RasGAP derived peptide for selective killing of cancer cells Description of the invention

1. Licensing Opportunity • The University of Lausanne is • proposing this technology for • exclusive or non-exclusive license. • Field: anti-cancer treatment. • This new technology is designed to selectively target tumor cells and to increase efficacy of genotoxins. • Development Phase • - In-vitro studies achieved • - Animal tumor models with very good results of tumor reduction / inhibition • Patent Status • US provisonal application 60/483,691 filed June 30th 2003 entitled “RasGAP derived peptide for selectively killing cancer cells”, naming C. Widmann, J. Yang and D. Michod as inventors • PCT/2004/002165 patent application filed on June 30th 2004 and published on January 6th 2005 under the number WO 2005/000887. • The patent application was granted in EP on May 28, 2008. • The patent application has been extended in Australia, Brazil, Canada, China, Europe, Israel, India, Japan, Korea, Mexico, Singapore, USA. • Innovative aspects • Design of a selective peptide-based sensitizer of tumor cells for enhanced efficacy of genotoxins. • Can be combined with various genotoxins used in clinical settings • Contact: • PACTT • Office of Technology Transfer • University of Lausanne & CHUV • 21, Rue du Bugnon • CH-1011 Lausanne, Switzerland • tel: +41 21 314 59 72 • fax: +41 21 314 49 57 • Pactt.info@chuv.ch • http:// www.pactt.ch • RasGAP derived peptide for selective killing of cancer cells • Description of the invention • The invention describes a new technology for treating cancers by improving the efficacy of currently used anti-tumor genotoxins. • The inventors have carried out a research program aimed at enhancing the selectivity and efficacy of current cancer treatments by associating a drug-sensitizer to currently used genotoxins to increase the sensitivity of cancer cells to drug-induced death. • By working on RasGAP, the inventors have shown that this caspase substrate can induce both anti and pro-apoptotic signals depending on the extent of its cleavage by caspases. A peptide derived from the N-terminal fragment of RasGAP fused to the TAT cell permeation sequence (TAT-RasGAP317-326) was then shown to enhance apoptosis of tumors cells induced by various genotoxins such as cisplatin, adryamycin and mitoxantrone. • The recombinant peptide TAT-RasGAP317-326 could inhibit the in vitro proliferation of 5 cancer cell lines: Hela cells (human adenocarcinoma), MCF-7 cells (breast cancer), U2OS (human osteosarcoma), H-Meso-1 (human malignant mesothelomia) and HCT116 (human colon carcinoma). • The recombinant peptide TAT-RasGAP317-326 in combination with cisplatin was further shown to inhibit the proliferation of established tumors in mice more efficiently than cisplatin alone at doses devoid of side effects and 100 times lower than lethal doses. • Applications • The results obtained with the RasGAP317-326 peptide offer the possibility to improve cancer treatments by: • 1.) enhancing the effect of genotoxins specifically in tumor cells • 2.) allowing efficient tumor treatment and reduced side-effects by • by using lower doses of genotoxins • 3.) preventing the outgrowth of genotoxin-resistant tumors • References • D. MICHOD et al. 2004. A RasGAP-derived cell permeable peptide potently enhances genotoxins-induced cytotoxicity in tumors cells. Oncogene Nov 25;23(55):8971-8978. • J.Y. YANG et al. 2004. Partial cleavage of RasGAP by caspases is required for cell survival in mild stress conditions. Mol Cell Biol. Dec;24(23):10425-36. • PITTET O. et al. 2007. Effect of the TAT-RasGAP(317-326) peptide on apoptosis of human malignant mesothelioma cells and fibroblasts exposed to meso-tetra-hydroxyphenyl-chlorin and light. • J Photochem Photobiol B. Jul 27;88(1):29-35. • D. MICHOD and C. WIDMANN. 2007. TAT-RasGAP(317-326) requires p53 and PUMA to sensitize tumor cells to genotoxins. Mol Cancer Res. May;5(5):497-507.

2. 1. RasGAP derived-peptides: a patent-protected new tool against cancer 2. Caspases are key effectors of apoptosis 3. RasGAP is cleaved by caspases during apoptosis TAT-RasGAP317-326: a potent sensitizer of tumors to genotoxins • The University of Lausanne is the owner of a patent protecting a set of peptides derived from the intracellular protein RasGAP. • These peptides have been characterized for their strong anti-tumor activity both in vitro and in vivo when used in combination with clinically-approved genotoxins such as platinum compounds. • The patent protects the use of these peptides in their use in combination with such genotoxins. • Caspases are proteases that are activated upon a death signal received by a cell. A strong activation of caspases is a lethal event that leads to apoptosis, a form of programmed cell death. • Most of the biochemical and cellular features of apoptosis are induced when executioner caspases (i.e. caspase-3, -6, and -7) cleave their substrates. • Inhibition of caspases with chemical compounds or with natural inhibitors efficiently blocks the death of cells in most situations. The inventors showed that RasGAP is a substrate of caspases. Their results support a model by which RasGAP functions as a sensor of caspase activity to determine cell survival or death.  RasGAP can be cleaved at low levels of caspase activity into a C-terminal fragment (fragment C) and an N-terminal fragment (fragment N) that generates potent anti-apoptotic signals.  As caspase activity increases, fragment N is cleaved into fragments N1 and N2 that have strong proapoptotic properties. The inventors further showed that apoptosis induced by genotoxins such as cisplatin is strongly potentiated by fragment N1 or N2 2

3. 6. Use of the Retro-inverso form of TAT-RasGAP317-326increases stability and efficacy The L or retro-inverso (RI) forms or the TAT-RasGAP molecule were incubated for the indicated time periods at 37°C in culture medium containing 10% Fetal Calf Serum HeLa cells were incubated at 37°C with cisplatin alone (control) or in the presence of the indicated compounds. Apoptosis was assessed 36 hours later. HeLa cells were then incubated at 37°C for 36h in the presence of cisplatin together with the collected media and the extent of apoptosis was assessed at the indicated time points. 4. Identification of 317-326 as the minimal genotoxin-sensitizing sequence within N2 sensitization to cisplatin HeLa cells were transfected with the indicated plasmids encoding the corresponding constructs. Cells were then treated with 15 mM cisplatin and the extent of apoptosis was determined 20 h later. The results are from 3 independent determinations. RasGAP: smallest N2 peptide sequence with genotoxin-sensitizing properties 317-326 5. Fusion of RasGAP317-326 to the HIV-TAT48-57 sequence allows entry into cells Phase FITC Identical results were obtained with various cancer and non-cancer cell lines U2OS H-Meso1 Hela cells were incubated for 3 h at 37°C 20M FITC-labelled Tat-RasGAP317-326 peptide. Phase contrast and epifluorescence (FITC) images are shown. HUVECs 3

4. 7. TAT-RasGAP317-326sensitizes cancer cells, but not non-cancer cells, to genotoxins CANCER CELLS NON-CANCER CELLS 8. In vivo toxicity and stability studies of TAT-RasGAP317-326 In vivo toxicity Plasma concentrations 3.3 mg/kg of TAT-RasGAP317-326 were injected i.p. into nude mice. The indicated compounds were injected i.p. into nude mice and lethality was assessed. All the mice that died did so in the first 24 hours. TAT-RasGAP317-326 concentration was measured by mass spectrometry in blood collected at the indicated time periods. Concentration (mM) Time after injection (min) Cancer and non-cancer cell lines were incubated with the indicated concentrations of cisplatin, adriamycin and mitoxantrone in the absence or in the presence of 20 mM HIV-TAT48-57 or TAT-RasGAP317-326 peptides for 20 h. The number of cells displaying pycnotic nucleus was then scored. The results correspond to the mean ± s.d. of three independent determinations. Asterisks denote significant differences between the genotoxin-treated cells incubated with TAT-RasGAP317-326 and those left untreated or incubated with the HIV-TAT48-57 peptide (**P<0.01; ***P<0.001). 4

5. 9. TAT-RasGAP317-326sensitizes HCT116 tumors to cisplatin In vivo chemo-sensitizing efficacy of RITAT-RasGAP317-326. (A) Nude mice were injected with 1.5x106 HCT116 cells intraperitoneally (i.p.). The following day and thereafter twice a week, mice were injected i.p. with PBS, 2.4 mg/kg of RITAT-RasGAP317-326, 1 mg/kg cisplatin, or a combination of cisplatin and RITAT-RasGAP317-326. After 28 days, the mice were sacrificed and the development of the tumors scored as indicated on the right hand-side of the panel. Mice that did not show any tumors were removed from the analysis (about 20%). The dots in the figure represent individual mice. Statistical analysis was performed with a Wilcoxon two-sample test. (B) Nude mice were injected sub-cutaneously with 250'000 HCT116 cells on the left and right upper flanks. Seven days later, the mice having developed visible tumors were injected each day for 7 consecutive days (in red on the figure) with PBS, RITAT-RasGAP317-326 (1.65 mg/kg), cisplatin (0.5 mg/kg), or RITAT-RasGAP317-326 and cisplatin. Tumor development was plotted as a function of time (left panel). The orthogonal dimensions of the tumors were measured at the indicated times with a caliper. The volume of the tumors was calculated as the (largest orthogonal dimension)2 * (smallest orthogonal dimension) * (/6). The number of tumors analyzed is indicated on the figure. The results correspond to the mean ± SEM. The right panel depicts representative examples of tumors from mice sacrificed at the end of the experiment. 5

6. A B 11. Applications and market: a new era for cancer treatment with platinum compounds 10. Therapeutic window of TAT-RasGAP: in vivo efficaceous doses of TAT-RasGAP317-326 are more than 100 fold less than lethal doses In vivo efficacious doses of TAT-RasGAP317-326 are more than 100 fold less than lethal doses (A) Nude mice were treated and analyzed as in panel B of the previous Figure. (B) Nude mice were injected with the indicated doses of RITAT-RasGAP317-326 peptide. The percentage of mice surviving was then recorded (3-4 mice per condition). Alternatively, the mice were injected sub-cutaneously with 250'000 HCT116 cells on the upper flanks. Seven days later, the mice having developed visible tumors were injected each day for 7 consecutive days with the indicated doses of TAT-RasGAP317-326 with 0.5 mg/kg cisplatin. Tumor volumes were determined 15 days later. The anti-tumor efficacy was calculated as 100% minus the ratio (in %) of the volume of the tumors with TAT-RasGAP317-326 over the volume of the tumors without peptide (7-13 mice per condition). (B) In vivo efficacy and lethality (based on cumulated experiments) are shown as a function on injected TAT- RasGAP317-326 amount. • In the next two decades, the world is expected to experience about 20 million new cases of cancer. • Genotoxins such as the platinum(II) complex known as cisplatin [cis-(NH3)2PtCl2] play a central role in cancer chemotherapy. With annual sales of about $500 millions, cisplatin is still one of the most effective drugs to treat testicular, ovarian, bladder and neck cancers. • However, genotoxins exhibit toxicity. Cisplatin and carboplatin have shown ototoxicity and nephrotoxicity while oxaliplatin has been associated with neurological symptoms. Most of these side effects are correlated with the cumulative-dose of the platinum drugs. • Moreover, limited efficacy and more severe adverse events are associated with the use of platinum compounds used as single agents. Therefore, combinations with synergistic treatments are needed. • To circumvent this toxicity and limited efficacy and to enhance their therapeutic index, several thousand platinum analogues with clinically more manageable side-effect profile, such as the orally bioavailable satraplatin, have been synthesized. Moreover, various treatments that combine platinum compounds are currently under investigation. • Combination of TAT-RasGAP317-326with genotoxins will result in two main advantages: • Use of lower amounts of genotoxins may decrease overall treatment toxicity • Increased sensitization of cancer cells to platinum by RasGAP may overcome genotoxin- • refractory tumors that are a major issue in cancer treatments. 6