“Bionano” research lines Prof. Nicola Rosato Dr. Massimo Bottini

NAST Workshop, July 3, 2013, Rome “Bionano” research lines Summary Nanotechnology, nanodrugs and carbon nanotubes “Bionano” research lines: the past “Bionano” research lines: the present “Bionano” research lines: nanodrugs for intratumor Treg targeting “Bionano” research lines: the future Conclusions Acknowledgments NAST Workshop, July 3, 2013, Rome

Nanotechnology, nanodrugs and carbon nanotubes “Bionano” research lines 1. Nanotechnology, nanodrugs and carbon nanotubes NAST Workshop, July 3, 2013, Rome

Information Technology “Bionano” research lines Nanotechnology The science of manipulating matter at the atomic and molecular level to obtain materials with specifically enhanced chemical and physical properties. Energy Information Technology More efficient and cost effective technologies for energy production Solar cells Fuel cells Batteries Bio fuels Smaller, faster, more energy efficient and powerful computing and other IT-based systems Consumer Goods Medicine Foods and beverages Advanced packaging materials, sensors, and lab-on-chips for food quality testing Appliances and textiles Stain proof, water proof and wrinkle free textiles Household and cosmetics Self-cleaning and scratch free products, paints, and better cosmetics Cancer treatment Bone treatment Drug delivery Appetite control Drug development Medical tools Diagnostic tests Imaging NAST Workshop, July 3, 2013, Rome

NAST Workshop, July 3, 2013, Rome “Bionano” research lines Nanodrugs NAST Workshop, July 3, 2013, Rome

Single-walled carbon nanotubes (SWCNTs) “Bionano” research lines Single-walled carbon nanotubes (SWCNTs) NAST Workshop, July 3, 2013, Rome

“Bionano” research lines: the past 2. “Bionano” research lines: the past NAST Workshop, July 3, 2013, Rome

Chemically-modified nanoparticles “Bionano” research lines Chemically-modified nanoparticles NAST Workshop, July 3, 2013, Rome

Chemically-modified nanoparticles “Bionano” research lines Chemically-modified nanoparticles NAST Workshop, July 3, 2013, Rome

PEG-modified carbon nanotubes in bio-medicine “Bionano” research lines PEG-modified carbon nanotubes in bio-medicine NAST Workshop, July 3, 2013, Rome

“Bionano” research lines: the present 3. “Bionano” research lines: the present NAST Workshop, July 3, 2013, Rome

NAST Workshop, July 3, 2013, Rome “Bionano” research lines Present projects Protein corona of PEG-modified carbon nanotubes Biodegradation of PEG-modified carbon nanotubes NAST Workshop, July 3, 2013, Rome

NAST Workshop, July 3, 2013, Rome “Bionano” research lines Present projects Biocompatibility of PEG-modified carbon nanotubes Intratumor Treg targeting by PEG-modified carbon nanotubes NAST Workshop, July 3, 2013, Rome

Nanodrugs for intratumor Treg targeting “Bionano” research lines 4. Nanodrugs for intratumor Treg targeting NAST Workshop, July 3, 2013, Rome

Background: Treg in tumor development “Bionano” research lines Background: Treg in tumor development CD4+CD25highFoxP3+ regulatory T cells (Treg) are harnessed by tumors to protect themselves from host anti-tumor responses. Immune cell relative abundance Spleen (healthy) Spleen (tumor) Tumor NAST Workshop, July 3, 2013, Rome

Background: Treg in tumor development “Bionano” research lines Background: Treg in tumor development The manipulation of Treg function selectively into tumors might be the next frontier of cancer immunotherapy. RES PEG-modified carbon nanotubes Tumor CD4 = carbon nanotube TCR/CD3 = monoclonal antibody CD25 = PEG = fluorochrome Regulatory T cell = cargo NAST Workshop, July 3, 2013, Rome

PEG-SWCNTs for intratumor Treg targeting “Bionano” research lines PEG-SWCNTs for intratumor Treg targeting 10141 nm Spleen = SWCNT = PEG = targeting agent = fluorochrome = Treg-specific receptor Tumor NAST Workshop, July 3, 2013, Rome

Choosing the target marker “Bionano” research lines Choosing the target marker GITR FR4 CD39 CD103 Spleen (healthy) Spleen (tumor) Tumor NAST Workshop, July 3, 2013, Rome

Ex vivo PEG-SWCNT-DTA1 Treg targeting efficiency and selectivity “Bionano” research lines Ex vivo PEG-SWCNT-DTA1 Treg targeting efficiency and selectivity Effect of number of DTA-1 mAb/SWCNT Effect of incubation time Effect of dose * * * * * * * NAST Workshop, July 3, 2013, Rome

Ex vivo PEG-SWCNT-DTA1 trafficking “Bionano” research lines Ex vivo PEG-SWCNT-DTA1 trafficking 1 hour 1 hour + 5 hours 6 hours NAST Workshop, July 3, 2013, Rome

Ex vivo PEG-SWCNT Treg targeting efficiency and selectivity “Bionano” research lines Ex vivo PEG-SWCNT Treg targeting efficiency and selectivity Anti-FR4 mAb Anti-CD39 mAb Anti-CD103 mAb * * * * * * * * NAST Workshop, July 3, 2013, Rome

Ex vivo PEG-SWCNT Treg targeting efficiency and selectivity “Bionano” research lines Ex vivo PEG-SWCNT Treg targeting efficiency and selectivity Treg targeting efficiency vs. receptor Treg targeting selectivity vs. receptor Treg vs. Unstained Treg vs. Teff * * * NAST Workshop, July 3, 2013, Rome

In vivo studies “Bionano” research lines Targeting efficiency (spleen of healthy mice) Targeting efficiency (B16-bearing mice) Pharmacokinetic profile * * NAST Workshop, July 3, 2013, Rome

“Bionano” research lines: the future 5. “Bionano” research lines: the future NAST Workshop, July 3, 2013, Rome

Nanodrugs for Treg-specific cancer immunotherapies “Bionano” research lines Nanodrugs for Treg-specific cancer immunotherapies Aim 1. Assess tumor killing elicited by PNT-DTA1. We will assess attenuation of intra-tumor Treg function and efficient tumor-killing by PNT-DTA1 systemically administered to B16-bearing mice. Aim 2. Assess increased intra-tumor delivery of PNT-DTA1 elicited by co-administered iRGD peptides. We will assess whether co-administration of PNT-DTA1 with iRGD peptides leads to a tumor-specific increase in nanoparticle accumulation and Treg targeting, and more efficient tumor killing in B16-bearing mice. NAST Workshop, July 3, 2013, Rome

NAST Workshop, July 3, 2013, Rome “Bionano” research lines Nanodrugs for RA 2,5ug 5ug 10ug NAST Workshop, July 3, 2013, Rome

NAST Workshop, July 3, 2013, Rome “Bionano” research lines Conclusions  Treg-targeting efficiency and selectivity of PEG-SWCNT-DTA1 depend on number of ligands per nanotube, incubation time, dose, and targeted surface marker.  PEG-SWCNT-DTA were internalized by Treg through receptor-mediated endocytosis and transported into the cytoplasm and nucleus ex vivo and in vivo.  Injection of PEG-SWCNT-DTA1 in animals carrying tumors enabled very good targeting of Treg residing in the tumor microenvironment, while much less efficiency and almost no selectivity was evident in the spleen. Preferential penetration of nanoparticles into the tumor microenvironment compared to other tissues (i.e. spleen) was a consequence of EPR effect naturally increased intra-tumor Treg vs. Teff ratio use of markers that are enriched in intra-tumor vs. peripheral Treg (i.e. GITR)   PEG-SWCNTs were degraded by activated neutrophils in approximately 3 hours NAST Workshop, July 3, 2013, Rome

NAST Workshop, July 3, 2013, Rome “Bionano” research lines Acknowledgements The Prof. Massimo Cristiano Novella Prof. N. Bottini’s Lab Prof. A. Magrini Prof. M. Mattei Dr. A. Pietroiusti Dr. L. Campagnolo Prof. E. Ruoslahti Prof. B. Fadeel Arthritis National Research Foundation Prof. A. Star NAST Workshop, July 3, 2013, Rome