Vascular Zip Codes in Nanoparticle Nanoparticle Targeting Targeting - PowerPoint PPT Presentation

Vascular Zip Codes in Nanoparticle Nanoparticle Targeting Targeting Vascular Zip Codes in Erkki Ruoslahti Erkki Ruoslahti Burnham Institute at UCSB Burnham Institute at UCSB and and Department of Molecular Cellular and Developmental Biology, UCSB Department of Molecular Cellular and Developmental Biology, UCSB

Ruoslahti lab Collaborators Former lab Doug Hanahan, UCSF Santa Barbara members Enrico Giraudo Hector Biliran Maria Akerman Priya Karmali Sven Christian Sangeeta Bhatia, MIT, UCSD, V.Ramana Kotamraju Markus Essler Austin Derfus, David Peters Pirjo Laakkonen Todd Harris Geoffrey von Maltzahn Barry Rowan Kimmo Porkka La Jolla Michael Sailor, UCSD Austin Derfus Ji HO Park Valentina Fogal Support Michael Schwartz Tero Jarvinen NCI, NHLBI, DoD, Komen Jan Pilch Robert Hoffman, UCSD Dmitri Simberg Kazuki Sugahara Matthew Tirrell, UCSB Tambet Teesalu Miriam Wankell Patrick Daugherty, UCSB Lianglin Zhang

Targeting Nanoparticles into Tumors Modular Approach to Multi- functional Nanoparticles Physically target: - vascular cells - tumor cells - extracellular matrix Trigger/engineer: - tissue penetration - RES avoidance - subcellular targeting - diagnostic signal(s) - release of therapeutic agent - amplification of targeting Ruoslahti, Cancer Cell, 2002

Isolation of homing peptides by ex vivo/in vivo phage screening Ex vivo Incubate Rescue phage Wash phage with and amplify cells from target tissue Inject (enriched) In vivo phage library into a mouse iv Repeat in vivo screening, Rescue and sequence individual phage amplify phage from enriched pool from target tissue Phage bind to blood vessels in target tissue Homing peptides

The blood vessels in individual tissues are distinct 350 Normal tissues targeted with Heart-homing phage 300 vascular homing peptides: 250 Relative phage titer Brain Kidneys 200 Lungs Prostate Skin 150 Breast Muscle 100 Pancreas Adrenals Uterus 50 Liver Heart 0 Muscle Lung Kidney Heart Brain - Bacterial 2-hybrid screen for receptors L. Zhang et al . Circulation , 2005

Metastasis Lymphangiogenesis (lymphatics) Angiogenesis (blood vessels)

Tumor-homing Peptides Peptide Sequence Specificity Receptor Reference Blood Vessels RGD-4C CDCRGDCFC Angiogenic endothelium α v β 3 and α v β 3 Arap et al., Science , 1998 integrins CAR Nonapeptide Angiogenic endothelium Jarvinen et al . F3 34-amino acid Angiogenic endothelium, Cell surface Porkka et al., basic peptide tumor cells nucleolin 2002; Christian et al., 2003 CGKRK CGKRK Angiogenic endo- Heparan Hoffman et al., thelium, tumor cells sulfate? 2003 Lymphatic Vessels LyP-1 CGNKRTRGC Breast ca lymphatics, Not known Laakkonen et tumor cells al., 2002; 2004 LSD Melanoma lymphatics CXCR4? Zhang et al, submitted REA Prostate ca lymphatics, Zhang et al, tumor cells submitted Vessel/Tumor ECM CREKA CREKA Breast ca extracellular Not known Essler et al., matrix unpublished Plasma Clot Pilch et al., CLTI/2 10-amino acid Blood clots, fibrin Fibro l., nectin cyclic peptides deposits in tumors PNAS, 2006

Tumor Blood Vessels

F3 is a cell-penetrating peptide specific for tumor endothelial cells and tumor cells FITC F3 PEPTIDE Inject i.v. Add to tumor cell culture F3 i.v.- green F3 + anti- Blood vessels - red nucleolin Nuclei - blue F3 F3, HL-60 tumor Anti-nucleolin i.v.- red Control IgG i.v.- red Blood vessels - green Blood vessels - green

Tumor-homing peptide CAR recognizes tumor vessels and penetrates into tumor parenchyma FITC-CAR peptide Blood vessels Nuclei Merge Jarvinen et al.

Fluorescein-labeled vascular homing peptides from in vivo phage display reveal blood vessel changes that parallel progression in tumor development Intravenously injected peptide Normal islet Angiogenic islet Tumor 1 2 3 Joyce et al., Cancer Cell, 2003; Hoffman et al., Cancer Cell, 2003

Tumor Lymphatics

LyP- LyP -1 peptide co 1 peptide co- -localizes with tumor lymphatics localizes with tumor lymphatics FITC-LyP-1 Podoplanin FITC-LyP-1 Lyve-1 Laakkonen et al.,Nature Med., 2002

LyP-1 and LSD phage have mutually exclusive tumor type specificities LyP-1 LSD recombinant phage 100 50 recombinant phage 80 Fold over non- 40 Fold over non- 60 30 20 40 10 20 0 r 5 r e 1 r 1 B o 0 C8161 3 o c 6 C m I 4 1 m n R - P B - 8 a u B K u P C c M t M t p n - T - A m i A M k D D a s y r M M P 5 V T 3 P 4 H Anti-podoplanin Anti-LYVE-1 Anti-CD31 Lymphatic homing peptides for Breast cancer Prostate cancer Zhang et al., Cancer Res., 2006 Melanoma

Selective tissue damage by homing peptide-targeted pro-apoptotic peptide D (KLAKLAK) 2 RGD-4C = c CDC RGD CFC Ellerby et al. Nat. Med., 1999 Receptor: integrins α v β 3 and α v β 5 Target: angiogenesis Arap et al. PNAS, 2002 Gerlag et al. Arthr. Res., 2001

Inhibition of C8161 tumor growth by D (KLAKLAK) 2 -LSD conjugate 1600 1400 D (KLAKLAK) 2 -LSD 40 D (KLAKLAK) 2 -LSD D (KLAKLAK) 2 plus LSD D (KLAKLAK) 2 plus LSD 35 1200 PBS PBS Vessels per field 30 1000 25 800 * 20 600 15 ** ** 400 10 5 200 0 0 Blood vessels Lymphatic vesse 16 20 24 28 32 36 days post implantatio L. Zhang et al.

Tumor imaging with a homing peptide for tumor lymphatics 140 Control peptide 120 LyP-1 LyP-1b 100 Fluorescence 80 60 40 20 0 Laakkonen et al., PNAS, 2004 S T B H Lu Li K

Extracellular Matrix

A Homing Peptide Binds to Clotted Plasma Proteins in a Tumor B A C liver tumor spleen CLT1 heart lung brain D F E spleen lung Control tumor heart peptide liver kidneybrain G H I CLT1/ Normal J L K tissues Pilch et al., PNAS, 2006

Nanomedicine

Specific in vivo homing of quantum dots PEG e d T Peptide i t p e P ZnS L CdSe Peptide PEG e d P i E t p G e P PEG F3-qdots Lung-homing LyP-qdots Tumor blood vessels qdots Tumor blood vessels F3 (34 aa peptide) cCGFECVRQCPERC cCGNKRTRGC Akerman et al. PNAS, 2002

Peptide-coated magnetic particle FITC F I T C PEPTIDE P E P T I D E Dextran Iron oxide PEPTIDE FITC PEPTIDE FITC E D P I E T P P T E I P D E C T I F F I T C

Proteolytically actuated self-assembly Harris, von Maltzahn et al. (Bhatia laboratory) Angew. Chem., 2006

Role of PEG length and characterization of assembly Harris, von Maltzahn et al. Angew. Chem., 2006

Triggered self-assembly of nanoparticles by tumor cells HT-1080 HT-1080 + inhibitor HT-1080 HT-1080 + Inhibitor Harris, von Maltzahn et al. Angew. Chem., 2006

Amplification of in vivo homing Tissue distribution CREKA-coated iron oxide particles vs CREKA peptide lymph nodes tumor liver tumor bladder bladder CREKA-IO CREKA peptide D. Simberg, T. Duza et al. (Ruoslahti laboratory)

Treatment of mice with decoy Ni-liposomes enhances tumor homing by CREKA peptide-targeted IO nanoparticles D. Simberg, T. Duza et al.

Ni-liposomes prolong blood half life of CREKA-IO nanoparticles D. Simberg, T. Duza et al.

CREKA-IO accumulation in tumor vessels after decoy particle treatment A x200 D. Simberg, T. Duza et al.

CREKA-IO nanoparticles accumulate in a fibrin meshwork in vivo and in vitro Tumor Plasma clot in vitro Tumor Fibrin CREKA-IO DAPI CREKA-IO DAPI CREKA-IO - Inhibited by blood clotting inhibitor (heparin, hirudin) D. Simberg, T. Duza et al.

Intravital imaging confirms clot formation in tumor vasculature in situ 20x Vessel wall D. Simberg, T. Duza et al.

Tumor imaging with decoy liposome-enhanced targeting of CREKA-IO nanoparticles D.Simberg et al.

Amplification of tumor targeting: CREKA-IO particles induce homing of non-targeted Cy7-IO particles A B C D.Simberg et al.

CREKA-IO particles may induce necrosis in tumor tissue D. Simberg, et al.

• Blood and Lymphatic Vessel Zip codes • Tumor blood vessels differ from vessels in normal tissues - angiogenesis-related - tumor type-related - tumor stage-related • Tumor lymphatics differ from lymphatics in normal tissues - tumor type-related - tumor stage-related • Vascular and tumor extracellular matrix differs from normal ECM - Clotted plasma proteins - ECM proteins • The markers of tumor vessels enable new therapies - guided drugs - druggable targets • Vascular zip code molecules are useful as guidance elements in nanoparticle targeting

CREKA-IO nanparticles may extravasate from some tumor vessels Simberg et al.

Homing peptide for Extracellular Matrix in Tumor Vessels and Tumor Stroma Tumor/FITC-CREKA Tumor /FITC-CSG Green= FITC conjugates; red=blood vessels; blue=nuclei M. Essler, M., H. Biliran et al.,

Multistage tumorigenesis in RIP-Tag transgenic mice Normal stage Hyperplastic/ Angiogenic Tumor (onc + ) Dysplastic stage stage stage <5 wks <5 wks 5- -7 wks 7 wks 7- -12 wks 12 wks 12- -14 wks 14 wks 5 7 12 100% 100% 2- -4% 4% ~50% ~50% ~10% ~10% 2