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Engineered Mesenchymal Stem Cells in Cancer Therapy: Optimizing Suicide Gene Approach

This research focuses on utilizing engineered mesenchymal stem cells to combat cancers by optimizing suicide gene therapy. The study aims to enhance the efficiency of suicide gene therapy by metabolizing prodrugs into cytotoxic compounds, promoting bystander effects, and triggering immune responses against tumor cells. By introducing mutations in the CYP2B6 enzyme sequence, the catalytic efficiency is increased to convert Cyclophosphamide (CPA) into toxic metabolites. The study involves constructing a fusion gene, CYP2B6TM-RED, to improve the metabolism of CPA. In in vivo mouse models, efficient tumor eradication and bystander effects were observed with the engineered stem cells. The results demonstrate the potential of this approach in developing effective cancer treatments.

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Engineered Mesenchymal Stem Cells in Cancer Therapy: Optimizing Suicide Gene Approach

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  1. Use of engineered mesenchymal stem cells against cancers Isabelle de Waziers INSERM U1147 Personalized Medicine, Pharmacogenomics, Therapeutic Optimization Faculté de Médecine, 45 rue des Saints Pères 75270 Paris Cedex 06, France

  2. Vector Tumoral cells Transgene Transgene Enzyme Toxic metabolites Prodrug Bystander effect Suicide gene therapy

  3. MAJOR GOALS FOR AN EFFICIENT SUICIDE GENE THERAPY 1) A SUICIDE GENE ABLE TO METABOLIZE VERY EFFICIENTLY THE PRODRUG INTO CYTOTOXIC METABOLITES 1) A SUICIDE GENE ABLE TO METABOLIZE VERY EFFICIENTLY THE PRODRUG INTO CYTOTOXIC METABOLITES 2) AN IMPORTANT BYSTANDER EFFECT 3) AN IMMUNE RESPONSE DIRECTED AGAINST TUMORAL CELLS 4) AN EFFICIENT METHOD FOR DELIVERY SUICIDE GENE INTO THE TUMOR

  4. Cytochrome P450 2B6 (CYP2B6) acrolein Prodrug: Cycloposphamide (CPA) 4’OH-CPA Cyclophosphamide (CPA) (Endoxan ) NADPH-cytochrome P450 reductase (RED) Phosphoramide mustard R - nitrogen mustard alkylating agent from oxazaphosphorines group • used to treat various types of cancer (breast, ovarian and leukemia) and some disorders of the immune system (such as lupus) - is a prodrug converted in the liver into cytotoxic metabolites

  5. Introduction of mutations in CYP2B6 sequence to increase its catalytic efficiency to metabolize CPA Relative low affinity of CYP2B6 for CPA (Km = 3 à 5 mM) CNRS UMR 8601, (Dr. P. Dansette, Pr. D. Mansuy) URA 2096 CNRS, DBJC, CEA-Saclay, Gif sur Yvette (Dr. T-A. Nguyen, Dr. F. André)

  6. Human CYP2B6 4,9 62,5 12,8 Rat CYP2B1 1,45 35,9 24,9 Rabbit CYP2B4 5,28 13,5 2,6 Rabbit CYP2B5 4,17 2,8 0,7 Canine CYP2B11 0,16 28,2 174,7 Sequence alignment of the different isoforms Identification amino acids exclusively present in the CYP2B11 substrate recognition sequences (SRS) 4-OH-CPA hydroxylation Km Vmax Vmax/Km min-1 mM

  7. Vmax/Km (% of wt) 2B6wt 100 I114V 221 CYP2B6 triple mutant (I114V + L199M + V477W) 411 V477W 278 790 L199M 205 CPA hydroxylase activity CYPB6 triple mutant 140 Vmax = 107,5 ± 3,4 min-1 Km = 0,51 ± 0,08 mM 130 120 CYP2B6wt 110 Vmax = 107,3 ± 3,74 min-1 Km = 4,33 ± 0,5 mM 100 90 80 70 60 50 40 30 20 10 0 0 5 10 15 20 25 30 CPA mM CYP2B6TM metabolizes CPA 8-fold more efficiently than CYP2B6 wt

  8. human Reductase (-171 pb en 5 ’) CYP2B6 TM (-TGA) Ser-Thr Ser-Ser linkers (Ser)2 à 7-Thr Optimisation ofCYP2B6 triple mutant (CYP2B6TM) • NADPH CYP reductase (RED) essential for electron transfer to CYP • Weak or no expression of RED in tumoral cells • Construction of a fusion gene CYP2B6TM-RED

  9. CPA hydroxylase activity Reductase activity (nmol/min/mg) (Vmax/Km) 2B6wt 12,7 2B6TM 100,5 2B6TM-RED fusion 75,4 342 1 Ser + Thr 72 80,0 2 Ser 81 345 2 Ser + Thr 104,1 653 3 Ser + Thr 76,8 99 4 Ser + Thr 121,9 292 5 Ser + Thr 97,27 314 6 Ser + Thr 91,45 120 7 Ser + Thr

  10. IC50 in infected cells (A549) Vector: Recombinant lentivirus (LV- CYP2B6TM-RED) Pr. F. Lemoine UMR-S-CR7, INSERM U1135

  11. SUICIDE GENE OPTIMISATION 3 Ser + 1 Thr CYP2B6 TM (-TGA) RED (-171 pb en 5 ’) linker CYP2B6TM-RED13-fold more efficient than CYP2B6wt-RED for CPA metabolization Brevet: PCT/EP2012/058219 le 4/5/2012 avec extension internationale US-2014-0127180A1 le 8/5/2014

  12. 9 8 7 IC50 in infected cells 6 CPA mM 5 LV-CYP2B6wt-RED 4 3 2 LV-CYP2B6TM-RED 1 0 TC1 murine pulmonary cell line A549 A549 ANIMAL MODELS • Immunocompetent C57Bl/6 mice • TC1 cell line derived from a C57Bl/6 lung epithelial-cell line expressing the HPV16 E6-E7 proteins (Dr. TC Wu , John Hopkins University, Baltimore, USA) • TC1-Luc2 with stable expression of luciferase • (Pr. E. Tartour, INSERM U970, PARCC) • TC1-Luc2 are insensitive to CPA Collaboration: DR. J. Seguin INSERM U1022, CNRS UMR8151),

  13. In vivo Immunocompetent mice C57Bl/6 Tumors TC1-CYP2B6TM-RED + TC1-Luc2 (expressing luciferase) + CYP2B6TM-RED 25%TC1-Luc2 + CYP2B6TM-RED 75 % TC1-Luc 2 1st injection of CPA + MIX J13 after 2 injections of CPA J25 after 4 injections of CPA - CPA Tumor volume (mm3) (n=6) J35 + CPA CPA 140 mg/Kg ip Days after cell injections Complete tumor eradication Efficient in vivo bystander effect

  14. MAJOR GOALS FOR AN EFFICIENT SUICIDE GENE THERAPY 1) A SUICIDE GENE ABLE TO METABOLIZE VERY EFFICIENTLY THE PRODRUG INTO CYTOTOXIC METABOLITES 1) A SUICIDE GENE ABLE TO METABOLIZE VERY EFFICIENTLY THE PRODRUG INTO CYTOTOXIC METABOLITES 2) AN IMPORTANT BYSTANDER EFFECT 3) AN IMMUNE RESPONSE DIRECTED AGAINST TUMORAL CELLS

  15. PROTECTION RECOVERY 2800 2.3x10 9 2600 2.0x10 9 2400 1.8x10 9 TC1-CYP2B6TM-RED - 140 mg/kg (n=8) 1.5x10 9 TC1-Luc2 - 140 mg/Kg (n=5) 2200 1.3x10 9 2000 1.0x10 9 1800 7.5x10 8 1600 5.0x10 8 CPA injection 1400 2.5x10 8 1.0x10 6 1200 8.0x10 5 7.0x10 5 Rechallenge 1000 treated anti-CD8 (n=4) 6.0x10 5 Rechallenge 800 5.0x10 5 untreated (n=4) 3 Rechallenge (250.10 Controls (n=5) 600 TC1-Luc2) 4.0x10 5 3.0x10 5 400 After s.c. injection of tumor cells in mice in complete remission spontaneous tumor eradication. 2.0x10 5 200 1.0x10 5 0 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Jours Anti-CD8 injections Collaboration: Pr. E. Tartour INSERM U1022, PARCC This CD8-dependent immune response constitutes a major advantage of our strategy and could prevent the development of metastasis (Touati et al. Curr Gene Ther 14:236-246, 2014)

  16. 1) A SUICIDE GENE ABLE TO METABOLIZE VERY EFFICIENTLY THE PRODRUG INTO CYTOTOXIC METABOLITES 1) A SUICIDE GENE ABLE TO METABOLIZE VERY EFFICIENTLY THE PRODRUG INTO CYTOTOXIC METABOLITES 2) AN IMPORTANT BYSTANDER EFFECT 3) AN IMMUNE RESPONSE DIRECTED AGAINST TUMORAL CELLS MAJOR GOALS FOR AN EFFICIENT SUICIDE GENE THERAPY 4) AN EFFICIENT METHOD FOR DELIVERY SUICIDE GENE INTO THE TUMOR

  17. PROJECTS Suicide gene delivery in tumoral cells • intratumoral injection of recombinant LV-CYP2B6TM-RED • intratumoral injection of mesenchymal stem cells • infected by recombinant LV-CYP2B6TM-RED Subventions of SATT Ile-de-France Innov (2014-2015) and the Ligue contre le cancer 2013, 2015

  18. Schematic presentation of mesenchymal stem cells (MSCs) expressing a suicide gene in cancer therapy Insertion of therapeutic gene and cell amplification Bone-marrow MSCs Isolation of MSCs from bone-marrow or adipose tissue Injection of transduced MSCs (intraveinous, intraperitoneal or intratumoral) in mice bearing tumors White adipose tissue Prodrug administration Effects on tumor size and animal survival Death of transduced MSCs and tumoral cells Tumoral tropism of MSCs Amara et al, Biochimie, 105: 4-11, 2014)

  19. Generation of MSCs clones expressing CYP2B6TM-RED IC50

  20. Cocultures of tumoral cells (TC1-Luc2) and MSCs CYP2B6TM-RED

  21. Generation of MSCs clones expressing luciferase

  22. INTRATUMORAL INJECTIONS OF MSCs-LUC D1 D3 D3 D1 D14 D6 D14 D6

  23. Immunocompetent mice C57Bl/6 40 mice Injection s.c 5.105 cellules TC1-Luc2 When the tumor volume was about 400 mm3 6 mice 17 mice 17 souris Intratumoral injection of PBS Intratumoral injection of 106MSCs non infected (NI) (controls) Intratumoral injection of 106 MSCs infected with LV-CYP2B6TM-REDcl.42 Once a week during four weeks 2 days later 2 days later 2 days later 3 mice 3 mice 5 mice 12 mice 5 mice 12 mice without CPA without CPA withpout CPA + CPA i.p. (140 mg/kg) + CPA i.p. (140 mg/kg) + CPA i.p. (140 mg/kg) Tumor growth evaluated by Vernier Caliper measurement

  24. Tumoral volume in mice treated with CPA CSM CYP2B6TM-RED CSM PBS CPA (90 mg/Kg) injections n=3 n=6 n=12 Volume tumoral mm3 n=3 Injection de PBS or CSMs days

  25. Rechallenge experiments

  26. Suicide gene Multiplication of MSCs containing the suicide gene + Mesenchymal stem cells (MSCs) • Destruction of tumoral cells • Memory of immune system Intratumoral injection Tumor Production of cytotoxic lymphocytes CD8 directed against tumoral cells + CPA Metabolization of CPA into cytotoxic metabolites in MSCs that kill MSCs and neighbouring tumoral cells Immune system alert

  27. POTENTIAL CLINICAL APPLICATIONS MSCs lack major histocompatibility complex MHCII and show minimal MHCI expression Allogenic mesenchymal stem cells delivered locally • well tolerated • currently used in Phase III clinical trials • (chrohn disease, rheumatoid arthritis, severe sepsis…)

  28. PERSPECTIVES • Increase in amounts of therapeutic MSCs delivered to the tumor using intra-arterial administration • (in Vx2 hepatic tumor models) - Co-expression of IFN in therapeutic MSCs to stimulate immune response - Association of CPA with other prodrugs bioactivated into cytotoxic metabolites by RED (MMC, …) - Enhancement of MSCs tropism by association with radiotherapy

  29. Philippe Beaune CNRS UMR 7211 Ikrame Amara François Lemoine Géraldine Lascaille Claude Baillou Walid Touati Marina Tychopoulos Elodie Pramil Jean-Pierre Flinois Monique Diry INSERM U970 Eric Tartour Thi Tran CNRS UMR 8601 Patrick Dansette URA 2096 CNRS Daniel Mansuy Thien-An Nguyen CNRS UMR 8151-INSERM U1022 François André Plateforme d’imagerie du petit animal Johanne Seguin Michel Bureau INSERM U1147

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