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Avacta Life Sciences Exhibits Affimers at Global Protein Engineering Summit Avacta Life Sciences exhibited recently at the Global Protein Engineering Summit ("PEGS") where it presented its Affimer technology. You can read more about Affimer technology here http://www.avactalifesciences.com PEGS is considered to be the essential protein engineering meeting where commercial and academic progress in protein engineering is showcased and this year it attracted over 1800 delegates from across the globe to Boston. Avacta Life Sciences presented its Affimer technology for the first time at a PEGS meeting with technical exhibits and a presentation by the CSO, Paul Ko Ferrigno, entitled "Biological Recognition: Beyond the Antibody."* The exhibition booth was busy with over 80 delegates talking to the Avacta Life Science management team over the four days of the summit. The feedback on the Affimer technology was very positive, in particular, the short development times and excellent stability were highlighted by delegates as key advantages of Affimers over antibodies. There was also a strong interest in Affimers from the management of companies developing biological therapeutics who were keen to learn more about the potential of Affimers as novel therapeutics. In addition, several companies were interested in the use of Affimers as an alternative to antibodies in diagnostic devices, mainly because they could generate binders against new biomarkers much more quickly and evaluate them in higher numbers. The benefits of Affimer microarrays for biomarker discovery also resonated with diagnostic developers who appreciated the advantage of being able to evaluate significantly larger numbers of potential biomarkers more cost and time effectively than by mass spectrometry. The potential of the arrays for multiplexed solutions for clinical diagnosis and monitoring during drug trials was also something that generated interest amongst those delegates. Matt Johnson, Chief Technical Officer of Avacta Life Sciences commented: "It was great to experience face to face the level of interest in Affimers. The majority of people I spoke to were either having problems raising antibodies to their target of interest or just couldn't use antibodies because of the type of assays they wanted to perform. Many of the presentations focused around the use of antibody fragments for intra-cellular studies which is a rapidly growing area that holds great interest for drug and diagnostics developers. It is an area where there are clear advantages for Affimers over antibody fragments which don't behave well in the cytoplasm. "The general enthusiasm around Affimers was very encouraging and the amount of interest generated by the potential of Affimers as therapeutics and by the Affimer arrays for biomarker discovery only reinforces my excitement around this new technology."
Citation preview
Biological Recognition:Beyond the Antibody
Paul Ko Ferrigno Chief Scientific Officer
This talk
• Antibodies and the need for new scaffolds
• Historical perspective- scaffolds
• Next generation scaffolds- design considerations
• Affimer performance
Biological Recognition- Antibodies
Antibodies
• Are fantastic diagnostic tools
• Are pretty good reagents for research
• Can be made to be good drugs
Why?
• Optimisation
• Large binding surface area
• Constant region allows generic protocols
But antibodies…
• Can suffer from cross-reactivity
• Can be difficult to express
• Can be difficult to engineer
• Can be difficult to use
• Generating a functional binder for each element of proteome is a huge challenge
What we need to do- Human Protein Atlas
Berglund et al (2008) Proteomics 2008, 8, 2832–2839
Validation score
Validation category
Criteria Number of antibodies
Fraction%
1. High Supportive Two independent antibodies with similar staining pattern consistent with experimental and/or bioinformatics data
687 7
2. Medium Supportive Staining pattern consistent with experimental and/or bioinformatics data
1414 15
3. Low Uncertain Staining pattern partly consistent with experimental and/or bioinformatics data
1853 20
4. Very low Uncertain No literature or bioinformatics data available 861 9
5. Failed Failed Staining pattern not consistent with experimental and/or bioinformatics data
4543 49
Where we are- Catalogue reagents and HPA
TABLE II Validation of 9358 internally generated antibodies
Berglund et al (2008) A Genecentric Human Protein Atlas for Expression Profiles Based on Antibodies. Molecular & Cellular Proteomics, 7, 2019-2027.
5436 external antibodies from 51 different antibody providers were obtained and subsequently validated. Of these, 1410 mono -clonal antibodies and 1316 polyclonal antibodies were approved by a standardized validation using Western blotting and IHC on tissue microarrays as described above. The success rates (…) showed (…) an average success rate of 49%.
If half of all research antibodies tested fail…
Research: huge amount of time wasted in finding the right reagent
Proteomics: limited coverage, limited understanding
Pharma: new target, but no tools= no assays = no drugs
First Gen Scaffolds
Binz, Amstutz & Plückthun, Nature Biotech 23, 1257 - 1268 (2005)
Why do we need more scaffolds?
• Early scaffolds started as academic research projects
• Later ‘fit for purposes’ scaffolds may rely on untested assumptions
• Purpose may dictate scaffold choice-but what happens if the experimental paradigm changes?
• Multiplexing represents unique challenges, but is needed for personalised medicine/CDx
Next Gen Scaffolds
A successful non-Ab scaffold protein should be:
(1) of known structure: informed choice of the site for peptide insertion or replacement
(2) stable: to constrain the folding of a broad range of peptides
(3) flexible: folding not affected by peptide inserts
(4) biologically neutral: lacking interactions with irrelevant proteins
(5) well-expressed in prokaryotic and eukaryotic environments, data obtained in one system informs experiments performed in the other
Woodman et al, J Mol Biol, 2005
Woodman et al, J Mol Biol, 2005
Affimers start from a robust, neutral and versatile scaffold
Small- 98 aa
Lysosomal resident
Protease inhibitor
No cys
Well understood interaction surfaces: very similar to Ab
Capable of 1 fM KD
Affimers are robust to extremes of pH
θ (d
eg c
m2 d
mol
-1)
wavelength (nm)
Peptide presentation and protein yields
100
50
0
200
protein yield (mg l-1)
SQM SQT
Affimers: non-antibody binding proteins derived from Stefin A
Multiple libraries
CIS display: 1012
Phage display: 1010
Y2H: >108
Microarray: >104
Speed of Screening
-5
0
5
10
15
20
25
30
35
40
-200 -140 -80 -20 40 100 160 220 280 340 400 460 520 580 640 700
Time sRe
spon
se
RU
ka (1/Ms) kd (1/s) Rmax (RU) RI (RU) Conc of analyte KA (1/M) KD (M) Req (RU) kobs (1/s) Chi26.76e3 2.15e-5 44.1 -1.28 1000n 3.15e8 3.18e-9 44 6.78e-3 0.0331
0
0.5
1
1.5
2
2.5Direct ELISA of Ψ F6 against Human Recombinant Proc-
alcitonin
No Ψ Ψ F6
Coat Concentration (µg/mL)
Abso
rban
ce O
D 48
0 nm
Binders generated in 7 weeks. LOD ~ 0.16 μg PCL /mL in a non-signal amplified direct ELISA.
Biacore trace
Kd= 3 nM
Baseline ELISA- No signal amplification 1:1:1 binding between Target, Affimer and Secondary
0 0.2 0.4 0.6 0.8 1 1.2 1.40
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Direct ELISA against TargetX with ΨE3
Gal7 ΨE3 Secondary Control
Target Coating Concentration (μg/mL)
Abs
orba
nce
OD
450
nm
0 0.2 0.4 0.6 0.8 1 1.2 1.40
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Direct ELISA against IL-2 with 2 Ψ's
IL-2 ΨA3 IL-2 ΨF3
Secondary Control
Target Coating Concentration (μg/mL)
Abs
orba
nce
OD
450
nm
0 0.2 0.4 0.6 0.8 1 1.2 1.40
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Direct ELISA against IL-6 with ΨA7
IL-6 ΨA7 Secondary Control
Target Coating Concentration (μg/mL)
Abs
orba
nce
OD
450
nm
X
0 0.2 0.4 0.6 0.8 1 1.2 1.40
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
0.05Anti-rabbit IgG Affimer D1 is highly specificrIgG
mIgG1
mIgG2a
mIgG2b
mIgG3
rat IgG
donkey IgG
sheep IgG
chicken IgG
goat IgG
mouse IgM
canine IgGAntibody coat concentration (μg/mL)
Ab
so
rba
nc
e (
45
0-6
20
nm
)
Western Blotting
Anti-SAAClone E12 Lysate
Lanes 1, 4 & 7 – Life Tech SeeBlue Plus 2 Marker, 5μlLanes 2, 5 & 8 – HeLa RIPA lysate, 10 μg, reducedLanes 3, 6 & 9 – Recombinant SAA, 0.1 μg, reduced
BinderLanes 1, 2 & 3 – Anti-SAA Ψ, 0.5 μg/mlLanes 4, 5 & 6 – Anti-SAA Ψ, 5 μg/mlLanes 7, 8 & 9 – No binder
DetectionAll Lanes – ab1187 anti-6xHis Rabbit Polyclonal, HRP conjugated, 1/5000
Affimers to Small Molecules
1 2 3 4 5 6 7 80
0.2
0.4
0.6
0.8
1
1.2
Phage ELISA of candidate Posaconazole binders from a screen
Posaconazole+Linker
Posaconazole+Linker & Free Posaconazole
Posaconazole+Linker & Free Voriconazole
No Coat Control
Clone ID
Ab
sorb
ance
O
D 4
80n
mPosaconazole
Voriconazole
Isoform Specificity and Intracellular Use
inactive active
p85 regulates p110 lipid kinase activity via its SH2 domain.
In the presence of a p85 SH2 binding protein the inhibition is released, activating p110
Active p110 inhibits phosphorylation of Akt
Isoform Specificity and Intracellular Use
Binders were generated to the SH2 domains of PI3K subunits.
PI3K p85A N-termPI3K p85B N-termPI3K p55G N-term
consensus
PI3K p85A N-termPI3K p85B N-termPI3K p55G N-term
consensus
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 240
0.2
0.4
0.6
0.8
1
1.2
1.4p85A C p85A N p85B C p85B N p55G C p55G N control
Clone ID
Ab
so
rba
nc
e O
D 4
50
nm
Phage ELISA results of a screen against the PI3K P85A N-terminal SH2 domain
Isoform Specificity and Intracellular Use
Candidate Affimers were expressed in 3T3 cells and shown to increase pAKT.
Inhibition does not correlate with expression level ie some Affimers are more potent
Affimer Arrays for Protein Detection
25,000 features
LLOD currently pg/mL in human serum
Signatures of Drug Action- DMSO 0.5 1 2 4 hours Staurosporine
Up-regulated Down-regulated
Panel A: Cells treated with staurosporine undergo apoptosis.
A
B
Panel C: highlights of some of the 336 other Affimers also gave altered signals- some proteins decreasing (probably represent-ing proteins cleaved in apoptosis) while others are increasing (possibly reflecting the detection of cleaved products)
Panel B: At 4 hours, after treatment with drug or carrier (DMSO) cells were lysed and proteins fluorescently labelled with Cy3 or Cy5. 17k Arrays were challenged with a mixture of these labelled lysates. Affimers binding proteins such as BAD, known to be in-creased in apoptosis, showed increased signals from cells treated with staurosporine. Affimers recognising proteins cleaved in ap-optosis, such as CDK2, showed decreased signals. Affimers bind-ing BCL6, a protein whose levels have not been investigated in apoptosis, suggest this protein is down-regulated.
Sumoylation
Yeast-SUMO-_smt3_ MSESPSANISDADKSAITPTTGDTSQQDVKPSTEHINLKVVGQDNNEVFFKIKKTTEFSKHuman-SUMO1 --------MSDQEAKPSTEDLGD------KKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKHuman-SUMO2 --------MADEKPKEGVKTENN----------DHINLKVAGQDGSVVQFKIKRHTPLSK ::* . . . .: ::*:*** ***.. : **:* * :.* Yeast-SUMO-_smt3_ LMKIYCARQGKSMNSLRFLVDGERIRPDQTPAELDMEDGDQIEAVLEQLGGCTHLCLHuman-SUMO1 LKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGHSTV--Human-SUMO2 LMKAYCERQGLSMRQIRFRFDGQPINETDTPAQLEMEDEDTIDVFQQQTGGVY---- * : ** *** .*..:** .:*: * .** :* **: * *:. :* **
Yeast SUMO binders and Species Specificity
ITC: Ψ-Clone 10
Ψ-Clone 10
Ψ-Clone 15
Ψ-Clone 19
Ψ-Clone 22
Yeas
t SU
MO
Hum
an S
UM
O1
Hum
an S
UM
O2
Ψ-C
lone
10
Ψ-C
lone
15
Ψ-C
lone
19
Ψ-C
lone
22
cont
rol
Yeast SUMOHuman SUMO1Human SUMO2
Abso
rban
ce O
D 4
80nm
0
0.5
1.0
1.5
Binders were generated that were specific to yeast SUMO. Data shown are direct ELISA and western blot results for 4 candidates against yeast SUMO and human SUMO 1 & 2. ITC for one candidate shows a Kd of ~30 nM.
Summary
• Affimers are small, robust and versatile proteins
• The scaffold has been engineered to lack partners in human systems
• Very large libraries can be quickly screened against a broad range of targets
• The resulting binders are high affinity and exquisitely specific
Acknowledgments
Screening/Validation
Christina RäuberLaura Dicker
Davinia FernandezRob Ford
Lindsay McMorran
Protein Expression
Paul ShadboltEmma BransonAmanda Evans
Katarzyna GorczakTony KwokRuth Lunn
Graham Spence
Affimer Arrays
Kit-Yee TanVincent Puard
University of Leeds
Mike McPhersonDarren Tomlinson
Christian TiedeAnna Tang
Presentation by Paul Ko Ferringo
Head of R&D: Matt Johnson
For more info visit Avacta Life Sciences or contact [email protected]