2 α-pPKC-θ (T538) Delivery via Cell Penetrating Peptide Mimics as a Novel Treatment of Aplastic Anemia E. Ilker Ozay 1 , Gabriela Gonzalez-Perez 2 , Joe Torres 2 , Gregory N. Tew 1,2,3 , and Lisa M. Minter 1,2,3 1 Molecular and Cellular Biology Graduate Program, 2 Department of Veterinary and Animal Sciences, 3 Department of Polymer Science and Engineering, UMass Amherst, Amherst, MA, 01003 MCB Retreat Spring 2014 Aplastic Anemia (AA) Factory for all blood cells Bone marrow In aplastic anemia (bone marrow failure), Bleeding Infection Autoimmune disorder “Aberrant T helper-1 (Th1) lymphocytes” Immune-mediated destruction Th1 Th1 Th1 Current Treatments Bone marrow transplantation Anti-thymocyte globulin administration Blood transfusion Molecular Signaling in Aberrant Th1 Cells IFN-γ T-BET IL-2 NOTCH1 IC PKC-θ Protein kinase C-θ(PKC-θ) gets phosphorylated in activated Th1 cells (Threonine 538 residue for full activation) Roderick et al., JEM 210, 1311-1329, 2013; Young, N.S. & Maciejewski, J., N Engl J Med 336, 1365-1372, 1997 1 Rationale behind targeting PKC-θ • Intact PKC-θ signaling is necessary to facilitate disease progression in the mouse model of aplastic anemia. • The disease can be completely rescued using Rottlerin by inhibiting PKC-θ function in the mouse model of aplastic anemia. • The viral immunity can still be protected in PKC-θ knockout mice. • Current treatment for aplastic anemia includes Anti-thymocyte globulin administration for Th1 cells targeting surface markers. However, 30-40% of the cohorts do not respond to this therapy. Inhibits the phosphorylation of PKC-θ, thereby its activation Matsumoto et al., Immunity 23, 2005; Springael et al., Biochem Pharma, 2007; Solomou et al. Blood, 2006; Scheinberg et al., The Journal of Pediatrics 153, 2008. 2 Targeting PKC-θ: Using An*bodies to Inhibit Its Func*on PKC-θ IS A POTENTIAL THERAPEUTIC TARGET FOR BONE MARROW FAILURE TREATMENT. INHIBITING PKC-θ FUNCTION IS BENEFICIAL TO TURN DOWN ABERRANT TH1 CELL ACTIVITY IN APLASTIC ANEMIA. STRATEGY PKC-θ T538 α-pPKC-θ PKC-θ T538 α-pPKC-θ Interfering with the function Cell Penetrating Peptides (CPPs) Peptides which have the ability to internalize a cargo into cells. HIV-1 TAT RKKRRQRRR 49- -57 Guanidinium-rich domain Covalently attached to cargo Responsible for cellular uptake More efficient than other cationic CPPs Preferable to lysine Polyarginine PEP-1 Better uptake Both hydrophobic and lysine-rich Also non-covalent interactions Sgolastra,F., deRonde, B.M, Sarapas, J.M., Som, A., Tew, G.N.; Accounts Chem. Res. 2013. ASAP; Stanzl et al., Accounts Chem. Res., 2013, ASAP; Kurzawa et al., Biochimica et Biophysica Acta 1798, 2010, 2274-2285; Morris et al., Nature Biotech. 19, 2001, 1173-1176 3 T-cell Master Key CPPM Inspired by; HIV-1 TAT, Pep-1, and Polyarginine Design of Cell Penetrating Peptide Mimics (CPPMs) CPPMs are able to enter and deliver a bioactive cargo to T cells Research Aim and Questions Forming a complex of CPPMs and α-pPKC-θ (T538) and delivering them into T cells (hPBMCs) to neutralize PKC-θ activity both in vitro and in vivo with the eventual goal of treating AA Can we deliver α-pPKC-θ (T538) by using our CPPM? Can cell-penetrating α-pPKC-θ (T538) neutralize the actions of PKC-θ in vitro? Can in vivo delivery of α-pPKC-θ (T538) attenuate immune- mediated bone marrow failure in a “humanized” mouse model? 4 Delivery of CPPM/α-pPKC-θ into T Cells Blank Agent1/FITC-IgG CPPM/FITC-IgG Comparison between commercial agent and CPPM 5 Population shifted to higher fluorescence with CPPM αpPKCθ WITHOUT CPPM αpPKCθ WITH CPPM Acknowledgments & Funding Lisa M. Minter Gregory N. Tew Barbara A. Osborne Tew Lab Federica Sgolastra Brittany M. deRonde Bob Fu Michael Lis Katie Gibney Cathy Walker Joel Sarapas Madhura Pawar Coralie Backlund Minter Lab Gabriela Gonzalez-Perez Christina Arieta Kuksin Joe Torres Karthik Chandiran Wesley Rossiter Victoria Mello Jessica Jarmolowicz Osborne Lab Rebecca Lawlor Anushka Dongre Furkan Ayaz Joseph Homsi Manit Munshi Charles H. Hood Foundation for Child Health Research 12 We are able to successfully deliver α-pPKC-θ (T538) via our CPPM into hPBMCs Our novel CPPM design has much better uptake of the antibody with compared to commercially available antibody delivery reagent We are able to interfere with the actions of PKC-θ by observing reduction of T cell immune response marker expressions AA model of mice treated with CPPM/α-pPKC-θ complex had longer survival compared to control mice In vitro and in vivo studies of α-pPKC-θ (T538) delivery into hPBMCs have promising results for the treatment of aplastic anemia for clinical studies Conclusions 11 In Vivo Humanized Mouse Model of Aplastic Anemia 10 9 6 7 8 In Vivo Humanized Mouse Model of Aplastic Anemia NSG (NODscidIL2Rγc null ) female mice CPPM treated human PBMCs Rest mice for 4 hours Human PBMCs CPPM/ α-pPKC-θ Wait for 17 days γIR CPPM treated mice lived LONGER (up to 36 days) Gabriela Gonzalez-Perez & Joe Torres 41% Mouse CD45 Human CD45 86% 92% 56% 88% 94% Bone Marrow Spleen Peripheral Blood DMSO treated hPBMCs: Bone Marrow Spleen Peripheral Blood CPPM/αpPKCθ treated hPBMCs: 11% Human CD4 Human CD8 9% 20% 18% 15% 25% Bone Marrow Spleen Peripheral Blood DMSO treated hPBMCs: Bone Marrow Spleen Peripheral Blood CPPM/αpPKCθ treated hPBMCs: 54% 27% 70% 53% 28% 63% Engraftment of Human CD45 Cells in Peripheral Tissues Infiltration of Human CD4 and CD8 T Cells in Peripheral Tissues 0 10 20 30 40 0 20 40 60 80 100 Days Survival (%) DMSO Rottlerin CPPM/α-pPKC-θ CPPM treated Humanized Mouse Model of AA SURVIVAL STUDY Gabriela Gonzalez-Perez & Joe Torres T-cell Activation and Disease Markers In Vitro Experimental Design Cytokine Expression CELLULAR VIABILITY AFTER 24 H OF TREATMENT CD25-MFI 24h 48h 72h 0 2000 4000 6000 8000 10000 12000 ** * * Hours after Treatment CD25 (MFI) ns ns ns NOTCH1 IC -MFI 24h 48h 72h 0 1000 2000 3000 4000 * * * Hours after Treatment NOTCH1 IC (MFI) ns ns ns CD69-MFI (24 h) Unstimulated DMSO Rottlerin CPPM/α-pPKC-θ Treatment 0 200 400 600 800 1000 * CD69 (MFI) ** T-BET-MFI 24h 48h 72h 0 500 1000 1500 2000 *** * * Hours after Treatment T-BET (MFI) ns ns ns IFN-γ 24h 48h 72h 0 10000 20000 30000 40000 * ns *** Hours after Treatment IFN-γ concentration (pg/mL) ns ns ns IL-2 24h 48h 72h 0 1000 2000 3000 4000 5000 ** ns * Hours after Treatment IL-2 Concentration (pg/ml) n/a ns ns 0 20 40 60 80 100 ns * Cellular viability (%) Unstimulated DMSO Rottlerin CPPM/α-pPKC-θ Treatment Unstimulated DMSO Rottlerin CPPM/α-pPKC-θ Treatment