1. Biological Recognition:
Beyond the Antibody
Paul Ko Ferrigno
Chief Scientific Officer

2. This talk
• Antibodies and the need for new scaffolds
• Historical perspective- scaffolds
• Next generation scaffolds- design considerations
• Affimer performance

3. 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

4. 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

5. What we need to do- Human Protein Atlas
Berglund et al (2008) Proteomics 2008, 8, 2832–2839

6. 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 monoclo
nal 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%.

7. 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

8. First Gen Scaffolds
Binz, Amstutz & Plückthun, Nature Biotech 23, 1257 - 1268 (2005)

9. 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

10. 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

11. 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

12. Affimers are robust to extremes of pH

13. wavelength (nm)
Peptide presentation and protein yields
100
50
0
200
SQM SQT

14. Affimers: non-antibody binding proteins derived from Stefin A
Multiple libraries
CIS display: 10
12
Phage display: 10
10
Y2H: >10
8
Microarray: >10
4

15. Speed of Screening
ka (1/Ms) kd (1/s) Rmax (RU) RI (RU) Conc of analyte KA (1/M) KD (M) Req (RU) kobs (1/s) Chi2
6.76e3 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
2.5
2.50 1.25 0.63 0.31 0.16 0.08 0.04 0
AbsorbanceOD480nm
Coat Concentration (µg/mL)
Direct ELISA of Ψ F6 against Human Recombinant
Procalcitonin
No Ψ Ψ F6
Binders generated in 7 weeks.
LOD ~ 0.16 μg PCL /mL in a non-signal amplified direct ELISA.
Biacore trace
Kd= 3 nM

16. 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
1.4
1.6
1.8
0 0.2 0.4 0.6 0.8 1 1.2 1.4
AbsorbanceOD450nm
Target Coating Concentration (μg/mL)
Direct ELISA against TargetX with ΨE3
Gal7 ΨE3 Secondary Control
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 0.2 0.4 0.6 0.8 1 1.2 1.4
AbsorbanceOD450nm
Target Coating Concentration (μg/mL)
Direct ELISA against IL-2 with 2 Ψ's
IL-2 ΨA3 IL-2 ΨF3 Secondary Control
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 0.2 0.4 0.6 0.8 1 1.2 1.4
AbsorbanceOD450nm
Target Coating Concentration (μg/mL)
Direct ELISA against IL-6 with ΨA7
IL-6 ΨA7 Secondary Control
X
-0.5
0
0.5
1
1.5
2
2.5
3
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Absorbance(450-620nm)
Antibody coat concentration (μg/mL)
Anti-rabbit IgG Affimer D1 is highly specific
rIgG
mIgG1
mIgG2a
mIgG2b
mIgG3
rat IgG
donkey IgG
sheep IgG
chicken IgG
goat IgG
mouse IgM
canine IgG

17. Western Blotting
Anti-SAA
Clone E12 Lysate
Lanes 1, 4 & 7 – Life Tech SeeBlue Plus 2 Marker,
5μl
Lanes 2, 5 & 8 – HeLa RIPA lysate, 10 μg, reduced
Lanes 3, 6 & 9 – Recombinant SAA, 0.1 μg,
reduced
Binder
Lanes 1, 2 & 3 – Anti-SAA Ψ, 0.5 μg/ml
Lanes 4, 5 & 6 – Anti-SAA Ψ, 5 μg/ml
Lanes 7, 8 & 9 – No binder
Detection
All Lanes – ab1187 anti-6xHis Rabbit Polyclonal,
HRP conjugated, 1/5000

18. Affimers to Small Molecules
0
0.2
0.4
0.6
0.8
1
1.2
1 2 3 4 5 6 7 8
AbsorbanceOD480nm
Clone ID
Phage ELISA of candidate Posaconazole binders from a screen
Posaconazole+Linker
Posaconazole+Linker & Free
Posaconazole
Posaconazole+Linker & Free
Voriconazole
No Coat Control
Posaconazole
Voriconazole

19. 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

20. Isoform Specificity and Intracellular Use
Binders were generated to the SH2 domains of PI3K subunits.
PI3K p85A N-term
PI3K p85B N-term
PI3K p55G N-term
consensus
PI3K p85A N-term
PI3K p85B N-term
PI3K p55G N-term
consensus
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
AbsorbanceOD450nm
Clone ID
p85A C p85A N p85B C p85B N p55G C p55G N control
Phage ELISA results of a screen against the PI3K P85A N-terminal SH2 domain

21. 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

22. Affimer Arrays for Protein Detection
25,000 features
LLOD currently
pg/mL in human
serum

23. 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 representin
g proteins cleaved in apoptosis) while others are increasing (po
ssibly 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 C
y5. 17k Arrays were challenged with a mixture of these labelled l
ysates. Affimers binding proteins such as BAD, known to be incre
ased in apoptosis, showed increased signals from cells treated wi
th staurosporine. Affimers recognising proteins cleaved in apopt
osis, such as CDK2, showed decreased signals. Affimers binding B
CL6, a protein whose levels have not been investigated in apopto
sis, suggest this protein is down-regulated.

24. Sumoylation
Yeast-SUMO-_smt3_ MSESPSANISDADKSAITPTTGDTSQQDVKPSTEHINLKVVGQDNNEVFFKIKKTTEFSK
Human-SUMO1 --------MSDQEAKPSTEDLGD------KKEGEYIKLKVIGQDSSEIHFKVKMTTHLKK
Human-SUMO2 --------MADEKPKEGVKTENN----------DHINLKVAGQDGSVVQFKIKRHTPLSK
::* . . . .: ::*:*** ***.. : **:* * :.*
Yeast-SUMO-_smt3_ LMKIYCARQGKSMNSLRFLVDGERIRPDQTPAELDMEDGDQIEAVLEQLGGCTHLCL
Human-SUMO1 LKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGHSTV--
Human-SUMO2 LMKAYCERQGLSMRQIRFRFDGQPINETDTPAQLEMEDEDTIDVFQQQTGGVY----
* : ** *** .*..:** .:*: * .** :* **: * *:. :* **

25. Yeast SUMO binders and Species Specificity
ITC: Ψ-Clone 10
Ψ-Clone 10
Ψ-Clone 15
Ψ-Clone 19
Ψ-Clone 22
YeastSUMO
HumanSUMO1
HumanSUMO2
Ψ-Clone10
Ψ-Clone15
Ψ-Clone19
Ψ-Clone22
control
Yeast SUMO
Human SUMO1
Human SUMO2
AbsorbanceOD480nm
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.

26. 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

27. Acknowledgments
Screening/Validation
Christina Räuber
Laura Dicker
Davinia Fernandez
Rob Ford
Lindsay McMorran
Protein Expression
Paul Shadbolt
Emma Branson
Amanda Evans
Katarzyna Gorczak
Tony Kwok
Ruth Lunn
Graham Spence
Affimer Arrays
Kit-Yee Tan
Vincent Puard
University of Leeds
Mike McPherson
Darren Tomlinson
Christian Tiede
Anna Tang
Presentation by Paul Ko Ferringo
Head of R&D: Matt Johnson
For more info visit Avacta Life Sciences or contact chris.miller@avacta.com