The Affimer® Scaffold: a Flexible Platform for the Generation of Renewable Affinity Reagents

The Affimer® scaffold: a flexible platform for the generation of renewable affinity reagents.

Dr Matt Johnson, CTO 2nd Annual Biomarkers & Precision Medicine USA Congress, 10th October 2017 Avacta Life Sciences

• Avacta Life Sciences (part of AIM listed Avacta Group plc) was established in 2012 to exploit Affimer IP acquired from the University of Leeds and MRC.

• Developing Affimer reagents for research and diagnostics alongside in-house and partnered therapeutic Affimer pipelines.

• Sites in Cambridge (~20 staff) and Wetherby (~40 staff).

• Raised £21m ($34m) in July 2015 for Affimer biotherapeutics with a focus on immuno- oncology and immuno-inflammation.

• Signed research collaboration and license deal with Moderna Therapeutics in May 2015.

• Small single domain proteins (14 kDa) with no disulphide bonds or post translational modifications. • Derived from the Cystatin family protein fold. • 2 scaffolds developed. • Mammalian scaffold based on Stefin A. • Plant scaffold based on Cystatin A consensus sequence. • Engineered to no longer bind Cathepsin.

• Two surface loops and N-terminus can be engineered to create vast peptide libraries (1x1010).

• Utilise phage display to identify binders.

• Simple protein structures versus multi-domain antibody. 14 nm 4 nm • Expression in simplest possible system means low production costs and robust processes. • No dependency on post-production modifications to function so no batch-to-batch variation. • Easily engineered.

3 3 nm • Fusions to other functional proteins are simple to generate and manufacture.

• Generation of multimers is comparatively 11 11 nm straightforward. • Easy to orientate. • Site-specific chemistries allow production of high- capacity surfaces for target protein capture or Affimer labelling. • Highly stable. • pH tolerant over a broad range (pH2-12). • Thermally stable. • Broad tolerance to organic solvents. 4 Avacta Life Sciences © 2017 4 Applications of Affimer Reagents

Affimers can be engineered to specifically Affimers have been used as: recognise: • Immunoassay reagents. • Ligand-receptor complexes. • Immuno-precipitation reagents. • Antibody-antigen complexes. • Imaging reagents. • Very homologous protein sequences. • Very specific motifs/epitope. • Crystallisation chaperones. • Antibody idiotypes. • Biosensor capture reagents. • Affinity purification reagents. We have functionalised Affimers with: • Protein-protein inhibitors. • Biotin. • Terminal cysteines to enable malemide chemistries. • Various protein fusion (GFP…). • Fluorophore labelling. • Resins and beads. 5 Avacta Life Sciences © 2017 Generating Robust Critical Reagents - Anti-Trastuzumab Affimers Anti-idiotypic Affimer Binder to Trastuzumab for PK assays

• Availability of capture reagents for Assay format therapeutic antibodies PK assays can be rate limiting in product development:

Anti-idiotype antibodies development can Fluorescence Signal / Detection be a long and difficult process. Ligand capture reagents not always reliably available and can be expensive. AP - Conjugate

Specificity of capture reagents can Anti-Human IgG sometimes be problematic (matrix effect). Trastuzumab /Test mAb

• Affimer binders can provide a fast, reliable Immobilised Her2 solution for therapeutic antibody capture. Proof of principle project: development of anti-trastuzumab binders in collaboration with Covance.

Target Protein Primary Screen ELISA Phage Display QC & Sequencing Validation

12 – 14 weeks

• Target protein (Trastuzumab) and counter-selection targets biotinylated and QC-ed by Western Blot and SDS-PAGE.

Target Protein Primary Screen ELISA Phage Display QC & Sequencing Validation

12 – 14 weeks

• 3-round phage display experiment

• Selection against biotinylated Trastuzumab. • Phage output deselected against therapeutic antibody cocktail. • Off-rate selection step. • 5000-fold enrichment observed for phage titre against Trastuzumab versus control.

Target Protein Primary Screen ELISA Phage Display QC & Sequencing Validation

12 – 14 weeks

• Affimer repertoire from phage selection subcloned into a prokaryotic expression vector fused with C-terminal tags and purified by IMAC. • 360 clones evaluated for Trastuzumab binding in a high throughput bead based assay. • Top 96 clones showing best sensitivity and specificity for Trastuzumab vs. unrelated human IgG were selected and sequenced. • 16 unique clones were identified and selected or further ELISA validation.

Target Protein Primary Screen ELISA Phage Display QC & Sequencing Validation

12 – 14 weeks

T ra s tu z u m a b c a p tu re E L IS A 3 8 6 _ 7 3 7 _ B 3 m o n o m e r , d im e r / B 3 -G F P -B 3 c o m p a ris o n

5 3 8 6 _ 7 3 7 _ B 3 5

3 8 6 _ 7 3 8 _ G 5 3 8 6 _ 7 3 7 _ B 3 m o n o m e r 4 4

) 3 8 6 _ 7 3 7 _ B 3 (G G S ) d im e r

m )

n 3 8 6 _ 7 4 0 _ D 6

2 3 3 B 3 -G F P -B 3

0 6

- H e r 2

4 (

5 3 8 6 _ 7 3 7 _ B 3 d im e r 4

( 2 m o u s e Ig G 2 b G 1 2 2

O O 1 1

0 0 0 .1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 0 .1 1 1 0 1 0 0 1 0 0 0 T r a s tu z u m a b c o n c (p M ) T a r g e t p r o te in (n g /m L )

Target Protein Primary Screen ELISA Phage Display QC & Sequencing Validation

12 – 14 weeks

Target Protein Primary Screen ELISA Phage Display QC & Sequencing Validation

12 – 14 weeks

• Matrix Testing: • <16.3 %CV across LLOQ of 60ng/ml to ULOQ of 1600 ng/ml (LQC outlier, remainder <6%).

• Intra-assay precision • <12.4 %CV across LLOQ of 60ng/ml - ULOQ of 2000ng/ml (n=6).

• Inter-assay precision • <4.6 %CV across LLOQ of 40ng/ml - ULOQ of 3000ng/ml (n=9).

• Broader dynamic range than existing bioanalysis assay (60-3000ng/ml compared to 40-750ng/ml with anti-ID Ab).

• anti-Trastuzumab Affimer binders identified in 13 weeks.

• Candidate Affimer proteins perform comparably to Her2 as a capture reagent in terms of specificity and LOD, and have similar IC50 values.

• Affimer binders compete with Her2 for Trastuzumab binding in competition ELISA format.

• No matrix affect could be detected.

• Minimal batch-batch variation was obtained on four different production batches.

• Validation of the reagents by Covance demonstrate that Affimer binders can be qualified as critical reagent in a clinical PK assay.

• Performance Improvements: Low Matrix Affect, broader dynamic range, No batch inconsistency, constant supply, ease of use.

• Clostridium difficile is now the most common cause of nosocomial hospital acquired infections in the US (15- 25% of cases)1.

• Toxin B is a glycosyltransferase with cytotoxic activity.

• Toxin B induces opening of tight junctions in intestinal epithelial cells leading to vascular permeability and hemorrhaging resulting in pseudomembranous colitis.

• Toxin A-/B+ strains maintain virulence, the reverse is not the case2.

• Annual case load/annual cost estimated $1b annually US alone, $3,600 per patient.

• A growing proportion of cases are not treatable by antibiotic therapy alone. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4885049/

• Current Dx assays and reagents are cross- reactive between Toxin A and Toxin B.

• Determination of detection limit of C. difficile Tox A/B II for toxin A and B: the kit detects toxin A up to 1 ng/ ml while it detects toxin B to 10 ng/ml.

• The goal of this project was to develop an Affimer binder specific for Toxin B which could be used as a capture reagent to help improve specificity of a clinical assay whilst maintaining sensitivity of detection.

• The Affimer library was screened against ToxinB and the output characterised for binding to Toxin A, Toxin B and both toxoid forms. • 10 unique clones identified that bind Toxin B and not Toxin A. • Clone 45 selected as lead clone.

Thermal unfolding of Clone B-45 Aggregation testing of Clone B-45 using OpTim using OpTim

• Clone 45 performs at least as well as the commercial diagnostic ELISA in terms of sensitivity in a hybrid assay using the kit detection antibody. • The hybrid assay is specific for ToxinB. • Requires no deviation from the commercial kit protocol. • Affimer coated at 200nM per well (~2.5μg/ml). • LODs Toxoid B: Kit: ~200 - 300 pg/ml Affimer surface: ~75 - 100 pg/ml) Toxoid A: Kit: ~100 – 200 pg/ml

• Affimer binders absolutely specific to ToxB were identified. • Affimer coated surfaces used for antigen capture, demonstrate similar sensitivity performance compared to an optimised commercial diagnostics kit. • Affimer proteins robust biophysical and manufacturing characteristics make them ideal candidates for the development of stable and reliable diagnostics tests.

Target Protein Primary Screen ELISA Phage Display QC & Sequencing Validation

12 – 14 weeks

• 360 clones picked, top 96 sequences, 24 unique Affimer clones. • ELISA Pair discovery: • 1st stage: identify best capture Affimer by capture of biotinylated target using passively absorbed Affimer surface => Picked top 5 performing Affimers. • 2nd stage: pairwise testing of all 24 candidate Affimers against top 5 Affimer surfaces (N-terminally biotinylated detection Affimers => Top 2 Affimer pairs selected. • 3rd stage: titration of best pairs in PBS and 10% CRP-depleted serum.

2 2 3 7 - h C R P - 2 2 4 0 - B 0 1 ( b u ffe r ) • Clone 2237 performed well as a capture h C R P s a n d w ic h E L IS A 2 2 3 7 - h C R P - 2 2 4 0 - B 0 1 ( s e r u m ) surface with 2240 as a detection reagent. 5 2 2 3 7 - h C R P - 2 2 4 8 - B 0 1 ( b u ffe r )

2 2 3 7 - h C R P - 2 2 4 8 - B 0 1 ( s e r u m ) • Clone 2254 behaved similarly. 4

2 2 5 4 - h C R P - 2 2 4 0 - B 0 1 ( b u ffe r )

)

m n

3 2 2 5 4 - h C R P - 2 2 4 0 - B 0 1 ( s e r u m )

• Matrix influences observed with 2248 as 0

5 4 ( 2 2 5 4 - h C R P - 2 2 4 8 - B 0 1 ( b u ffe r )

a detection reagent. 2 D

O 2 2 5 4 - h C R P - 2 2 4 8 - B 0 1 ( s e r u m ) • 2237/2240 chosen as development pair. 1 m Ig G 2 b - G 1 2 - h C R P - 2 2 4 0 - B 0 1 ( b u ffe r )

m Ig G 2 b - G 1 2 - h C R P - 2 2 4 0 - B 0 1 ( s e r u m ) 0 0 .1 1 1 0 1 0 0 1 0 0 0 m Ig G 2 b - G 1 2 - h C R P - 2 2 4 8 - B 0 1 ( b u ffe r )

h C R P c o n c (n g /m L ) m Ig G 2 b - G 1 2 - h C R P - 2 2 4 8 - B 0 1 ( s e r u m )

C R P T itra tio n Intra- LOQ Accuracy Inter-assay Precision assay (ng/mL) 50ng/mL 25ng/mL 125ng/mL 0.5ng/mL 0ng/mL

4 Precision

)

0 1.88 84.8% 109.1% 6.1% 8.3% 8.5% 9.2% 3

6 3

4 (

e 2

a b

r 1

o s

b • Avacta assay was optimised and validated using A 0 1:10 dilution of serum, therefore the range of the 0 5 0 1 0 0 1 5 0 C R P (n g /m L ) assay is 20 - 500ng/mL CRP in undiluted serum samples

DasherGFP EcMBP 26.6 kDa 43.4 kDa total: 40.3 kDa total 54.5 kDa pI6.7 pI5.5

5 5 5 2 2 3 7 _ 2 2 4 0 d e p s e ru m 2 4 6 6 _ 2 2 4 0 d e p s e ru m 2 4 8 4 _ 2 2 4 0 d e p s e ru m

4 2 2 3 7 _ 2 2 4 0 n o s e ru m 4 2 4 6 6 _ 2 2 4 0 n o s e ru m 4 2 4 8 4 _ 2 2 4 0 n o s e ru m

)

0 0 3 0

3 3 3

( (

2 ( 2 2

O O 1 1 1

0 0 0 1 0 1 0 0 1 0 0 0 1 0 1 0 0 1 0 0 0 1 0 1 0 0 1 0 0 0 L o g n g /m L L o g n g /m L L o g n g /m L 2237_2240 dep serum 2237_2240 no serum IC50 82.52 87.83 2466_2240 dep serum 2466_2240 no serum 2484_2240 dep serum 2484_2240 no serum IC50 72.05 72.91 IC50 45.71 63.75

• Affimer-Affimer pairs developed in 11 weeks. • Fusion formatting and ELISA added ~4 weeks. • Affimer-based ELISA demonstrating excellent sensitivity in a 10% dilution of serum. • Affimer-fusion proteins express well and follow same purification process as monomers. • Performance in Lateral Flow now being evaluated with a 3rd party (Mologic)

• Quick to develop (including pairs) • Specificity can be controlled • Small, robust and stable • Consistent batch reproducibility • Various formatting options • Easily functionalised and orientated • Not limited by the immune system • No use of animals Acknowledgements • Trastuzumab • Rob Ford • Toni Hoffmann • Covance • Toxin B • Toni Hoffmann • Mike McPherson – University of Leeds • Christian Tiede – University of Leeds • Modupe Ajayi – University of Leeds • Public Health England • CRP • Rob Ford • Amanda Nicholl • James Nuttall • Alex Davidson • Mologic