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Heterodimeric Aptamers

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Matilde Trabuco

on 21 April 2016

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Transcript of Heterodimeric Aptamers

- Beneficial effect from the co-action of both therapeutic agents
DNA strand
18-21 nt
primer binding site
random region
20-80nt
primer binding site
Rheumatoid Arthritis
CDC
A new therapeutic approach targeting Rheumatoid Arthritis
Heterodimeric Aptamers
APTAMERS
Monoclonal ANTIBODIES
DEVELOPMENT
BATCH-TO-BATCH VARIATION
METHOD
PRODUCTION
SPECIFICITY
METHOD
PURIFICATION
METHOD
THE FACTS
CURRENT THERAPIES
APTAMERS: The new rivals of Antibodies
VS
1
2
3
4
Design
Production
Purification
Quality Control
References
In vitro (SELEX process)
Chemical Synthesis
None or very little
HPLC
What are Aptamers?
OLIGONUCLEOTIDE SEQUENCES (DNA or RNA) with the ability to recognize target molecules with high affinity and specificity

10-15 kDa in size (30-45 nucleotides)

Non-immunogenic and non-toxic

Well-defined THREE DIMENSIONAL STRUCTURE, that allows it to bind targets with sub-nanomolar affinity and discriminate against closely related targets
"Rheumatoid Arthritis is a chronic systemic disease that affects the joints, connective tissue, muscle, tendons, and fibrous tissue."
WHO
Affects 0,3 -1,0% of the Global Population
> Women affected 5x more often
> Developed Countries
WHO (2004)
EPIDEMIOLOGY
PHYSIOPATHOLOGY
SMARD's
Biological DMARD's
etanercept
infliximab
adalimumab
golimumab
certolizumab
anakinra
rituximab
abatacept
tocilizumab
Analgesics
Oral DMARD's
leflunomide
methotrexate
sulfasalazine
hydroxychloroquine
NSAID's
TNF-alpha blockers:
IL-1 blockers:
IL-6 blockers:
anti B-cell mab's:
T-cell co-stimulation blocker:
Aptamers combine many of the advantages of oligonucleotides and biologics - 'Chemical Antibodies'
Corticosteroids
methhylprednisolone
prednisolone
prednisone
In vivo (animal testing)
Cell Culture (bioreactors)
Affinity Chromatography
Is an issue
Can be an issue
Customized as desired
TARGETS
Limited
Virtually any molecule
Automated chemical solid-phase synthesis
KEY-FEATURES
TIME
COST
> 8 months
4-6 months
$20.000 - $30.000
<$20.000
TIME
COST
~3 months (grams)
2 weeks (grams)
~$300/gram
~$50/gram
W
S
o
T
TRENGTHS
EAKNESSES
PORTUNITIES
HREATS
METHOD
> More than a 'ME-TOO' Product
- Wider range of targets
> Time and Cost Advantage
> Promising results
> Well established products (e.g. Antibodies)
> Emergence of substitute products
> Failure of Pre-Clinical and Clinical Trials
> Technological development
> Need to decrease RA therapy cost
> Need for a faster generation of leads
> Easier regulatory approval
> Low popularity
> Quality Control methods not fully established
> Need of further testing
A cycle for each nucleoside addition performed on an insoluble solid support
Controlled pore glass
Deprotection step
Coupling step
Capping step
Oxidation step
Cleavage from the solid support
Deprotection
+
Background of the INNOVATION
Accuracy
Reproducibility
SELEX
Speed of synthesis
Systematic Evolution of Ligands by Exponential Enrichment
Purity and integrity of the final product
Keefe, A., Wilson, C. (2005) MULTIVALENT APTAMERS. WO/2005/052121A2
Minimal manual intervention
Optimized automated instrumentation
Zhang, Z., et al (2007) OLIGONUCLEOTIDE ANTAGONIST FOR HUMAN TUMOR NECROSIS FACTOR ALPHA. USP 7309786
random DNA library
randomized RNA pool
(10 to 10 )
13
15
incubation with TNF-alpha
or IL-1
SELEX cycle
partition
elution of bound fraction
bound /unbound
amplification
RT-PCR
conditioning
in vitro transcription
enriched
RNA pool
affinity chromatography
Low cost
Group of new oligonucleotides (DNA and RNA) sequences with human TNF-alpha inhibiting activity
Reversed Phase HPLC
METHOD
Depends on the hydrophobic nature of the oligonucleotide

More hydrophobic oligonucleotides are retained stronger on the purification media than less hydrophobic materials
Higher percentage of organic solvent is required to elute the more hydrophobic material
CONDITIONS
Column:
silica or polystyrene linked with carbon-18 C
Additives: acetate, formate, and carbonate and their corresponding ammonium salts
Organic component: acetonitrile (CH3CN)
Ion pairing reagents: TFA and HFBA at concentrations of 0.05–0.1% (v/v)
TGFßl/TGFß2 multivalent aptamer
Multivalent aptamers having binding specificity to multiple targets
Proposed INNOVATION

Heterodimeric aptamer
Anti IL-1/TNF-alpha
Anti IL-1aptamer
Anti TNF-alpha aptamer
+
SELEX modifications
synthetic nucleotides
sequence truncations
LNA
SPIEGELMER - technology
multi-stage SELEX
maximum affinity
achieved
6-20 cicles
Sequence characterization
Muller, J., et al. (2007) Chem Bio Chem. 8, 18: 2223-2226
Fusion aptamer that combines the functions of two aptamers in one molecule.
Excellent reproducibility
Heterodimeric Aptamer
High productivity
cloning into a bacterial vector
sequence individual colonies
1 to 1,000,000 aptamers obtained
KEY-FEATURES
Key Features
> The fusion aptamer possesses the functionality of its precursor molecules
Precise control of variables
Purity of 85% to 95%
High resolution
Sequence alignments
identifying homologous regions
Secondary structure analysis
Mobile Phase:
-
Act in a manner substantially equivalent to, or potentially more potent than, a combination of the individual compounds
Hundreds of grams of material at high purity
Competitive cost
(Compared with combination therapies in which multiple compounds need to be separately assessed)
Final aptamers
Connecting the aptamers
poli-A link
5 to 15 nt
provides flexibility
Phosporamidite approach
Incidence: 3 cases per 10,000 population per year
Prevalence rate: 1%
YIELD
Depends on: the chain length, scale of the synthesis, the applied purification method and the chain composition
40 - 60 bases
Yield= 70-80%
Selection
1. Mclnnes, I.B., Schett, G. (2011) The Pathogenesis of Rheumatoid Arthritis. N Engl J Med, 365, 2205-2219
2. Bingham, C.O. (2002) The Pathogenesis of Rheumatoid Arthritis: Pivotal Cytokines Involved in Bone Degradation and Inflammation. J Rheumatol, 29, Suppl 65, 3-9
3. Choy, E.H.S., Panayi, G.S.P. (2001) Cytokine pathways and joint inflammation in Rheumatoid Arthritis. N Engl J Med, 344, 907-916 4. http://www.roche.com/media/media_backgrounder/media_ra.htm (10.11.2012)
5. http://www.hopkinsarthritis.org/arthritis-info/rheumatoid-arthritis/ra-treatment/#tnf (10.11.2012)
6. Donahue, K.E. et al (2012) Drug Therapy for Rheumatoid Arthritis in Adults: An Update. Compared Effectiveness Rev, 55, 1-168
7. http://www.ema.europa.eu/docs/pt_PT/document_library/EPAR_Product_Information/human/000363/WC500042310.pdf (11.11.2012)
8. Jayasena, S.D. (1999) Aptamers: An Emerging Class of Molecules that Rival Antibodies in Diagnostics. Clin Chem, 45, 1628-1650
9. Zhang, Q., Landgraf, R. (2012) Selecting Molecular Recognition. What can existing Aptamers tell us about their Inherent Recognition Capabilities and Modes of Interaction? Pharm J, 5, 493 -513
10. Xu, D., Shi, H. (2009) Composite RNA aptamers as functional mimics of proteins. Nucl Acids Res. 5. 1-9 11. Keefe, A.D., Pai, S., Ellington, A. (2010) Aptamers as therapeutics. Nat Rev Drug Discov, 9, 7: 537–550
12. McKeague, M., DeRosa, M.C. (2012) Challenges and Opportunities for Small Molecule Aptamer Development. J Nucl Acids. 2012, 1-20
13. http://www.freepatentsonline.com/WO2005052121A2.html (12.11.2012)
14. http://www.google.com/patents/US20040254139 (08.11.2012)
15. Boltz, A. et al (2011) Bi-specific Aptamers Mediating Tumor Cell Lysis. J Biol Chem. 286, 24: 21896-21905
16. Muller, J., et al. (2007) Multidomain Targeting Generates a High-Affinity Thrombin-Inhibiting Bivalent Aptamer. Chem Bio Chem. 8, 18: 2223-2226
17. Ahmad, K.M., Xiao, Y., Soh, H.T. (2012) Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution. Nucl Acids Res. 40, 20:1-7
18. http://www.faqs.org/patents/app/20080207523 (09.11.2012)
19. Beaucage, S.L., Caruthers, M.H. (1981) Deoxynucleoside phosphoramidites-A new class of key intermediates for deoxypolynucleotide synthesis. Tet Lett. 22, 20:1859–1862
20. Beaucage, S.L., Iyer, R.P. (1992) Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tet Lett. 48, 12: 2223–2311
21. Beaucage, S.L., Iyer, R.P. (1993) The synthesis of modified oligonucleotides by the phosporamidite approach and their applications. Tet Lett. 49, 28:6123–6194
22. http://www.atdbio.com/content/17/Solid-phase-oligonucleotide-synthesis#Introduction (12.11.2012)
23. http://www.oligofactory.com/pdf/Purification_methods.pdf (13.12.2012)
24.Marshall, W. S., and R. J. Kaiser. 2004. Recent advances in the high-speed solid phase synthesis of RNA. Curr. Opin. Chem. Biol. 8:222–229.
A new therapeutic approach targeting Rheumatoid Arthritis
Heterodimeric Aptamers
Ana Filipa Louro | Joana Guerra | Joana Loureiro | Matilde Trabuco
Pharmaceutical Biotechnology
2012/2013
Sensitivity
IDENTIFICATION
EFFECTIVENESS
PURITY GRADE
> HPLC
QUANTIFICATION
> MALDI-TOF SPECTROMETRY
> HPLC
> UV QUANTIFICATION (260 nm)
> EMSA (binding ability)

> SURFACE PLASMON RESONANCE (Kd)

> ELISA and DOT-BLOT ASSAYS (affinity)

> IN-VIVO ASSAYS
- Bioassays (supernatant of cultured synovia in patients with RA)
- RA rat models
USP 5861254
USP 5660985
USP 7309786
Full transcript