1. “Forget Antibodies. Use Aptamers!”

2. Presentation Contents:
1. Introduction and Background
2. Aptamer Introduction
3. Diagnostic Applications
4. Drug Discovery Applications
5. Delivery Applications

4. B.S. in Biochemistry, minor in Mathematics
Founder Highlights:
B.S. in Biochemistry, minor in Mathematics
Philadelphia College of Pharmacy and Science
Ph.D. in Neuroscience
Hahnemann University (Drexel University College of Medicine)
Dissertation Thesis work
Yale University
Gaetano Tom Caltagirone, Ph.D.
Aptagen, a biotechnology company based in central Pennsylvania, offers aptamer custom-based services to replace antibodies in research, diagnostic platforms, drug discovery and therapeutics. The company was founded in 2004 by Dr. G. Thomas Caltagirone, and operations began at the current facility located in Jacobus, PA in Dr. Caltagirone has over 20 years of research and business experience in start-ups. A native of York, PA, he began his studies at The University of the Sciences in Philadelphia followed by Drexel University in Philadelphia and completed his thesis on “Proton-Sensitive Ribozyme Switches with Molecular Memory” at Yale University with a Ph.D. in Neuroscience. Aptagen has grown from a one-man operation with the help of local interns to a tight-knit developing business with clients ranging globally from research academics at top-tier institutions to BigPharma companies. Aptagen has been named as a finalist for the “Top Emerging Business of the Year” by Central Penn Business Journal. Aptamers are an emerging technology that is poised to become the next evolution in diagnostics and drug discovery. Aptagen continues to play a leading role in developing aptamer technology that will assist in the treatment and diagnosis of various diseases.
© 2007

5. Clients and Organizations We Served
Serving over 50 Companies, Organizations, and Universities globally.

7. Examples of Aptamer ShapesB. C. D.
A. Pseudoknot (ligand for HIV-1 reverse transcriptase)
B. G-quartet (ligand for thrombin)
C. Hairpin (ligand for bacteriophage for T4 polymerase)
D. Stem loop/bulge (ligand for ATP)
taken from McGown, et.al. (1995)

8. Apta-index™ pM to nM affinity
Engineer out cross-reactivity…eliminate false positives (10,000 fold specificity, e.g. Theophylline/Caffeine)
Ligand binding against unknown and undiscovered biomarkers
Manufacturing (pennies on the dollar)
Stability (long shelf-life; heat denature/refold)
Apta-index™
(database of aptamers)

9. Basic Concept of ‘Directed Molecular Evolution’
Heterogeneous
Population
of Molecules
’sloppy’ copy
to explore
mutations
collect
Target immobilized
on column surface
‘Fittest’
molecules
Molecules that Bind to Target
discard
Molecules that do not
Bind to Target

10. General Aptamer Selection Scheme
random oligonucleotide pool
determine oligo
sequence(s)
of aptamer(s)
1014 single-stranded molecules
Oligo synthesizer
(7 to 15 rounds)
target
Capture
ligand-target
complexes
Propagate
(i.e. amplify by PCR)
discard unbound
collect bound oligo ligands

11. Diagnostic Applications

12. Conventional Antibody-based Diagnostics (ELISA) Method
Plate coated with capture antibody
Add samples
Add detection antibody
Add substrate
Incubation steps and wash steps before detection = total time >>2 hours

13. Quantitate based on a titration of controls
Apta-beacon™ Diagnostic Assay (simple 1-step reaction, free in-solution)
Positive
Sample
Negative
Sample
No Incubation or wash steps = total time << 1 minute
Analyte
Quantitate based on a titration of controls

14. Biosensor and Biochip Platforms
Point mutation to inactivate switch function
Aptamers easily tethered to solid interface through a wide variety of conjugation chemistries.
Detection of binding event intact

15. FlashGel™ analysis (5 minute run)
Apta-sensors
Aptamers that produce an immediate output signal for detection of target analyte.
apta-beacons™
apta-switches™
F/Q removed Q F
FlashGel™ analysis (5 minute run)

16. (aptamer that produces a self-cleavage output signal)
Apta-switch™
(aptamer that produces a self-cleavage output signal)
Target
Example
Co2+, Ni2+, Cd2+
Zn2+, Mn2+
Metal Ions
Small Organics
Caffeine
Peptides
Rev Peptide
Phosphorylated ERK2,
Unphosphorylted ERK2
Proteins
100 mM Theophylline
Specificity Against Theophylline vs. Caffeine
100 90 80 70 60
Ladder
DEPC only
RXN Buffer only
1 mM Caffeine
100 mM Caffeine
10 mM Caffeine
10 mM Theophylline
1 mM Theophylline
1 minute reaction at 23oC, then stopped with stop buffer containing excess EDTA.
pH Profile of Selected Acid-Induced Allosteric Ribozymes A)pH profile of proton sensor. Internally 32P-labeled wildtype acid sensitive ribozymes were pulsed with a series of buffers (at constant ionic strength, I=.005) ranging from pH 2-12 for < 1 minute, followed by neutralization in 500 mMTris-HCl pH 7.5, 100 mM MgCl2 at 23oC for 15 minutes. Each buffer species was prepared for two different pH values (indicated by Buffer Series A & B). Reaction products were separated by denaturing 10% PAGE, and the bands were visualized and quantified using a phosphorimager and ImageQuant software (Molecular Dynamics). Open and filled arrowheads identify the precursor and 5’ cleavage, respectively. The 3’ cleavage products have greater electrophoretic mobility than the significantly larger precursor RNAs and 5’ cleavage fragment, and are therefore not present on the images. As indicated, two buffer sets were tested which allowed not only duplication of results within acceptable experimental error, but discounted any buffer species effect. The negative control consisted of RNA devoid of an acid pulse and simply exposed to standard neutral buffer conditions for 15 minutes. B) Numerical representation of the above gel cleavage activities under differing buffer pulses. The fraction cleaved at each pH pulsing event before neutralization and permissive reaction conditions. The filled and open circles represent buffer series A and B tested, respectively. C) Representation of an allosteric ribozyme. The secondary structure of the hammerhead motif (stems I,III, and catalytic core), as well as the short communication module (cm) sequence structurally linking the hammerhead motif to the aptamer-binding domain are indicated by heavy bars. The site of catalytic cleavage is indicated by the arrow. Numerouse RNA ribozymes for metal ions, small organics, peptides, and proteins have been reported in published work. * The following targets have been previously reported in Muller, S., D. Strohbach, and J. Wolf, Sensors made of RNA: tailored ribozymes for detection of small organic molecules, metals, nucleic acids and proteins. IEE Proc Nanobiotechnol, (2): p
500-fold sensitivity range

17. Apta-switch Selection Strategy5’
N55
Synthesized N55
random oligo library
Promoter
Random Region
Primer
Extension
RT-PCR 5’
N55
Transcription
Aptamer
RNA library
N55
Mg++ dependent
Cleavage site
pre
clv
Purify
Cleaved 5’
Fluorophore
Hammerhead
ribozyme motif
Apta-switch Selection Strategy
Refolding
PAGE
Partitioning
Positive
Selection
Negative/Counter
Selection
Selection
(-) Target
(Buffer alone
or Counter-target)
Library
(+) Target
Library
Optional:
RT-PCR
2. Transcription
Refolding
PAGE
Partitioning
pre
clv
Purify
Pre-cleaved

18. Apta-beacons™ vs. Competition
antibodies
aptamers
apta-beacons™
Chemistry
protein
DNA/RNA
RNA
Stable / Refolding
++++
++++ (with RNAse inhibitor)
HIGH affinity
HIGH selectivity + ++
Unknown or undiscovered biomarkers
Small targets
Targets which are difficult to immobilize
One-step detection: direct output signal from target binding
In-solution based detection
Biosensor implementation
Lower Cost to manufacture
Sequences provided
Client retains IP

19. Apta-switch™ Demonstration Kit (Theophylline/Caffeine)

20. Drug Discovery Applications

21. > 8 years >$1B Pharmaceutical Drug Development Process 5
Success
Rate 5
Enter human clinical trials
> 8 years
>$1B
Animal Testing
of Drug candidates
5000
In vitro or in vivo assays
on drug candidates
Because of all of these issues involved in drug development, the process is reduced to a trial and error approach inevitably making it unpredictable—on average, only one in a thousand drug candidates make it to clinical trials in humans. As a consequence, the cost and time associated with this traditional method of drug development is expensive and quite slow (>6 years and $800+M).
Knowledge of Target / Mechanism
Pharmaceutical Drug Development
(combinatorial, natural product screening, etc.)

22. Drug Discovery Process (time consuming and labor intensive)
MASS SCREENING
Drug Discovery Process
(time consuming and labor intensive)
Random
High Volume
Screening
In Vitro
Studies
In Vivo
Studies
Clinical Studies
Humans
A positive hit in a “test” tube environment does not necessarily translate into a success in an in vivo environment. Compound has to be re-engineered and tested again in test tube, then back to animal. Back and
forward through this iterative process costs time
and money.
Combinatorial
Chemistry

23. X X Aptagen’s Drug Discovery in Whole-Animal Models
(Saving Time and Money)
Random
High Volume
Screening X
In Vitro
Studies
In Vivo
Studies
Clinical Studies
Humans
By eliminating the “test” tube step, and performing drug discovery ‘directly’ in an animal model, we are one step closer to human clinical trials, thereby saving time and money.
Combinatorial
Chemistry 23

24. DELIVERY is always an issue!
Reasons for Failures of Aptamer Drug Candidates
Typical Aptamer Strategy: Develop aptamers in vitro against a
known protein target of interest to block disease pathway.
however…
In vitro selected aptamers do not necessarily operate/function
in vivo as therapeutic candidates.
Aptamers are sensitive to the environmental conditions in
which they are selected.
In Vitro
Studies
In Vivo
Studies
Clinical Studies
Humans
The Conventional Paradigm in preclinical development is deficient.
DELIVERY is always an issue! 24

25. W H O L E - A N I M A L S E L E C T I O N
Animal Model of disease or condition
Molecular Library
(bolus injection,
nasal, or oral
administration)
Narrative:
If the molecules under question are chemically stable enough to survive in the blood stream of an organism, then selection can be performed in a whole animal model. We can bypass in vitro selection and do selection directly in vivo to generate potential drug candidates. Here, a library of molecules is administered to an animal model exhibiting the disease or condition of interest. Depending on the chemical nature and stability of the library of molecules, administration of the library can be an injection, nasal or oral delivery. The tissue or organ exhibiting the pathology is harvested, and any molecules associated are recovered and amplified for another round of selection.
[INCOMPLETE- search old records for text]
Scene Description:
Professional female voice for narration.
Music in background.
ANIMATION and/or video clips based on narration content.
General Comments:
Introduction to technology of drug discovery and Aptagen’s drug discovery methods.
Replicate (Amplify), enrich,
and reselect MOLECULES
associated with
pathological marker
Isolate and
process tissue
or organ of
pathological
interest
Pathological
Marker
Normal Tissue
Area
Tissue Selection

26. In drug development, DELIVERY is always an issue!
Selection in Whole-Animals solves DELIVERY issues.
(Use molecular bullet to attach known drug to increase specificity)
Chemical Diversity solves drug-like effects.

27. Potential for ‘smart’ molecular bullets with Drug-like properties
Initial round
Progression of
Selection
with gradual
disappearance
of pathological
marker…
Normal tissue -
no sign of
pathology
Nth round
of ‘natural’
selection…

28. Key Requirements for Successful Selection:
1) Self-replicating molecules
2) Animal Model
3) Characteristic Phenotype for Visualization
(of Target or Biomarker)
Disease, Infection (bacterial or viral), etc...
Could possibly Influence behavior?
Enhanced cognitive abilities? etc…

29. Delivery Applications

30. Preliminary Experiment: Targeting Major Organs & In Vivo Stability
Tail vein injection
2’-F-RNA library
(-) Library
nanomolar amounts
40 minutes
post-IV
Isolate various
organs/tissue
Tissue Harvesting
Purification of Rare 2’-F-RNA species
RT-PCR
Lane: 1 2 3
gel
DNA
Ladder
no band

31. 2’-F-RNA Targeting to Major Organs of the Mammalian Anatomy

32. 2’-F-RNA LUNG Targeting focused on LUNG enrichment...
Enrichment Ratio = qPCR of ‘extracted’ library relative to ‘input’ library

33. * * * LIBRARY C L O N E S Family # of Clones 12 7 2 Secondary
Enrichment RATIO *
3.00E-02
2.00E-02 *
1.00E-02
0.00E+00 G9
G9A2
G9B1
G9C4
LIBRARY
C L O N E S
Secondary
Structures
(MFOLD)
[original legend notes for EndNote]
All tissue samples were normalized at 0.5 mg/ul for enrichment ratio analysis.
(*) The following tissue samples that could not be analyzed by qRT-PCR because of insufficient data points to meet the standards for quantitative measurement: G9 library (brain & spleen), G9A2 (brain), G9C4 (brain & heart). Rejected data points include samples with a significant primer-dimer formation as indicated by melting curve analysis or a correlation coefficient of less than 0.98 for linear regression analysis and interpretation of the enrichment ratio as defined.
Also, note, the bar graph for G9C4 represents a second trial analysis. The first trial analysis exhibited an elevated liver background for enrichment ratio. Subsequent trials have not yet been attempted to reproduce the results shown.
The underlined guanine bases indicate possible G-quartet structure according to….
ΔG kcal.mole-1
Tm 73.6oC
ΔG kcal.mole-1
Tm 75.6oC
ΔG kcal.mole-1
Tm 65.9oC
Family
# of Clones
5’- gggcgacccugaugag [Consensus Sequence] cgaaacggugaaagccguagguugccc -3’
Group A 12
[UGACUGCUCCGUUCCGUUAUGACAGCUGCACCCAGUUAAAGC:GGUUCUGGGUCCGGA]
G9A2
Group B 7
[CCUUUUUGAACAACUGUGCGAUUUGAUUG:AAAAUUCUCUCUGAUCCCACCGUGACG]
G9B1
Group C 2
[UCUAGAGCGCAGAAACUUCUCUCAACGAUUCCCCACGUCCUCGCCCCGCCCGGU]
G9C4 33

34. Fluorescence Microscopy
1) 5’-end labeled G9C4 RNA aptamer
with ADO™550/570
2) Washed with PBS & Fixed tissues
with acetone
3) In situ bound (~4 mg) aptamer for
40 minutes at room temperature,
and wash
1/6 sec exposure
Lung
1/3 sec exposure
Note:
brain, spleen, heart, kidney were NEGATIVE
Liver 34

35. Aptamer Selection for Surface Binders
Template
LCR
Negative Selection
G6-Gx
Circular DNA
PCR
Amplification of bound aptamers
PC3
Positive Selection
(G0-G5)
(Optional) PCR Amplification of unbound aptamers
Bound
aptamers
PC3-PSMA
Unbound aptamers
WASTE
Figure 1. Schematic of Strategy. Linear template will undergo circularization via LCR (Ligation Chain Reaction). The circularized aptamers will be incubated with PC3-PSMA cells for positive selection. Aptamers specific for PSMA will be amplified; the selection process will be repeated for approximately five generations, before beginning a negative selection process with parental PC3 cells.

36. Flow Cytometry of Enriched Aptamer Library on (-) Parental Cells
Figure 7. Enrichment of the circular ssDNA library specific for PC3 monitored by flow cytometry. 5 x 105 PC3 cells were
incubated with G0 (scrambled), G19 (enriched), or unlabeled (binding buffer only) library for 30 min at 4°C.
a. Flow cytometry dotplot results of unlabeled (left), G0 (center), and G19 (right) labeled PC3 cells. The top row
represents side scatter (y-axis) and forward scatter (x-axis) cell morphology by identification of the cells, and
excluding any debris and dead cells from the PC3 cells. The bottom row shows fluorescence (x-axis) and side scatter
(y-axis) of the FITC fluorescently-library that has bound to the PC3 cells.
Ref:[Notebook, AN Priya Book 3, ]
b. Histogram of flow cytometry Fluorescence intensity (x-axis) as a function of the number of viable cells (y-axis)
analyzed with Flowing Software v The G19 library (blue) is shifted to the right of the G0 (red) and unlabeled library
(black) after incubation with PC3 cells.
Ref:{Notebook, AN Priya Book 3, ]

37. Flow Cytometry of Enriched Aptamer Library on (+) Cells
Figure 6. Enrichment of the circular ssDNA library specific for PSMA-PC3 monitored by flow cytometry. 2.5 x 105 PSMA-PC3 cells were incubated with G0 (scrambled), G19 (enriched), or unlabeled (binding buffer only) library for 30 min at 4°C.
a. Flow cytometry dotplot results of unlabeled (left), G0 (center), and G19 (right) labeled PSMA-PC3 cells. The top row represents side scatter (y-axis) and forward scatter (x-axis) cell morphology by identification of the cells, and excluding any debris and dead cells from the PSMA-PC3 cells. The bottom row shows fluorescence (x-axis) and side scatter (y-axis) of the FITC-labeled library that has bound to the PSMA-PC3 cells.
Ref:{Notebook, AN Priya Book 3, ]
b. Histogram of flow cytometry Fluorescence intensity (x-axis) as a function of the number of viable cells (y-axis) analyzed with Flowing Software v The G19 library (blue) is shifted to the right of the G0 (red) and unlabeled library (black) after incubation with PSMA-PC3 cells.

38. Cell-based Selection for Intracellular-targeting Aptamers
Circular-ssDNA library
Capture
ligand-target
complexes
discard unbound
isolate intracellular bound oligo ligands
Fig. 5. PCR amplification of Comma-D PDK and Comma-D parental immunoprecipitated material. These data show the results of a serial dilution series comparing pull-down material from transfected (PDK) Comma-D and Comma-D parental cells. The dilutions of the PDK material appear much earlier than the parental samples (pink, undiluted PDK; dark green, 1:10 PDK; blue, 1:100 PDK). The CT for all parental samples is greater than 35, meaning that the difference is considerably greater than 100-fold. There is no signal from the no-template control. The tall, sharp peak associated with the undiluted PDK sample, and the minimal amount of heteroduplex formation associated with this particular sample (see “Discussion”), suggest greatly increased library homogeneity. At this time we are testing these samples for reproducibility before moving into the final phase of this project. Ref: [Notebook NC 6-107]
Fig. 4. PAGE analysis of ligated material to ensure circularization with biotinlyated aptamer library. Following ligation and exonuclease treatment, the single band observed in lane three indicates successful circularization. This material is now being used to validate the PDK-specific nature of the enriched aptamer pool. The samples were run on a 10% detnaturing gel. Lane 1, MW ladder; lane 2, ligated material, exonuclease (-); lane 3, ligated material, exonuclease (+). Ref: [Notebook NC 7-35]
>100-fold preference for cells expressing intracellular target versus control cells 38

39. Microscopy of Internalized Polyclonal Aptamer Library
(-) counter cells expressing mutant receptor
Figure 2B. Phase contrast and fluorescent images of (-) Mutant receptor cell line following exposure to the TAMRA labeled G12 library. Mutant receptor cells, grown to 100% confluency in a 100 mm TPP tissue culture dish, were exposed to 0.06 µM TAMRA labeled G12 library in 3ml of binding buffer (0.1mg/ml yeast tRNA, 1mg/ml BSA in wash buffer) for 30 minutes at 370C. The unbound library was aspirated from the dish (transferred to Positive target cells); cells were washed twice with 5 mL wash buffer, scraped from their plate into 1 mL of wash buffer. A 20 ul aliquot was placed on a glass slide for microscopy. Both the phase contrast (left image) and fluorescent (right image) images were taken at 40X magnification of the same field using a Tsview 1.4 MP CCD COOLED camera. These images suggest the library does not bind to the (-) Mutant receptor cell line. [Ref: Notebook, NSR 3 – 43]

40. Microscopy of Internalized Polyclonal Aptamer Library
(+) target receptor expressing cells
Figure 2A. Phase contrast and fluorescent images of Target receptor cell line following exposure to TAMRA labeled G12 library. Target cells, grown to 100% confluency in a 60mm TPP tissue culture dish, were exposed to TAMRA labeled G12 library (3 ml of the unbound fraction after (-) Mutant selection), for 30 minutes at 370C. The excess library was aspirated from the dish; cells were washed twice with 5 mL wash buffer (1X PBS supplemented with 4.5 mg/mL glucose and 5mM MgCl2); scraped from their plate into 1 mL of wash buffer. A 20 ul aliquot was placed on a glass slide for microscopy. Both the phase contrast (left image) and fluorescent (right image) images were taken at 40X magnification of the same field using a Tsview 1.4 MP CCD COOLED camera. The images suggest that the G12 library was internalized. [Ref: Notebook, NSR 3 – 43]

41. Aptagen’s Capability Against a Wide Range of Targets

43. 43

44. The AptabodyTM Technology

45. Conceptual Relationships Conjugated nucleic acid
aptabodyTM
aptamer
Functionalized
nucleic acid
Naked nucleic acid
Conjugated nucleic acid
Effective Drug
Delivery
Improve PK/PD 45

46. AptabodyTM Library (>1014 molecules)
unique SupraMolecular structures
(activity arises from the precise positioning
of functional groups within scaffold)
Aptabodies™ are single-stranded DNA or RNA molecules with conjugated functional groups, which like antibodies, can bind to targets with high affinity and specificity. Aptabodies™ are small molecules – less than 1/3 the size of a typical antibody. Thus, aptabodies™ can circulate through the body and penetrate tumors and other disease targets. The high binding affinity of aptabodies™ results from the functional group attachments and a very large surface area available for the binding. The synthetic production of aptabodies™ permits a wide range of functionality to be incorporated, and can be easily modified with cytotoxic and radioactive materials to destroy tumors; moreover, the functional group attachments provide sufficient chemistry to allow for enzymatic activity and processing of diseased targets.
Diversity of Functional Groups
organics
metals
* fatty acids
* sugars
* amino acids
* small molecule drugs
*molecular sizes are not relatively proportional 46

47. Comparison of Pharmaceutical Drug Formats
Largest
organics & Biologics Nucleic Acid Aptabody™
natural products Peptides & Proteins Aptamer (postulated)
Yes
Small
Chemical Diversity
Moderate
Yes
Large
Yes
Serum stability
Yes
None
Yes
n/a
DELIVERY
Moderate
Yes
Moderate
One
Drugs on the Market
Small
Largest
Largest
Yes
‘In Vivo Selection’ Capability
Yes
Large (<60 KD) No
Flexibility to Improve PK/PD properties
Moderate
Large (<30 KD)
Molecular Size
Smallest
Moderate to Largest (up to 180 KD for Antibody)
Moderate
Small ( D) 3’
pro C
tyr C
Most favorable condition A
ser
leu G T
Small molecule drugs
val 5’ N 47

48. Aptagen 48

49. 49