Anti-androgen peptides and uses thereof in cancer therapy

Abstract

The present invention relates to isolated or purified or partially purified peptide derived molecules having the following general formula (S1): X-[(Pro).sub.n-His-Pro-His-Ala-Arg-Ile-Lys].sub.m-Y. The peptides are for medical use, in particular as anti-tumoral agents.

Claims

1. A synthetic, isolated or purified or partially purified peptide which is effective to inhibit or prevent the interaction of the androgen receptor (AR) with the SH3 domain of the tyrosine kinase Src, wherein the synthetic, isolated or purified or partially purified peptide consists of the amino acid sequence of SEQ ID NO: 1.

2. The synthetic, isolated or purified or partially purified peptide according to claim 1 having an anti-tumor activity.

3. The synthetic, isolated or purified or partially purified peptide according to claim 1 further comprising one or more additional proline moieties on the N-terminal portion.

4. The synthetic, isolated or purified or partially purified peptide according to claim 1 for use as a medicament.

5. The synthetic, isolated or purified or partially purified peptide according to claim 1 for use as anti-tumoral agent.

6. The synthetic, isolated or purified or partially purified peptide according to claim 5 for use against cancers expressing androgen receptor alone or together with estradiol receptors.

7. A pharmaceutical composition comprising a pharmaceutically acceptable and effective amount of the synthetic, isolated or purified or partially purified peptide according to claim 1.

8. The composition according to claim 7 wherein said peptide is linked to a carrier molecule and/or is comprised in a lipid composition.

9. The composition according to claim 7 further comprising at least a second anti-cancer agent.

Description

(1) The drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

(2) FIG. 1A. DNA synthesis inhibition of human prostate cancer cells by Src-SH3 binder peptide modeled from AR (SEQ.ID NO.1). LNCaP cells derived from human prostate cancer were grown in phenol red-free medium added with charcoal-treated serum as previously described (Migliaccio et al., 2000) and treated for 24 hrs with 10 nM R1881 synthetic androgen alone or in the presence of a 1000-fold excess of the anti-androgen Casodex or 1 nM of the SH3-binder peptide (SH3, SEQ.ID NO.1) or 1 nM Ss (shuffled sequence: SEQ.ID NO.7) peptide. At the end of hormone treatment BrdU incorporation in cells untreated (control) or treated with R1881 alone (R1881) or in the presence of casodex (Cdx) or SH3 binder peptide (SH3, SEQ.ID NO.1) or shuffled sequence of peptide SEQ.ID NO.1 (Ss, SEQ.ID NO.7) was assayed as reported (Castoria et al., 1999). Data are presented as percentage of cells incorporating BrdU, averaged from 6 experiments.

(3) FIG. 1B. DNA synthesis inhibition of estradiol-stimulated human breast cancer cells by SH3 binder peptides derived from AR (SEQ.ID NO.1). MCF-7 cells derived from human breast cancer were maintained for 3 days in phenol red-free medium containing charcoal-treated serum as previously described and treated for 24 hrs with 10 nM 17B estradiol alone or in the presence of a 1000-fold excess of the anti-estrogen ICI 182,780 or 1 nM of the SH3-binder peptide SEQ.ID NO.1 or the shuffled sequence (Ss, SEQ.ID NO.7) of peptide SEQ ID NO.1. At the end of the hormone treatment BrdU incorporation in the cells untreated (control) or treated with estradiol alone (E2) or in the presence of ICI 182,780 (ICI) or AR-derived (SH3) or Ss peptide was assayed. Data are reported as percentage of cells incorporating BrdU, averaged from 6 experiments.

(4) FIG. 1C. DNA synthesis inhibition of EGF-stimulated human prostate cancer cells by SH3 binder peptide modeled from AR (SEQ. ID NO.1). LNCaP cells were grown in phenol red-free medium added with charcoal-treated serum as previously described and treated for 24 hrs with 100 ng/ml EGF alone or in the presence of a 10 μM of the anti-estrogen ICI 182,780 or 1 nM of the AR-derived (SH3-binder) peptide (SEQ.ID NO.1) or the shuffled sequence of peptide SEQ.ID NO.1 (SEQ.ID NO.7). At the end of the growth factor treatment BrdU incorporation in the cells untreated (control) or treated with EGF alone (EGF) or in the presence of ICI 182,780 (ICI) or the SH3-binder (SH3) or the shuffled sequence of peptide SEQ. ID NO.1 (Ss) was assayed. Data are reported as percentage of cells incorporating BrdU.

(5) FIG. 1D. DNA synthesis inhibition of EGF-stimulated human breast cancer cells by SH3 binder peptides modeled from AR (SEQ. ID NO.1). MCF-7 cells were grown in phenol red-free medium added with charcoal-treated serum as previously described and treated for 24 hrs with 100 ng/ml EGF alone or in the presence of a 10 μM of the anti-estrogen ICI 182,780 or 1 nM of the AR-derived (SH3-binder) peptide or the shuffled sequence of peptide SEQ ID NO. 1(SEQ.ID. NO.7). At the end of the growth factor treatment, BrdU incorporation in the cells untreated (control) or treated with EGF alone (EGF) or in the presence of ICI 182,780 (ICI) or SH3-binder (SH3) or shuffled sequence of peptide SEQ.ID NO.1 (Ss) was assayed. Data are reported as percentage of cells incorporating BrdU.

(6) FIG. 2A. Inhibition of androgen-stimulated AR association with Src by the AR-derived (Src-SH3 binder) peptide (SEQ.ID NO.1). Human prostate cancer LNCaP cells were left unstimulated or stimulated for 2 min with 10 nM R1881 alone or in the presence of 1000-fold excess of Casodex or 1 nM of the SH3-binder peptide or the shuffled sequence of peptide SEQ.ID NO.1 (SEQ.ID NO.7). Cell lysates were incubated with anti-Src antibodies, and the immunoprecipitated proteins resolved on SDS-PAGE and transferred onto nitrocellulose filters. Filters were then blotted with either anti-Src or anti-human AR antibodies to detect AR associated with Src. Immunocomplexes were revealed using ECL detection kit. Lane 1: unstimulated cells; lane 2: R1881 treated cells; lane 3: cells treated with R1881 in the presence of Casodex (Cdx); lane 4: cells treated with R1881 in the presence of the SH3 binder peptide (SH3, SEQ.ID NO.1); lane 5: cells treated with R1881 in the presence of the shuffled sequence of peptide SEQ.ID NO.1 (Ss, SEQ.ID NO.7).

(7) FIG. 2B. Inhibition of estrogen-stimulated AR association with Src by the AR-derived Src-SH3 binder peptide (SEQ.ID NO.1). Human breast cancer MCF-7 cells were left unstimulated or stimulated for 2 min with 10 nM 17ß estradiol alone or in the presence of a 1000 fold excess of ICI 182,780 or 1 nM of the SH3 binder peptide or the shuffled sequence of peptide SEQ.ID NO.1 (SEQ.ID NO.7). Cell lysates were incubated with anti-Src antibodies, immunoprecipitated proteins were resolved on SDS-PAGE and transferred onto nitrocellulose filters. Filters were then blotted with either anti-Src or anti-human AR antibodies to detect AR associated with Src. Immunocomplexes were revealed using ECL detection kit. Lane 1: unstimulated cells; lane 2: estradiol (E2) treated cells; lane 3: cells treated with E2 in the presence of ICI 182,780 (ICI); lane 4: cells treated with E2 in the presence of SH3 binder peptide (SH3); lane 5: cells treated with estradiol in the presence of the shuffled sequence of peptide SEQ.ID NO.1 (Ss)

(8) FIG. 3A. Inhibition of androgen-induced cyclin D1 expression by AR-derived Src-SH3 binder peptide (SEQ.ID NO.1). Quiescent MCF-7 and LNCaP cells were left untreated or treated for 8 hrs with 10 nM R1881 alone or in the presence of 5 μM PI3-kinase inhibitor LY294,002 or 1 nM AR-derived SH3 binder or the shuffled sequence of peptide SEQ.ID NO.1 (SEQ.ID. NO.7). Protein from cell lysates were resolved on SDS-PAGE, then transferred to nitrocellulose filters. Endogenous Cyclin D1 was revealed using appropriate antibodies. Lanes 1: unstimulated cells; lanes 2: R1881 treated cells; lanes 3: cells treated with R1881 in the presence of LY294,002 (LY); lanes 4: cells treated with R1881 in the presence of AR-derived (SH3-binder) peptide (SH3); lane 5: cells treated with R1881 in the presence of the shuffled sequence of peptide SEQ.ID NO.1 (Ss).

(9) FIG. 3B. Inhibition of estrogen-induced cyclin D1 expression by AR-derived SH3 binder peptide (SEQ.ID NO.1). Quiescent MCF-7 and LNCaP cells were left untreated or treated for 8 hrs with 10 nM 171 estradiol alone or in the presence of 5 μM PI3-Kinase inhibitor LY294,002 or 1 nM of the SH3 binder or the shuffled sequence of peptide SEQ.ID NO.1 (SEQ.ID NO.7). Protein from cell lysates were resolved on SDS-PAGE, then transferred to nitrocellulose filters. Endogenous Cyclin D1 was revealed using appropriate antibodies. Lanes 1: unstimulated cells; lanes 2: estradiol treated cells; lanes 3: cells treated with estradiol in the presence of LY294,002 (LY); lane 4: cells treated with estradiol in the presence of SH3-binder peptide (SH3); lane 5: cells treated with estradiol in the presence of the shuffled sequence of peptide SEQ.ID NO.1 (Ss).

(10) FIG. 4A. Ineffectiveness of AR-derived (SH3 binder) peptide (SEQ.ID NO.1) on androgen receptor regulated gene transcription in prostate cancer (LNCaP cells). LNCaP cells were transfected with a reporter gene encoding the luciferase gene under the control of an androgen responsive element (ARE 3416) (Castoria et al., 2003). Six hrs after transfection, cells were kept for further 24 hrs in the absence or in presence of 10 nM R1881 alone or with a 1000 fold excess of Casodex or with 1 nM SH3 binder peptide (SEQ.ID NO.1) or 1 nM shuffled sequence of peptide SEQ.ID NO.1 (SEQ.ID. NO.7). Luciferase activity was then assayed in cell lysates. Bar1: unstimulated cells; Bar 2: R1881 treated cells; Bar 3: cells treated with R1881 in the presence of Casodex (Cdx); Bar 4: cells treated with R1881 in the presence of the SH3 binder peptide (SH3); Bar 5: cells treated with R1881 in the presence of the shuffled sequence of peptide SEQ.ID NO.1 (Ss).

(11) FIG. 4B. Ineffectiveness of AR-derived (SH3 binder) peptide (SEQ.ID NO.1) on estrogen receptor regulated gene transcription in breast cancer (MCF-7 cells). MCF-7 cells were transfected with a reporter gene encoding the luciferase gene under the control of an estrogen responsive element (vt-tk-LUC) (Castoria et al., 2003). Six hrs after transfection, cells were kept for further 24 hrs in the absence or in presence of 10 nM 17ß-estradiol (E2) alone or with a 1000 fold excess of ICI 182,780 or with 1 nM SH3 binder peptide (SEQ.ID NO.1) or 1 nM shuffled sequence of peptide SEQ.ID NO.1 (SEQ.ID. NO.7). Luciferase activity was then assayed in cell lysates. Bar 1: unstimulated cells; Bar 2: E2 treated cells; Bar 3: cells treated with E2 in the presence of ICI 182,780 (ICI); Bar 4: cells treated with E2 in the presence of the SH3 binder peptide (SH3); Bar 5: cells treated with E2 in the presence of the shuffled sequence of peptide SEQ ID NO.1(Ss).

(12) FIG. 5A Effect of SH3-binder peptide on human prostate cancer cells in vivo. LNCaP prostate cancer cells were grown subcutaneously in nude male mice. After tumor reached the size of 200-400 mm.sup.3, mice were treated with intra-peritoneal injection of 200 μl of control (ctrl) solution (circles) or the same solution containing 2 μM of SH3 (SH3) binder peptide SEQ.ID NO.1 (squares). Treatment started at beginning of week 0 and peptides were given in alternate days for 4 weeks, using 5 animals per group.

(13) FIG. 5B Effect of SH3-binder peptide on human breast cancer cells in vivo. MCF-7 human breast cancer cells were grown subcutaneously in nude male mice. When tumors were about 1000 mm.sup.3 in size, mice were treated with intraperitoneal injection of 200 μl of control solution (ctrl, circles) or 200 μl of the same solution containing 2 μM of the SH3 binder peptide SEQ.ID NO.1 (SH3, squares). Treatment started at beginning of week 0 and peptides were given in alternate days for 5 weeks, using 5 animals per group

(14) FIG. 6A. Effect of SH3-binder peptide on Ki-67 antigen expression and apoptosis in human prostate cancer cells. LNCaP cell xenografts in male nude mice are the same as the one used in the experiment presented in FIG. 5A. At the end of the treatment, tumor specimens were assayed for Ki-67 antigen expression and apoptosis. The left panel shows the expression of Ki-67 antigen as percentage of Ki-67 positive cells in untreated (ctrl) and SEQ.ID NO.1 peptide treated (SH3) mice. The right panel shows the TUNEL assay positive cells observed in representative fields.

(15) FIG. 6B. Effect of SH3-binder peptide on Ki-67 antigen expression and apoptosis in human breast cancer cells. MCF-7 cells xenografts in male nude mice are the same as the one used in the experiment of FIG. 5B. At the end of treatment tumor specimens were assayed for Ki-67 antigen expression and apoptosis. The left panel shows the expression of Ki-67 antigen as percentage of Ki-67 positive cells in untreated (ctrl) and SEQ.ID NO.1 peptide treated (SH3) mice. The right panel shows the TUNEL assay positive cells observed in representative fields.

EXAMPLES

(16) The following examples are included to demonstrate preferred embodiments of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1: Preparation of AR-Derived Src-SH3 Binder Peptide (SEQ. ID. No. 1) by Chemical Synthesis

(17) Peptides 1 through 4 can be conveniently manually prepared by applying the solid phase method (Bodansky M and Bodansky A, 1995) and the Fmoc/tBu (Fmoc: 9-fluorenyl-methoxycarbonyl) chemistry that is largely described in the scientific literature (Carpino and Han, 1972; Fields and Noble, 1990) and is well known to those skilled in the art. To expedite and facilitate the preparation, automatic multiple peptide synthesizers can be utilized. Any kind of chemical method or mechanic synthesizer with single or multiple channels can be also conveniently used to carry out the synthesis, without affecting the biological properties of the final compounds.

(18) The synthesis of the peptide is performed on a scale of 50 μmoles using a resin suitably derivatized with a RINK linker capable to give C-terminal amide peptides (Rink, 1987). One of such resins is the product 4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin, 100-200 mesh; copoly-styrene 1% Di-vinylbenzene), substitution 0.50 mmol/g (Novabiochem cat. N. 01-64-5026), also known as RINK AMIDE resin to those skilled in the art. An amount of 100 mg of resin is used. The resin is placed in a 5 ml polypropylene reaction vessel (RV) endowed with a filtration septum at the bottom (Shimadzu Corp. cat. N. 292-05250-02). In a typical protocol, the resin is swollen for 10 minutes under stirring and then rinsed several times (at least 4) with 1.0 mL of dry N,N-dimethylformamide (DMF, Peptide synthesis grade, LabScan, cat. N. H6533) by removing the solvent from the bottom applying a slight vacuum. The resin is then treated with 1.0 mL of a 20% v/v solution of piperidine (BIOSOLVE LTD, cat. N. 16183301) in DMF for 15 minutes at room temperature under stirring to remove the initial Fmoc group and washed several times (at least 4) with 1.0 mL of dry DMF for 2 minutes to remove the excess of reagent.

(19) Then the following 6 steps are carried out subsequently:

(20) 1. 250 μmoles (117 mg) of Fmoc-L-Lys(Boc)-OH (Novabiochem, cat. 04-12-1026) are dissolved in 500 μL of dry DMF.

(21) 2. The protected aminoacid is preactivated with 400 μL of solution A and 400 μL of solution B for 4 minutes under stirring at room temperature, where:

(22) Solution A Contains:

(23) 0.5 M 2-(1H-Benzotriazol-yl)-1,1,3,3-tetramethyl-uronium tetrafluoroborate (TBTU, >99%, Chem-Impex Intl, cat. N. 02056) and 0.5 M of 1-Hydroxybenzotriazole (HOBt, SIGMA-ALDRICH, cat. N. H2006) in DMF.

(24) Solution B Contains:

(25) 1 M Di-isopropyl-ethylamine (DIEA, SIGMA-ALDRICH, cat. N.D-3887) in DMF.

(26) 3. The solution is transferred on to the resin and stirred for 30 minutes.

(27) 4. The reagent is removed under vacuum and the resin washed 4 times with 1.0 mL of dry DMF.

(28) 5. The resin is again treated with 1.0 mL of 20% v/v piperidine in DMF for 15 minutes at room temperature under stirring to remove the N-terminal Fmoc.

(29) 6. The resin is washed 3 times with 1.0 mL of DMF.

(30) Steps 1 through 6 are then repeated, by changing in step 1 the corresponding protected amino acid requested in the sequence. The protected derivatives used are reported in the following Table II.

(31) TABLE-US-00006 TABLE II Common protected amino acids used for the chemical synthesis of peptide SEQ. ID NO. 1 and amounts required. AA Protected deriv. Amount Code Ile Fmoc-L-Ile-OH  88 mg (250 μmol) Novabiochem, cat. 04-12-1024 Arg Fmoc-L-Arg(Pbt)-OH 161 mg (250 μmol) Novabiochem, cat. 04-12-1145 Ala Fmoc-L-Ala-OH  78 mg (250 μmol) Novabiochem, cat. 04-12-1006 His Fmoc-L-His(Trt)-OH 155 mg (250 μmol) Novabiochem, cat. 04-12-1065 Pro Fmoc-L-Pro-OH  84 mg (250 μmol) Novabiochem, cat. 04-12-1031 Lys Fmoc-L-Lys(Boc)-OH 117 mg (250 μmol) Novabiochem, cat. 04-12-0069 Fmoc-L-Lys(Boc)-OH indicates the protected lysine derivative: N-α-Fmoc-N-ε-t-Boc-L-lysine and the symbol “t-Boc”, indicates the protecting group t-butyloxycarbanyl; Fmoc-L-Ile-OH indicates the protected isoleucine derivative: N-α-Fmoc-L-isoleucine; Fmoc-L-Arg(Pbf)-OH indicates the protected arginine derivative: N-α-Fmoc-N.sup.G-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine; Fmoc-L-Ala-OH indicates the protected alanine derivative: N-α-Fmoc-L-alanine; Fmoc-L-His(Trt)-OH indicates the protected histidine derivative: N-α-Fmoc-N-im-trityl-L-histidine and the symbol “trityl”, indicates the protecting group t-phenylmethyl; Fmoc-L-Pro-OH indicates the protected proline derivative: N-α-Fmoc-L-proline.

(32) After removal of the last Fmoc group, the resin is acetylated by treatment with a 1.0 M solution in dry DMF of Acetic Anhydride (Fluka, cat. 45830), containing 1.0 M DIEA for 30 minutes at room temperature under stirring. After extensive rinsing with DMF, the resin is submitted to the following washings:

(33) TABLE-US-00007 Solvent N.sup.o of washes Volume (mL) DMF 3 4.0 MeOH* 3 4.0 Et.sub.2O** 3 4.0 *Methanol (MeOH, LabScan, cat. N. A3513), **Ethyl Ether (Et.sub.2O, LabScan, cat. N. A3509E).

(34) The resin is dried applying a Nitrogen stream and then weighted. The final weight of the resin is 280.0 mg.

(35) 3.0 mL of a TFA-H.sub.2O-TIS (100:5:5, v/v/v) mixture (TIS, tri-iso-propylsilane, SIGMA-ALDRICH cat. N. 23,378-1) are freshly prepared and added to the resin. After stirring for 3 hours, the resin is filtered off and the acidic solution is directly collected in a 15 ml polypropylene tube containing 15 mL of cold Et.sub.2O. The white precipitate is separated by centrifugation at 3000 rpm for 10 minutes and the organic solvents are discarded. The precipitate is washed once with 5.0 mL of cold Et.sub.2O and after centrifugation is dissolved in 2.0 mL of deionized H.sub.2O and lyophilized. The white solid is weighted: 102 mg.

(36) The lyophilized peptide can be conveniently purified by reverse phase chromatography using commercially available High Performance Liquid Chromatography (HPLC) systems, such as, for example, the LC-8 system provided by Shimadzu Corp., equipped with commercially available preparative reversed phase C18 columns, such as the Jupiter 25×2.1 cm ID C18 columns (Phenomenex cat. N. 00G-4053-P0) provided by Phenomenex. Typical gradients for the peptide purification make use of solvents such as deionized H.sub.2O, 0.1% trifluoroacetic acid (TFA, Sigma-Aldrich, Cat N. 91700) and acetonitrile (CH.sub.3CN, LabScan, Cat. NO C2503, 0.1% TFA and are from a typical concentration of 5% CH.sub.3CN, 0.1% TFA to 60% over 35 minutes with a typical operating flow rate of 20 mL/min. These methods are very well known to those skilled in the art and will provide purified peptides with purity levels of up to 95-99% as determined by analytical HPLC analysis on a commercially analytical RP18 columns, such as the Phenomenex Jupiter column, 250×4.6 mm ID RP-18 (Phenomenex cat. N. 00G-4053-E0). Gradients are from 5% CH.sub.3CN, 0.1% TFA to 60% over 35 minutes, with a typical flow rate of 1.0 mL/min. Monitoring is typically achieved by using UV-vis detector implemented on to the HPLC systems and set at a typical wavelength of 214 nm.

(37) The final peptide is conveniently characterized by mass spectrometry methods, such as MALDI-TOF and ESI-MS. In a typical example, the experimental molecular weight (MW) determined with an ESI-MS mass spectrometer is 1189.5 atomic mass units (amu), in agreement with the theoretical value of 1189.68 amu (monoisotopic species).

Example 2: “In Vitro” Anti Tumor Effect of the AR-Derived Src-SH3 Binder Peptide SEO. ID NO.1

(38) It has been shown that treatment of human prostate cancer LNCaP with 10 nM R1881 synthetic androgen strongly stimulates DNA synthesis. Therefore, LNCaP cells are routinely grown in 5% CO.sub.2 in air in RPMI 1640 medium (GIBCO) supplemented with phenol-red, 2 mM L-glutamine (GIBCO), penicillin (100 U/ml), gentamicin (50 μl/ml), insulin (Humulin I 0.2 U.I/ml, Roche) and 10% fetal calf serum. Cells are then seeded onto gelatine-precoated coverslips at about 40% confluence and maintained in phenol red-free RPMI-1640 containing insulin and charcoal stripped calf serum to assure minimal to no steroid contamination (Shi et al., 1994) for 3 days. LNCaP cells are then treated for 24 hrs with 10 nM of the synthetic androgen R1881 (Astra-Zeneca) alone or in the presence of a 1000-fold molar excess of the anti-androgen Casodex (Astra-Zeneca) or with the indicated peptides.

(39) Results presented in FIG. 1A, averaged from six different assays show that androgen increases from 20 to 54% BrdU incorporation rate. The hormone stimulated incorporation is strongly reduced (25% of residual BrdU incorporation) by SH3 binder peptide at a concentration of 1 nM (SH3). The effect of SH3 peptide was compared with that of the pure anti-androgen Casodex, which reduced by a similar extent DNA synthesis (21% of residual BrdU incorporation). A synthetic peptide corresponding to the shuffled sequence of SEQ.ID NO.1 (SEQ.ID NO.7) was also used (Ss). Addition of this peptide during androgen stimulation of cells has only a negligible inhibitory effect on hormone-dependent DNA synthesis (48% of residual BrdU incorporation). In addition to the above mentioned peptides, peptides SEQ.ID NO.2-SEQ.ID NO.4 were tested. The inhibitory effect was not stronger than that of the peptide SEQ.ID NO.1.

(40) The effect of peptide SEQ.ID NO.1 was also tested on estradiol-stimulated DNA synthesis of MCF-7 human breast cancer cells. MCF-7 cells were grown in 5% CO.sub.2 in air in Dulbecco modified Eagle medium (DMEM, GIBCO) supplemented with phenol-red, 2 mM L-glutamine (GIBCO), penicillin (100 U/ml), gentamicin (50 μl/ml), hydrocortisone 3.75 ng/ml, insulin (Humulin I 0.2 U.I/l, Roche) and 5% fetal calf serum. Cells are then seeded onto gelatine-precoated coverslips at about 40% confluence and maintained in phenol red-free DMEM containing insulin and charcoal stripped calf serum for 3 days. MCF-7 cells are then treated for 24 hrs with the 10 nM 17ß estradiol (SIGMA, Mo.) alone or in the presence of a 1000-fold molar excess of anti-estrogen ICI 182,780 (Astra-Zeneca) or with the indicated peptides.

(41) Results presented in FIG. 1B, averaged from six different assays show that estradiol (E.sub.2) increases from 8 to 61% BrdU incorporation rate. The hormone stimulated incorporation is significantly reduced (23% of residual BrdU incorporation) by the SH3 binder peptide (SH3) at a concentration of 1 nM. The effect of SH3 peptide is comparable with that of pure anti-estrogen ICI 182,780 that abolished the estrogen-induced DNA synthesis (7% of residual BrdU incorporation). The shuffled sequence of peptide SEQ.ID NO.1 (Ss) has not significant effect on estrogen-induced DNA synthesis.

(42) The peptide SEQ.ID NO.1 is finally assayed for its ability to reduce or inhibit EGF induced DNA synthesis in human prostate cancer LNCaP (1C) and MCF-7 (1D) cells. The LNCaP and MCF-7 cells are cultured as described above, then seeded onto coverslips. Cells are then stimulated with 100 ng/ml of highly purified EGF (Boheringer, Calif.) in the absence or in presence of 1 nM peptide SEQ.ID NO.1.

(43) In FIG. 1C, it can be observed that EGF stimulates BrdU incorporation from 8 to 21%. Addition of the peptide reduces to 5,8% BrdU incorporation, similar to the pure antiestrogen, ICI 182,780 (7% of residual incorporation). The shuffled sequence of peptide SEQ.ID NO.1 (Ss) has a little effect on BdrU incorporation (15% of residual incorporation). In MCF-7 cells, as shown in FIG. 1D, EGF also stimulates BrdU incorporation (from 6 to 36%); addition of the peptide reduces to 13% BrdU incorporation, and ICI 182,780 to 9% of residual incorporation. In contrast, the shuffled sequence of peptide SEQ.ID NO.1 (Ss) does not reduce BdrU incorporation (48% of incorporation)

(44) Methods

(45) DNA synthesis is assayed in single cells by a 6 h pulse with 100M (final concentration) BrdU (Boeheringer). Cells on coverslips are fixed, and incubated with diluted (1:1 in PBS) fluorescein-conjugated mouse anti-BrdU mAbs (clone BMC9318 from Boheringer Mannheim Co., Ind.), then washed three times with PBS. Mouse antibodies are revealed using diluted (1:200 in PBS) Texas-red conjugated goat anti-mouse antibodies (Calbiochem, Calif.). All coverslips are washed three times in PBS, inverted and mounted in Moviol (Calbiochem, Calif.) on glass slides. Slides are analyzed using an Axiophot fluorescent microscope (Zeiss).

Example 3: Inhibition of Androgen-Stimulated AR Association with Src by AR-Derived (Src-SH3 Binder) Peptide (SEQ.ID NO. 1

(46) It has been demonstrated that hormone bound AR interacts with SH3 domain of Src kinase probably through a proline stretch (Migliaccio et al., 2000). As a consequence of this interaction, the kinase and the downstream signaling pathways are activated and, finally, DNA synthesis is activated. Use of a small peptide sequences mimicking the domain of AR involved in this interaction should be able to inhibit by competition the AR-Src association and block DNA synthesis. To test this hypothesis LNCaP cells are routinely grown in 5% CO.sub.2 in air in RPMI 1640 medium (GIBCO) supplemented with phenol-red, 2 mM L-glutamine (GIBCO), penicillin (100 U/ml), gentamicin (50 μl/ml), insulin (0.2 U.I./ml) and 10% fetal calf serum. The cell are then kept in RPMI 1640 without phenol red and supplemented with glutamine, penicillin, gentamicin and insulin as above and containing 10% charcoal-stripped fetal calf serum for further 4 days. Cells are then stimulated with 10 nM R1881 alone or in the presence of a 1000-fold excess of the anti-androgen Casodex or 1 nM of the SEQ.ID NO.1 peptide or 1 nM of the shuffled sequence of peptide SEQ.ID NO.1 (SEQ.ID NO.7) for 2 min., and lysed. Cell lysates are submitted to immuno-precipitation using mouse monoclonal anti-Src antibodies (Clone 327, Oncogene Science, Manhasset, N.Y.). The immuno-precipitated proteins are resolved on a 12% SDS-polyacrylammide gel and thereafter transferred onto nitrocellulose filters. The filters are incubated with either anti-Src antibodies or mouse monoclonal anti AR antibodies. The immuno-complexes on nitrocellulose filters are revealed using peroxidase-linked anti-mouse antibodies with a chemiluminescent substrate (Pierce Chemicals, Ill.).

(47) As expected, in hormone treated cells, AR is co-immuno-precipitated by anti Src antibodies (FIG. 2A, Lane 2). The association between AR and Src is abolished when LNCaP cells are stimulated with hormone in the presence of Casodex (Lane 3).

(48) Similarly, no AR is co-immunoprecipitated by anti-Src antibodies in cells treated with androgens and SH3 binder peptide. In contrast, the hormone stimulated Src-AR association is only slightly affected by treatment with the same concentration of the shuffled sequence of peptide SEQ ID NO.1(Ss, SEQ.ID NO.7).

(49) Methods

(50) Preparation of cell lysates. Cells are suspended in 1 ml of lysis buffer: 50 mM Tris-HCl, pH 7.40, containing 5 mM MgCl2, 150 mM NaCl, 0,5% Triton X-100 and left under gentle shaking for 2 min, at 4° C. Suspensions are then centrifuged at about 800 g for 30 min and supernatant collected and used for immunoprecipitation.

Example 4: Inhibition of Estrogen-Stimulated ER Association with SRC by AR-Derived (Src-SH3 Binder) Peptide (SEO.ID NO. 1)

(51) It has previously shown that ER together with AR forms a ternary complex with Src (Migliaccio et al., 2000). Inhibition of interaction of either ER or AR with Src leads to disruption of this ternary complex and inhibition of Src-mediated signaling. Therefore inhibition of AR-Src interaction by SH3 binder peptide(s) should also abolish the ERα association with Src and the estradiol induced signal transduction. To address this point mammary cancer derived MCF-7 cells are grown in 5% CO.sub.2 in air in DMEM medium (GIBCO) supplemented with phenol-red, 2 mM L-glutamine (GIBCO), penicillin (100 U/ml), gentamicin (50 l/ml), hydrocortisone (3.75 ng/ml) insulin (0.2 U.I./ml) and 5% fetal calf serum. The cells are then kept in DMEM without phenol red and supplemented with glutamine, penicillin, gentamicin and insulin as above and containing 5% charcoal-stripped fetal calf serum for additional 4 days. Cells are then stimulated with 10 nM 17ß estradiol alone or in the presence of a 1000-fold excess of the anti-estrogen ICI 182,780 or 1 nM of the SEQ.ID NO.1 peptide or the shuffled sequence of peptide SEQ.ID NO.1 for 3 min., and lysed. Cell lysates are submitted to immunoprecipitation using mouse monoclonal anti-Src antibodies (Oncogene Science, Manhasset, N.Y.). The immunoprecipitated proteins are resolved on a 12% SDS-polyacrylammide gel and thereafter transferred onto nitrocellulose filters. The filters are incubated with either anti-Src antibodies or mouse monoclonal anti AR antibodies. The immunocomplexes on nitrocellulose filters are revealed using peroxidase-linked anti-mouse antibodies with a chemiluminescent substrate as described above.

(52) As expected, in hormone treated cells ER is co-immunoprecipitated by anti Src antibodies (FIG. 2B, Lane 2). The association between ER and Src is abolished when MCF-7 cells are stimulated with hormone in the presence of ICI 182,780 (Lane 3). No ER is co-immunoprecipitated by anti-Src antibodies in cells treated with androgens and SH3 binder peptide at a concentration of 1 nM. Src-AR association is only slightly affected by treatment with the shuffled sequence of peptide SEQ ID NO.1 at 1 nM (Ss).

Example 5: Inhibition of Androgen-Stimulated Cyclin-d Expression by AR-Y (Src-SH13 Binder) Peptide (SEO.ID NO.1) in Prostate Cancer (LNCAP) and Mammary Cancer (MCF-7) Cells

(53) Estrogen and androgen receptors stimulate Src which in turn activates phosphatidyl-3-kinase (PI3-K) pathways (Castoria et al., 2000). Activation of PI3-K leads to PKB/Akt kinase phosphorylation that results in increased Cyclin D expression. This drives hormone dependent cells towards the cell cycle G1/S transition (Castoria et al., 2000). Inhibition of Src or PI3-K causes cell accumulation in G1 phase and block of DNA synthesis. Therefore SH3 binder peptides could be used to inhibit Src kinase and PI-3K dependent Cyclin D1 expression. To verify this possibility, SH3 binder peptide (SEQ.ID NO.1) is added to MCF-7 and LNCaP cells stimulated with the androgen. MCF-7 and LNCaP cells are routinely grown as described above. Cells are then kept for at least 4 days in red phenol-free medium supplemented as described above and added with dextran-coated charcoal treated fetal calf serum to minimize steroid concentration. Cells are then treated with 10 nM R1881 alone or in the presence of 1 nM SH3 binder peptide (SH3, SEQ.ID NO.1) or 1 nM of the shuffled sequence of peptide SEQ.ID NO.1 (SEQ.ID NO.7) (Ss) for 6 hrs, then lysed and submitted to western blot using anti-Cyclin D1 antibodies (FIG. 3A, upper and lower panels). As expected, androgen stimulates Cyclin D1 expression in MCF-7 and LNCaP cells. The PI3-K inhibitor, LY 294,002, at a concentration of 5 μM and the Src inhibitor, PP2 (not shown), abolishes the hormone stimulated cyclin expression. Addition of 1 nM SH3 binder peptide (SEQ.ID NO.1) (SH3) also suppresses Cyclin D induction by R1881 whereas the shuffled sequence of peptide SEQ.ID NO.1 (Ss) only partially reduces this effect.

Example 6: Inhibition of Estrogen-Stimulated Cyclin-d1 Expression by AR-Derived (Src-SH3 Binder) Peptide (SEQ.ID NO. 1) in Prostate (LNCAP) and Mammary (MCF-7) Cancer Cells

(54) To test the effect of SH3 binder peptide (SEQ.ID NO.1) on estrogen stimulated Cyclin D1 expression, this peptide is added to LNCaP and MCF-7 cells stimulated with 10 nM 17ß-estradiol (FIG. 3B). MCF-7 and LNCaP cells are routinely grown as reported above. Cells are then kept for at least 4 days in red phenol-free medium supplemented as described above and added with dextran-coated charcoal treated fetal calf serum to minimize steroid contamination. Cells are then treated with 10 nM 17ß estradiol alone or in the presence of either the PI-3K inhibitor LY 294,002 (5 μM) or 1 nM SH3 binder peptide (SEQ.ID NO.1) or 1 nM shuffled sequence of peptide SEQ ID NO.1 (SEQ ID NO.7) for 6 hrs (FIG. 3B, upper and lower panels), then lysed and submitted to western blot using anti-Cyclin DI antibodies as above. Like the androgen, 171 estradiol stimulates Cyclin DI expression. The PI3-K inhibitor, LY 294,002, also in this case abolishes the hormone stimulated cyclin expression. One nM SH3 binder peptide (SEQ.ID NO.1) (SH3) completely suppresses Cyclin D induction by estrogen whereas the shuffled peptide SEQ.ID NO.1 (Ss) has a little effect.

Example 7. Ineffectiveness on Androgen Receptor Dependent Gene Transcription of AR-Derived (Src-sh3 Binder) Peptide (SEQ.ID NO.1) in Prostate Cancer (LNCaP) and Breast Cancer (MCF-7) Cells

(55) Steroidal receptors are generally known as ligand activated transcription factors. Therefore, it is important to assess whether SH3-binder anti-androgen peptide effect involves the transcriptional activity of the androgen receptor. LNCaP cells, kept in 5% CO.sub.2 in air in RPMI 1640 medium (GIBCO) supplemented with phenol-red, 2 mM L-glutamine (GIBCO), penicillin (100 U/ml), gentamicin (50 μl/ml), insulin and 10% fetal calf serum, are transfected with a reporter gene engineered in a pSG5 expression vector with the luciferase gene under the control of an androgen responsive element (ARE3416) (Verrijdt et al., 2000). Six hrs after transfection, the medium was replaced by fresh medium and cells were left for further 24 hrs in the absence or in presence of 10 nM R1881 alone or with a 1000 fold excess of Casodex or with 1 nM SH3 binder peptide SEQ.ID NO. 1 or 1 nM shuffled peptide SEQ ID NO.1 (SEQ.ID NO.7). Cells lysates are assayed for luciferase activity. The same experiment is repeated to analyze the anti-androgen peptide effect on the transcriptional activity of estrogen receptor in human breast cancer cells. MCF-7 cells, kept in 5% CO.sub.2 in air in DMEM medium (GIBCO) supplemented with phenol-red, 2 mM L-glutamine (GIBCO), penicillin (100 U/ml), gentamicin (50 μl/ml), hydrocortisone (3.75 ng/ml), insulin (0.2 U.I./ml) and 5% fetal calf serum, are transfected with a reporter gene QUALE cloned in a pSG5 expression vector with the luciferase gene under the control of an estrogen responsive element. Finally, cells lysates are assayed for luciferase activity.

(56) FIG. 4A shows the luciferase activity in untreated LNCaP cells (bar 1), in cells stimulated with R1881 alone (bar 2) or in presence of either Casodex excess (bar 3), or SH3 peptide (SH3) (bar 4) or shuffled peptide SEQ.ID NO.1(Ss) (bar 5).

(57) FIG. 4B shows the luciferase activity in untreated MCF-7 cells (bar 1), in cells stimulated with 171 estradiol alone (bar 2) or in presence of either ICI 182,780 excess (bar 3), or the peptide SEQ. ID NO.1 (SH3) (bar 4) or the shuffled sequence of peptide SEQ. ID NO.1 (Ss) (bar 5).

(58) It can be observed that whereas the conventional anti-androgen completely inhibits androgen induced transcription of reporter gene neither the SH3 peptide nor the shuffled sequence of peptide SEQ. ID NO.1 affect transcriptional activity of AR.

Example 8. “In Vivo” Antitumor Effects of the AR Derived. Src-SH3 Binder Peptide (SEQ. ID NO:1

(59) Since results of in vitro and in vivo tumor growth studies can be divergent, it has been studied the growth response of LNCaP and MCF-7 cells to the antiandrogen-peptide in vivo. LNCaP cancer cells, grown under routine conditions above described, are suspended in 50% (vol/vol) Matrigel solution in sterile PBS (pH 7.4) and injected subcutaneously in the dorsal posterior region at 2.5×10.sup.6 cells/male athymic mice (CD mice, Charles-River) without hormone priming.

(60) After 14-21 days, animals with tumors of similar size are randomized to treatment with SH3 binder-peptide, SEQ.ID NO.1, or vehicle alone for an additional 5 weeks. The treatment is initiated with tumors at approximately 200-400 mm.sup.3 in size. Tumor volumes of LNCaP cells xenografts with or without treatments are measured by a caliper and recorded according to the formula D×d.sup.2×0.5, where D is the length and d is the width of tumor.

(61) For peptide treatment of each animal, 200 μl of 20 nM SH3 binder-peptide SEQ. ID NO.1 dissolved in 0.1% DMSO or the same amount of vehicle alone are intraperitoneally administered on alternate days to the mice. Such studies are especially important to assess the efficacy of peptides as potential therapeutic agents in human prostate and breast cancer cells, which express androgen receptor levels commonly found in human malignancies. The dose, the type and the size of these peptides (general formula Si and SEQ. ID NO.1 to SEQ. ID NO.6) and the route of delivery of peptides are all factors that may be determined using ordinary skill in the art.

(62) FIG. 5A shows the growth rate of LNCaP cell xenografts in nude male mice treated with SH3-binder peptide (SH3) or with vehicle alone (ctrl).

(63) The authors observed that in this model, tumor mass were significantly lower in the SH3 treated group when compared to the control group. No difference is found between weight of mice treated with vehicle solution or the peptide (data not shown).

(64) The effect of anti-androgenic peptides is also analyzed on breast cancer MCF-7 cell xenografts established in nude mice (FIG. 5B). In this case MCF-7 cells, previously grown as described above, are suspended in 50% Matrigel (vol/vol) solution in sterile PBS and injected subcutaneously at 2.5×10.sup.6 cells/animal in athymic male mice. After 14-21 days, animals with similar size tumors are randomized to treatment with SH3 binder-peptide, SEQ.ID. NO.1, or vehicle alone for additional 5 weeks. Tumors at beginning of the treatment measure approximately 1000 mm.sup.3. 200 μl/of 20 nM SH3 binder-peptide in 0.1% DMSO or the same volume of vehicle alone are administered on alternate days to the mice intraperitoneally. Tumor volumes of MCF-7 cancer cell xenograft with or without treatment are measured and recorded as reported above. No difference of body weight is found between control mice or peptide treated mice.

(65) Similarly to what was observed for the LNCaP cell xenografts model, the authors also found in the MCF-7 cell xenografts model that tumor mass was lower in the SH3 treated group when compared to the control group. No difference is found between weight of mice treated with vehicle solution or the peptide (data not shown).

(66) At the end of treatment the animals are sacrificed and tumor specimens are assayed for Ki67 antigen and apoptotic cells. Briefly, sections from each specimen are cut at 3-5 micron, mounted on glass and dried overnight at 37° C. All sections are then deparaffinized in xylene, rehydrated through a graded alcohol series and washed in PBS. This buffer is used for all the subsequent washes and for antibody dilution. Light-microscopic examination is performed after staining with hematoxylin/eosin and hematoxylin/Van Gieson. For immunohistochemistry, tissue sections are heated twice in a microwave oven for 5 min each at 700 W in citrate buffer (pH 6) and then processed with the standard streptavidin-biotin-immunoperoxidase method (DAKO Universal Kit, DAKO Corporation, Carpinteria, Calif., USA). Rabbit anti-human Ki67 from DAKO is used at a 1:100 dilution. The primary antibody was incubated for 1 hour at room temperature. Diamino-benzidine is used as the final chromogen, and hematoxylin as the nuclear counterstain. Negative controls for each tissue section are performed leaving out the primary antibody. Positive controls included in each experiment consist of tissue previously shown to express the antigen of interest. Two observers evaluate the staining pattern of the two proteins separately and score the protein expression in each specimen by scanning the entire section and estimating the number of positive cells visible for high-power-field 10×20. The level of concordance, expressed as the percentage of agreement between the observers, is 92%. In the remaining specimens, the score is obtained after collegial revision and agreement. TUNEL reaction is performed using the peroxidase-based Apoptag kit (Oncor, Gaithersburg, Md., USA). TUNEL positive cells are detected with diamino-benzidine and H.sub.2O.sub.2 according to the supplier's instructions. Two observers evaluate the staining pattern of the two proteins separately and score the protein expression in each specimen by scanning the entire section and estimating the number of positive nuclei visible for high-power-field 10×20. The level of concordance, expressed as the percentage of agreement between the observers, is 100%.

(67) The authors observed in LNCaP tumor xenograft specimens a significant reduction in the percentage of Ki-67 antigen positive cells (P<0.002, FIG. 6A, left panel) and a significant increase in the number of TUNEL-positive cells (P<0.009, FIG. 6A, right panel) in the SH3 treated group when compared to the control group. A similar result was found for the MCF-7 tumor xenograft specimens (FIG. 6B left and right panel).

Example 9: Preparation of Antibody Compositions

(68) The synthetic peptides and recombinant peptides described above may be used in the generation of an immune response in an animal or an human and for the preparation of antibodies specific for these epitopes. The preparation of vaccines and antibodies is well known to those of skill in the art as described herein above. Briefly, the novel peptides of the present invention may be used as antigens in animals in the following manner:

(69) Each peptide may be coupled to keyhole limpet hemocyanin (KLH) and used to subcutaneously immunize BALB/c mice. Initial injections contain 250 pg protein and the mice are boosted 7 weeks later with 250 μg of the respective KLH-coupled peptide and then bled 1 week later. The polyclonal antibodies produced by the injected mice are tested for their ability to recognize the peptide antigen in an ELISA assay. The Abs are also assayed for their ability to inhibit AR induced DNA synthesis.

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