Avacta Life Sciences Affimers Presentation Global Protein Engineering Summit (PEGS)
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Transcript of Avacta Life Sciences Affimers Presentation Global Protein Engineering Summit (PEGS)
![Page 1: Avacta Life Sciences Affimers Presentation Global Protein Engineering Summit (PEGS)](https://reader031.fdocuments.in/reader031/viewer/2022020217/54c5dc254a7959b0068b4595/html5/thumbnails/1.jpg)
Biological Recognition:Beyond the Antibody
Paul Ko Ferrigno Chief Scientific Officer
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This talk
• Antibodies and the need for new scaffolds
• Historical perspective- scaffolds
• Next generation scaffolds- design considerations
• Affimer performance
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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
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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
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What we need to do- Human Protein Atlas
Berglund et al (2008) Proteomics 2008, 8, 2832–2839
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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%.
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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
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First Gen Scaffolds
Binz, Amstutz & Plückthun, Nature Biotech 23, 1257 - 1268 (2005)
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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
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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
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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
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Affimers are robust to extremes of pH
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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
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Affimers: non-antibody binding proteins derived from Stefin A
Multiple libraries
CIS display: 1012
Phage display: 1010
Y2H: >108
Microarray: >104
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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
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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
)
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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
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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
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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
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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
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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
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Affimer Arrays for Protein Detection
25,000 features
LLOD currently pg/mL in human serum
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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.
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Sumoylation
Yeast-SUMO-_smt3_ MSESPSANISDADKSAITPTTGDTSQQDVKPSTEHINLKVVGQDNNEVFFKIKKTTEFSKHuman-SUMO1 --------MSDQEAKPSTEDLGD------KKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKHuman-SUMO2 --------MADEKPKEGVKTENN----------DHINLKVAGQDGSVVQFKIKRHTPLSK ::* . . . .: ::*:*** ***.. : **:* * :.* Yeast-SUMO-_smt3_ LMKIYCARQGKSMNSLRFLVDGERIRPDQTPAELDMEDGDQIEAVLEQLGGCTHLCLHuman-SUMO1 LKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGHSTV--Human-SUMO2 LMKAYCERQGLSMRQIRFRFDGQPINETDTPAQLEMEDEDTIDVFQQQTGGVY---- * : ** *** .*..:** .:*: * .** :* **: * *:. :* **
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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.
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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
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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]