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. 2 Avacta Life Sciences © 2017 What is an Affimer? • 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. 3 Avacta Life Sciences © 2017 Biophysical Properties • 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. 7 Avacta Life Sciences © 2017 Affimer Discovery Process 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. Western Blot with Strep-HRP detection SDS-PAGE 8 Avacta Life Sciences © 2017 Affimer Discovery Process 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. 9 Avacta Life Sciences © 2017 Affimer Discovery Process 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. 10 Avacta Life Sciences © 2017 Affimer Discovery Process 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 m 0 n 2 3 3 B 3 -G F P -B 3 0 6 - H e r 2 5 0 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 D D 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 ) 11 Avacta Life Sciences © 2017 Affimer Discovery Process Target Protein Primary Screen ELISA Phage Display QC & Sequencing Validation 12 – 14 weeks 12 Avacta Life Sciences © 2017 Affimer Discovery Process Target Protein Primary Screen ELISA Phage Display QC & Sequencing Validation 12 – 14 weeks 13 Avacta Life Sciences © 2017 Covance Data • 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). 14 Avacta Life Sciences © 2017 Trastuzumab project summary • 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. 15 Avacta Life Sciences © 2017 Increasing Assay Specificity of an Existing Clinical ELISA kit Using Affimers as a Capture Surface – C. Difficile ToxinB C. Difficile Toxin B • 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/ 17 Avacta Life Sciences © 2017 1. Magill et al, 2014. N.Eng.J.Med, 370:1198-1208 2. Bella et al, 2016. Toxins, 8(5):134 A View of the Current Dx Assay • 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. 18 Avacta Life Sciences © 2017 Identification of a specific anti-ToxB Affimer • 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 19 Avacta Life Sciences © 2017 Surface Performance Comparison • 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 20 AffimerAvacta Life Sciences surface: © 2017 n.d. C. Diff Toxin B project summary • 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. 21 Avacta Life Sciences © 2017 Rapid Generation and Formatting of Affimer Pairs to a Clinically Important Target - CRP Generating Affimer pairs to CRP Target Protein Primary Screen ELISA Phage Display QC & Sequencing Validation 12 – 14 weeks • 360 clones picked, top 96 sequences, 24 unique Affimer clones.