A Robotic Chemistry System for the Discovery of Cancer ... · A Robotic Chemistry System for the Discovery of Cancer-Specific Targeting Ligands John V. Frangioni, M.D., Ph.D. Associate

Frangioni LaboratorySeeing is Curing.™ Center for Molecular Imaging

Beth Israel Deaconess Medical Center

A Robotic Chemistry System for the Discovery ofCancer-Specific Targeting Ligands

John V. Frangioni, M.D., Ph.D.

Associate Professor of MedicineAssociate Professor of Radiology

Beth Israel Deaconess Medical CenterHarvard Medical School

Boston, MA

Frangioni Laboratory - Center for Molecular ImagingBeth Israel Deaconess Medical Center

Disclosures• This talk describes the off-label use of indocyanine green,

which is FDA-approved for other indications.

• The FLARE™ imaging system is investigational only andnot approved for the indications shown.

• All FLARE™ technology is owned by Beth IsraelDeaconess Medical Center, a teaching hospital ofHarvard Medical School. As inventor, Dr. Frangioni maysomeday receive royalties if products are commercialized.

• Dr. Frangioni is the founder and unpaid director of TheFLARE Foundation, a non-profit organization focused onpromoting the dissemination of medical imagingtechnology for research and clinical use.


Does this Man, Woman, or Child Have Cancer?

If so, how much and where is it?

If so, how can we cure it?


Frangioni JV. “New technologies for human cancer imaging.”J. Clin. Oncol. 2008; 26: 4012-21.

The “Big Picture” in Cancer

≈ 1 million cells

≈ 1 billion cells


‘Plug and Play’ Chemistry to See and Kill Cancer

NonradioactiveOptical Contrast Agents

ChemotherapeuticsMRI Contrast Agents

RadioactivePET RadiotracersRadiotherapeutics

SPECT Radiotracers

Homing Device Light Bulb or Bomb


JChem-Based Roboticized Combinatorial Chemistry

#1 - Library SynthesisInput: Reactants and solventsOutput: Single tube w/104-105 beads

Gilson Quad Z215Liquid Handler

CEMMicrowave Synthesizer

Tech ElanBead Sorter

Sciclone ALH3000Liquid Handler

w/ custom UV Cleavage

Twister IIRobot for384 well

Plate Moves

Nikon TE2000Computer-Controlled

6-Channel Microscope

Bruker Ultraflex IIIMALDI-TOF

HST PrecisionGrids

#2 - Bead to Plate WellsInput: Synthesized beadsOutput: One bead per well

#3 - Compound Cleavage/Plating Inputs: Beads in wells Output: Compounds in DMSO

#4 - Glass Slide MicroarrayingInputs: Compounds in DMSOOutput: Compounds on Glass Slides

#5 - Live Cancer Cell Screening Inputs: Cells & glass slides Output: Hit selection (well #)

#6 - Compound IdentificationInputs: Compounds, EnumerationOutput: Absolute compound ID

TechElan BeadSorter


Plate Moves

Genomic SolutionsMicroarraying

Robot


Plate Moves

Custom LargeArea Stage


Robotic Chemistry


Station 0 - Software Infrastructure

ChemAxon JChem

In silico enumerationChemical databases

Pharmacophore modeling2-D and 3-D viewing

Custom C++/C# (± iLink Pro)Barcode managementInstrument integration

Robot logsQuality controlLinks to JChem

Database querying

Oracle Databases

BIDMC PetabyteStorage System


Station 1 (Automated Split-Pool Synthesis)JChem: 1) In combination with KNIME, GUI-based construction of

combinatorial libraries.2) In silico enumeration of all possible stop-products and final

products.3) Creation of master data set for mass spec analysis (Station #6)

• Bead aspirator/dispenser• Liquid handler• Microwave heating• Conventional heating(+120˚C) and cooling (-20˚C)


Station 2 (Bead Sorting and Quality Control)

CaliperTwister II

Tech ElanBeadSorter

MicrovideoInstrumentsDark-Field

Imaging System

VelmexBislider

CaliperMS-3 Barcode

Reader

1 Bead 2 Beads


Station 3 (Liquid Handling and Bead Cleavage)

CaliperUV Cleavage Station

375 nm @ 20 mW/cm2

Variable Shaking

Caliper ScicloneALH3000

Caliper Twister IIw/ 3 pods

Caliper MS-3 BarcodeReader

1 mm

NH2

NH

O

NH

OH

O

OO

N+

O-

O

NH

O

NH2

1) Fmoc-6-Ahx-OH/HATU/DIEA/DMF

2) Piperidine/DMF

OH

O

O

OH

ON

+

O-

O

HATU/DIEA/DMF

1) COCl2/DCM

2) R-NH2/TEA

NH

O

NH

O

O

OO

N+

O-

O

NH

OR

h! (360 nm)

OOOH

NH2

COOH

= 4'-(aminomethyl)fluorescein

(4'-AMF)

5 5

R NH2

R

0 min

30 min


Station 4 (Slide Microarraying)

CaliperTwister IIGenomic

SolutionsMicroarrayer

Caliper MS-3Barcode Reader

Glass Slides for LiveCancer Cell Screening

High-Throughput (5,076 spots)MALDI Slide


Station 5 (Live Cancer Cell Screening)

Nikon TE2000Computer-Controlled,6-ChannelMicroscope

MicrovideoInstruments

CustomLarge-

Format Stage

Velmex Bislider

Automated quantitation of up to 56 1”x3” glass slides,2 cell types per slide, 6 fluorophores per cell with

absolute co-registration


PC-3 (800nm)LNCaP (700nm) 700 nm/800 nm Merge

1 mm

200 µm

Poly(allylamine) (PAAm)60 kDa; 1 mg/ml spotting



Cancer-Specific Model Ligands

PC-3 (800nm)LNCaP (700nm) 700 nm/800 nm Merge

1 mm

200 µm

GPI (9 nM)β-AG Trimer (80 nM) GPI Trimer (700 pM)



Station 6 (Non-Encoded MALDIIdentification of Compounds)

Bruker Ultraflex IIIMALDI-TOF

Calibration spots

Teaching spots

For spotting organic solvents

Hydrophobicregion

Hydrophilic region

SolventFocusing

Stainless steel

Custom 5,076 Spot Hydrophobic Patterned MALDI Plate (HST, Inc.)


Station 6 (Non-Encoded MALDI)Combined view of all MALDI-TOF spectra acquiredfrom 5076 position array for 36 spotted compounds

1436.3 Da121.2 Da


Station 0 - Software InfrastructureJChem: The key to making the system work.


Station 0 - Software Infrastructure

JChem: The key to making the system work.

...5 Reactions in Series with 2 scaffolds and 5, 5, 5, 5, 4 reactants each = 5,000 compounds

Enumeration Process Time (JChem Reactor w/ KNIME and Custom Node): 6 min


Summary of System Capabilities (all scalable)

Station # Unique Compounds1 Limited only by bead volume, likely ≈ 105 beads2 2 racks = 76 plates = 29,184 compounds3 1.4 racks/7 racks = 54 plates = 20,736 compounds4 2 racks = 29,184 compounds (6 1x3”slides)5 56 1”x3” slides (280,000 compounds)6 5,076 or 10,152 compounds per 13 hours

≈ Synthesis of 1 new 10,000 compound library every 2 days

≈ Screening of up to 100 cancer biopsies against 5,000 newcompounds in ≈ 2 hr


The Future is “Personalized Medicine”“Personalized Medicine” and the cure of cancer is going torequire cocktails (5-10 compounds per individual cancer) of

agents that target the population (stem, intermediate,differentiated) of cells that comprise a human tumor

Breast Cancer “Chip” (5,000 compounds)

Prostate Cancer “Chip” (5,000 compounds)

Patient #1

Patient #2

Patient #1

Patient #2


Discovery Engine

Young Investigators

The BIDMC Center for Molecular Imaging

BIDMC andLongwood

Medical AreaResearch

Community

ImagingClinicalTrials

(eINDs,Phase 0,

IDEs)

TranslationalCancer Imaging

Facility

LongwoodSmall Animal

Imaging Facility(Longwood SAIF)

LenkinskiLab

FrangioniLab

Choi Lab Vinogradov Lab

Grant LabGibbs-Strauss LabPatientsIn

Need

Who are We?


Translational Cancer Imaging Facility

• Funded by a $1.8M NIHStimulus Grant to construct thefacility

• Facilitates the clinicaltranslation of PET, SPECT, andNIR fluorescent targetedcontrast agents

...From Mouse to Human...


An Optical Imaging Platform for Image-Guided Surgery


NIR Fluorescent SLN Mapping in a Patient with Breast Cancer


Acknowledgments

Director, Robotic Chemistry Group

Hak Soo Choi, Ph.D.

MALDIJeong Heon Lee, B.S.

EngineeringConor J. Cross, B.E.

JChem/Oracle IntegrationRafiou Oketokoun, M.S.

Tejas Gajera, M.S.Gabe Tobon, B.S.

ChemistryKhaled Nasr, Ph.D.


Websites

www.centerformolecularimaging.org

www.frangionilab.org

www.longwoodsaif.org


Relevant ReviewsFrangioni JV. New technologies for human cancer imaging. J. Clin.Oncol. 2008; 26: 4012-21.

Gioux & Frangioni. Image-Guided Surgery with NIR Light: Fundamentalsof Clinical Translation. Mol Imaging., 2010, in preparation.

Choi & Frangioni. Nanoparticles for Biomedical Imaging: Fundamentalsof Clinical Translation. Mol Imaging., 2010, in preparation.

Frangioni JV. Translating In Vivo Diagnostics into Clinical Reality.Nature Biotechnol. 2006; 24: 909-13.

Frangioni JV. The impact of greed on academic medicine and patientcare. Nature Biotechnol. 2008; 26: 503-7.

Gibbs et al. First-in-human clinical trials of imaging devices: Anexample from optical imaging. Proc IEEE Eng Med Biol Soc. 2009; 1:2001-4.

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A Robotic Chemistry System for the Discovery of Cancer ... · A Robotic Chemistry System for the Discovery of Cancer-Specific Targeting Ligands John V. Frangioni, M.D., Ph.D. Associate