to induce to the basis so that the clone T47-D morphology that contains estrogen receptor is fast-changing.The T47-D cell grows in and does not contain in serum and the phenol red cell culture medium, has replenished transferrin in this substratum, the bovine serum albumin of low levels Regular Insulin and 1mg/ml (the Albumax I of Gibco).Inoculum density is 25,000-35,000 cell/cm ²Inoculate that cell is used for the MM determination of activity after 2 days.Similar to the ZR-75-1 cell to the T47-D cell of MM reaction (comparing with Figure 11 a), but the easy a bit and more processing of sensitivity slightly are because this cell combines more closely with substrate.Add an amount of solution that contains MM in the hole of containing cell to be detected, then culture dish is put back to the cell cultures incubator at once, the temperature that maintains the standard is carried out cell cultures for 37 ℃.After 10-15 minute, after 30-45 minute, amplify 200 times with phase microscope and check that cell is so that induce the formation of film shrinkage and [for the second time.Activity can be divided into five levels: a) non-activity (-), b) weak and of short duration, but conspicuous film shrinkage all appears in many cell spots forms (+), c) many cell spots have [formation and all cells spot film shrinkage (++) all to occur, d) all cells spot all has [to form and many cell spots have big [to form (+++), and e) all cells spot intensive film shrinkage occurs and big [forms (++ ++).The cytoactive situation of last 5 grades in 1: 2 diluent of this grade classification of measurement result and substratum that contains MM or chromatography component roughly conforms to.Specify (+) active extent of dilution to be defined as every milliliter and contain 1 MM of unit.

Measurement result also has some variation, depends primarily on the practical situation (cell density, incubation time) of cell culture.At other time point with measure active its difference of MM with other culture of T47-D cell and be not more than 50% to 200% usually.

Example 7

The antibody of anti-MM

MM has antigenicity for non-human mammal, therefore, just produces antibody after animal inoculation pvaccination MM.5 μ gMM just are enough to inducing mouse for two doses and produce antibody.From being isolated polyclonal antiserum the blood of inoculation animal.

Kohler-Milstein method with routine produces the MM monoclonal antibody: injection MM makes the animal immune of suitable kind, acquisition is to the antibody producing cells (being splenocyte) of MM sensitivity, merge to keep the function that goes down to posterity of antibody-producting cell with suitable myeloma cell line, from the selectivity immortal cell line of setting up thus, separate monoclonal antibody.Preferably use Titremax(CytRx, 150 Technology Parkway, Technology Park Atlanta, Norcross, Georgia 30092) to female Balb/c injected in mice MM, peritoneal injection administration.The back duplicate injection of two weeks is measured antibody, for example available protein blotting after all blood sample collections.The mouse that antibody horizontal is the highest kills, and uses PEG4000 to merge to keep its function and it is distributed in 24 * 24 holes of going down to posterity its splenocyte and mouse (Balb/c) myeloma cell.Screening is the hybridoma system of foundation thus, chooses to be used for producing.

Example 8

The clone of MM and expression

Poly (A) with the MDA-MB-231 cell ⁺RNA sets up and guides synthetic cDNA library at random, can therefrom separate MM cDNA.Form the MM fusion rotein with carrier (for example λ ZAP II carrier), use MM antibody screening expressed protein again.Another kind method is to screen the cDNA library with MM amino-acid sequence information, for example use the clone hybridization technology, expression library in expression system (preferably E.coli) for example, dissolving clone (for example on nitrocellulose filter), original position DNA sex change also makes it fixing on filter paper, with the mark that contains 24 base pairs at least (preferably radio-labeled) oligonucleotide probe hybridization, this probe has the cDNA base sequence consistent with all or part of MM amino-acid sequence, differentiate the hybridization clone, the corresponding carrier of reentrying from the library adopts the chromosome walking technical point from whole cDNA and analyze its characteristic in case of necessity.Just can in suitable expression system, express in case isolate cDNA, for example, the prokaryotic cell prokaryocyte system as E.coli.Separablely from the substratum of expression system go out MM, for example, adopt the method for above general introduction.

Example 9

With heparin as the MM inhibitor

Because heparin active inhibited for MM, so it is of great use for the treatment and the control of mammary cancer.Heparin application in this respect is the enteron aisle external administration preferably, and the most desirable mode is to place secular implantable pump intravenously to continue input.Although heparin is nontoxic relatively, the test dose of 1000 units also is to be higher than common therapeutic dose, therefore will guarantee that patient anaphylaxis can not occur.The application of heparin preparations need design the scheme of long term administration, for example has the former activity of lower anticoagulation and blood vessel.

Example 10

The therepic use of MM monoclonal antibody

The MM monoclonal antibody can be used to block the activity of MM aspect treatment, thus the transfer of may command or minimizing mammary cancer.Suitable dosage and mode are conspicuous for the technology expert.

Example 11

The diagnostic uses of MM polyclone or monoclonal antibody

Use the detection kit of MM polyclone or monoclonal antibody (following general designation antibody) can comprise following composition: (ⅰ) antibody, preferably freeze-dried preparation; (ⅱ) through MM or its segment of mark (preferably radio-labeled); (ⅲ) the known MM standard substance of MM content.If adopt radio-labeling MM, preferably ¹²⁵The MM of I-mark, the about 50-100 μ of labelled amount Ci/ μ g.

With antibody dissolving and with mark MM(or segment) and sample to be determined or MM standard substance be incubated.Insulation is preferably under the low temperature (as about 4 ℃) to be carried out, and continues at least 2 hours, preferably 4-6 hour.The pH of insulation mixed solution preferably remains in the scope of 5-8, and more suitable is 7 or 8, preferably uses such as buffer reagents such as Citrate trianion or Tris damping fluids and adjusts.After the insulation MM of mark or segment part are separated with binding fragment not, for example available such as with the dextran bag by charcoal isoreactivity charcoal.Bound fraction is not adsorbed on the gac, and available then filtration method or centrifuging are separated.The available standards technology is measured the amount of radioactivity in the component, for example uses liquid scintillation counting behind the auxiliary solute of adding.Mark MM or with the ratio of antibody-binding fraction and unknown blood plasma sample in the amount of MM be inversely proportional to.Can pass through the known MM formulations prepared from solutions of analytical concentration typical curve during quantitative analysis.

Example 12

Animal model

For the effect of research MM with the effect aspect of antagonist blocking-up MM effect, the exposed mouse that inoculates the mankind mastopathy cell is suitable animal model.For example, hormone is relied on breast cancer cell (as the MCF-7 cell) to be transplanted to exposed mouse, at MM blocker (for example heparin), in and treat the tumour that infusion is just forming with MM under MAbs and the MM receptor blocking agent condition that do not exist and exists, with evaluation MM in vivo to the effect of responsive cell.Another kind method is with non-hormone dependence breast cancer cell (as the MDA-MB-231 cell) inoculation nude mice, with heparin, MM is had the MAbs of blocking effect and the tumour that the MM receptor blocking agent is treated formation, to estimate MM in vivo to the effect of production cell proliferation.

Table 1:

Generation induce film shrinkage and the cell movement factor (MM, MM) or human tumor cell and normal breast cell that this factor is replied.

Cell and oestrogenic hormon be subjected to cellular activity because of to MDA-MB to purifying MM

The reaction (2) of the CM of the secretion of the expressor of clone body (1)-231

Reaction

MDA-MB-231 ATCC?HTB?26 - + - -

HBL-100 ATCC?HTB?124 - + - -

Hs578T( ³) ATCC HTB 126 - + - -

Hs578Bst( ⁴) ATCC HTB 125 - (+) - -

SK-BR-2?III ATCC?HTB?29 - + - -

MDA-MB-453 ATCC?HTB?131 - - - -

BT-20 ATCC?HTB?19 - - - -

MCF-7 ATCC?HTB?22 + - + +

ZR-75-1 ATCC?CRL?1500 + - + +

ZR-75-30 ATCC?CRL?1504 + - + +

T-47D ATCC?HTB?133 + - + +

MDA-MB-361 ATCC?HTB?27 + - + +

BT-474( ⁵) ATCC HTB 20 (+) - (+) +

Normal epithelium cell/organoid-(+)-

Normal mesenchymal cell ( ⁶)+--

(1) observation of morphological change behind restricted substratum (CM) the stimulation MM reacting cells ZR-75-1 of the next free listed cell of result and the T-47D.Fresh serum free medium is added in the fused cell culture of every kind of cell (serum-free growth), the collection substratum is measured after 3 days, and measuring method is as described in the example 6."+" represents with obvious visible cellular activity after the 100 μ g/ml effects of heparin;

The more weak cellular activity of (+) representative.

(2) the highly purified factor (component among Fig. 6 " m ").

(3) tumor cell line of same tumor specimen and (4) myoepithelical cell system.

(5) very low-level ER of BT-474 cell expressing and PR.

(6) take from dysplastic mammary tissue.

Hs578Bst, MDA-MB-453 and BT-20 are the estrogen receptor negative cells, the speed of growth is slow during cell cultures, only produces the very low MM activity that maybe can not measure.

The order inventory

(1) generalized case:

(ⅰ) applicant:

(A) name: F.HOFFMANN-LA ROCHE AG

(B) street: Grenzacherstrasse 124

(C) city: Basle

(D) continent: BS

(E) country: Switzerland

(F) postcode (ZIP): CH-4002

(G) phone: 061-6884256

(H) fax: 061-6881359

(I) fax: 962292/965542 hlr ch

(ⅱ) invention exercise question: novel organic compound and application thereof

(ⅲ) order quantity: 3

(ⅳ) computer-reader form

(A) media type: floppy disk

(B) computer: Apple Macintosh

(C) operating system: System 7.1(Macintosh)

(D) software: Word 5.0

(2) SEQ ID No:1 situation:

(ⅰ) ordinal characteristics:

(A) length: 8 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅴ) episode types: inside

(ⅹ ⅰ) order explanation: SEQ ID NO:1:

Leu?Val?Leu?Arg?Xaa?Xaa?Glu?Thr

1 5

(2) situation of SEQ ID No:2:

(ⅰ) ordinal characteristics:

(A) length: 7 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅴ) episode types: inside

(ⅹ ⅰ) order explanation: SEQ ID NO:2:

Ser?Glu?Leu?Arg?Ile?Asn?Lys

1 5

(2) situation of SEQ ID No:3:

(ⅰ) ordinal characteristics:

(A) length: 9 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅴ) episode types: inside

(ⅹ ⅰ) order explanation: SEQ ID NO:3:

Xaa?Leu?Xaa?Asn?Pro?Xaa?Xaa?Tyr?Leu

1 5

Claims (18)

1, the MM of purified form.

2, MM according to claim 1, wherein it is:

A) be that molecular weight is the protein of about 52-55KDa with the analysis of 10-15% gradient sds page method:

B) it is as follows to contain partial amino-acid order

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 Unidentified amino-acid residue;

C) unstable to heat and acid, to trypsinase and mercaptoethanol sensitivity; With

D) can from non-hormone dependence breast tumor cell, obtain.

3, the DNA or the cDNA sequence of coding claim 1 or the described MM of claim 2.

4, be used for claim 1 or the 2 described MM that polyclone or monoclonal antibody are produced.

5, be used to differentiate the claim 1 or the 2 described MM that can suppress its active compound.

6, the claim 1 that comprises the following steps or the production method of the described MM of claim 2:

A) cultivate the non-hormone dependence breast tumor cell; With

B) separating mammary regulin from substratum.

7, the claim 1 that comprises the following steps or the production method of the described MM of claim 2:

A) described DNA of claim 3 or cDNA are inserted in the carrier;

B) with carrier transfection protokaryon or eukaryotic cell lines;

C) cell that selection can be expressed MM from cells transfected system;

D) in suitable substratum, cultivate the cell of selecting; With

E) from biomass, extract MM.

Except the compound that 8, can suppress the MM biologic activity, heparin.

9, have the described compound of at least a following active claim 8:

A) expression, the especially breast tumor cell of inhibition tumour cell MM;

B) MM there is affinity;

C) the MM acceptor to tumour cell has affinity, especially breast tumor cell.

10, can discern the polyclone or the monoclonal antibody of at least one epi-position in claim 1 or the 2 described MM.

11, the described antibody of claim 10, it is a mono-clonal.

12, the medicinal compositions that is used for breast cancer treatment and control, it contains one or more following MM inhibitor alive:

A) the described compound of claim 8 or claim 9; And/or

B) the described MM antibody of claim 10 or claim 11; And pharmaceutical carrier or thinner.

13, be used to measure the test kit of MM level, it comprises polyclone or the monoclonal antibody that requires as in claim 10 or 11.

14, the discrimination method that suppresses the active compound of MM, this method comprises the determination step of measuring described compound effect, and the effect of described compound is to detect according to any one or a plurality of following parameters in the pilot system that comprises MM and hormone dependence breast cancer cell:

A) morphology of hormonal dependent cell;

B) mobility of hormonal dependent cell;

C) propagation of hormonal dependent cell;

D) the mitogen activity of hormonal dependent cell;

Or

E) the hormonal dependent cell is to the conversion of non-hormone dependent cells.

15, claim 1 or the 2 described MM application in polyclone or monoclonal antibody production.

16, claim 1 or the 2 described MM application aspect the active compound of discriminating inhibition MM.

17, claim 1 or the 2 described any MM that prepare with claim 6 and 7 desired methods.

18, whole product innovation described herein, new process and new the application.

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