Affimer Technology Nov 2017

Non-confidential Technical Introduction to the Affimer® Technology for Therapeutics and Reagents

Dr. Alastair Smith Chief Executive, Avacta Group plc Introduction Avacta Group plc AIM: AVCT

• 80 staff over two sites: • 1300 m2 of bespoke laboratory, production and logistics space in Wetherby. • 790 m2 of bespoke laboratory space in Cambridge. • Balance sheet to support existing plans. Wetherby • Experienced management team with interests aligned to shareholders. • Strongly supportive shareholder base. Cambridge London Shareholders >5% . IP Group plc 24.8% Lombard Odier 11.4% Aviva 9.6% Baillie Gifford 7.2% Ruffer LLP 7.1% Fidelity 5.9% J O Hambro 5.7%

Dr Alastair Smith, CEO Dr Matt Johnson, CTO Mr Tony Gardiner, CFO • Over 10 years experience as a public • Genetics & Microbiology Molecular • Joined Avacta from AHR, an company CEO Biology international architecture practice • Was a leading UK biophysicist - founded • 8 years at Abcam becoming global • Chief Financial Officer of AIM listed Avacta in 2006 Head of R&D Fusion IP plc 2007 – 2011 which was acquired by IP Group plc in 2014 • World class scientific and technical • Joined Avacta in 2014 knowledge with a highly commercial • Joined Avacta in 2016 mindset

Dr Philippe Cotrel, CCO Dr Amrik Basran, CSO • Over 20 years’ commercial experience in senior • Over 10 years’ experience of both the biotech and positions in Amersham Pharmacia Biotech, Oxford pharma industries Glycosciences, Affymetrix and Abcam • Director of Protein Biosciences at Domantis, Head of • Commercial Director of Abcam since 2008 – grew Topical Delivery (Biopharm) at GSK revenue from £36.7m to £144m over a 7-year period • Joined Avacta in 2013 • Joined Avacta in 2016

What is an Affimer? Binding Surface • Based on a naturally occurring proteins (cystatins) and engineered to stably display two loops which create a binding surface.

• Loops are randomised to create large libraries of diversity ~1010 and Affimers are selected by phage display.

Key Benefits

• Smaller (14 kDa), simpler (no disulphide bridges and no post- translational modifications), more robust (thermally and chemically) than antibodies.

• High affinity (single digit nM) Affimers generated for new targets in a few weeks

• Exquisite specificity obtained by control of phage selection process.

• Easily modified (chemically and as fusion proteins) and easily manufactured in bacterial, yeast or mammalian systems with high expression yields.

• Intracellular survival and activity.

• Core Affimer protein is non-immunogenic.

First Generation • Acquired from the Medical Research Council and Leeds University UK in 2012. • Based on human stefin A with multiple mutations to reduce dimerisation and prevent binding to cathepsin. • Patents granted in EU, US, Asia; Priority date: 2006. • Current technology for therapeutic programmes. Second Generation • Affimer technology based on plant cystatin consensus sequence; high stability suitable for challenging applications in research and diagnostics. • IP exclusively licensed to Avacta by Leeds University; Priority date: 2014. Third Generation • Developed in-house and based on human stefin A with improved biophysical properties and minimal mutations from human sequence for therapeutics; broad claims based on protein engineering and not on a specific sequence. • Priority date: July 2017. • New technology for future therapeutic programmes.

1 Reagents 2 Therapeutics

Building a profitable reagents Building a pipeline of Affimer business through licensing drug candidates in immuno- oncology for partnering

• Opportunities for Affimer Therapeutics • Avacta Therapeutics Strategy • Pipeline and Research Collaborations • Affimer Therapeutic Molecule Formats • PD-L1 Programme Update: In-vitro activity, in-vivo efficacy and Affimer/PD-L1 co-crystal structure • Affimer Technology Immunogenicity • Pharmacokinetics and Half-life Extension

7 Opportunities for Affimer Therapeutics A novel class of therapeutics with favourable drug properties

Affimer-Drug Respiratory Ocular Conjugates

o Small size (relative to antibodies) o Potential for a significantly lower dose, o Adaptable for topical delivery in front- favors extravasation/tissue lower cost of goods, lower systemic of-eye conditions and intravitreal & penetration. target engagement with potentially sub-retinal injection for back-of-eye fewer systemic side effects and therapeutics. May have differentiated PK/ADME properties particularly beneficial for treating: increased patient convenience. o Target tissues include cornea, vitreous humor, neurosensory retina (retinal o fibrotic diseases and fibrotic tumors o Can be engineered for high affinity ganglion and photoreceptors) and (breast and pancreatic tumors, uterine and specificity, and low/null systemic RPE/choroidal layers. fibroids). availability. o poorly or abnormally vascularized tissues o Ability to tune duration of action and and tissues with high interstitial pressures. o Stability allows longer duration of reduce frequency of injection for o Tunable serum half-life - can be used action than small molecules. protein therapeutics generally. to limit toxicity in peripheral tissues o Protease stability in diseased lung o Improved intra-ocular pharmacokinetics and improve therapeutic index. (protease inhibitor scaffold). through intravitreal half-life extension by fusion with HA-binding Affimers o Bi-paratopic and bispecific formats to o Non-toxic metabolic products (amino improve both specificity and acids). o Small molecular weight and internalisation. o Thermal stability for nebulisers. compactness of Affimers allows for higher molar loading and better/ o Site directed labeling and biophysics o Attributes suggest compatibility with consistent release kinetics over time to improve manufacturability. inhalable powders/particles. for implantable drug eluting devices. TM o New Approaches: AfDC program o With ability to optimize particle size, o Opportunity for gene delivery for exploring linker/warhead morphology, hygroscopicity, electrical charge and density per application. constitutively expressing recombinant combinations for TME drug release Affimer drug product in eye. with bystander effects. o Exemplars include anti-VEGF Affimers for o PD-L1 Affimer to target tumor and inhibit wet AMD, and bispecific anti-Factor D/C5 PD-1 axis coupled to highly potent drug and anti-b-amyloid Affimers for dry AMD. ablating tumor associated macrophages.

Gene Delivery Dermatological Oral/GI

o Small size means recombinant coding o Dermal and transdermal delivery o pH stability + enteric coating or sequence amenable to wide range of embodiments. suppository for intraluminal delivery viral or other coding sequence o Transdermal – skin is the most accessible to large or small intestines. delivery platforms. organ of the body with a large surface o pH stability for intraluminal or area. o Ease of cellular expression and submucusal (endoscopic) delivery to secretion. o Needle-free topical application routes esophagus. - small molecular weight, compactness o Flexible formatting. and stability of Affimers may be o Potential for selective GPCR agonist o Anticipate high specific activity of secreted exploited to enhance the dermal and antagonists for inflammatory gut recombinant Affimers. diseases. permeability of Affimers and o Potential for generating Affimers that associated proteins. o Potential for use in drug eluting alter intracellular targets. o Amendable to fusion protein formats devices (esophageal and intestinal o Modify epigenetics, mitotic activity, and/or use of penetrant enhancers. stents and collars). viability, or other characteristics. o May enable penetration of the stratum o Exploring engineered Affimers and o Cell surface retained embodiments for corneum and entry into the viable formulations strategies permitting epidermis. altering cell trafficking, cellular half- systemic exposure from orally dosed life, immunogenicity or other function. o Readily adaptable to Affimers. o Secreted IO Affimers from engineered microneedle/dermal rollers, CAR-T or ACTR cell therapies. noninvasive jet injectors and electroporation for delivery. o Targeting moieties for viral delivery to o Should be able to achieve delivery of specific cells. Affimers at >1mg/cm2.

In-house Pipeline Proof-of-Concept Research Collaborations

• Immune-checkpoint inhibitors (combinations, • Gene delivery (Moderna Tx Inc) bispecifics, biparatopics) • CAR-T (Memorial Sloan Kettering) • T-cell engagers • Drug conjugates (Glythera) • Agonists

Key Benefit of Affimers Key Benefits of Affimers

Ease of creating and manufacturing “multimers” Small size, stability and ease of production by cells that combine multiple Affimers

T-cell Engagers Agonists

T-cell Tumour Targeting Immune Activators GITR, CD27 CD3e CD19 Other Potential Targets: CD28, OX40, HVEM, CD137, ICOS, CD40 ”Basic” T-cell Engager

CD3e CD19 CD22 Fc Formatted Dual Targeting

CD3e CD19 CD22 Albumin Dimers

Dual Targeting Half Life Extended

Trimers

Other Potential Tumour Targets: 5T4, CD33, CD20, EGFR, HER2, FOLR1

Lead Programme Discovery Pre-clinical Phase I Optimisation Immunoncology

AVA-004 PD-L1 Antagonist

AVA-017 LAG-3 Antagonist

AVA-014 CD27 Agonist

AVA-018 GITR Agonist

AVA-008 CD19 T-cell engager

AVA-002 CD3e T-cell engager

AVA-012 CD22 Tumour targeting

AVA-020 5T4 Tumour targeting

Technology Development

AVA-003 HSA Half-life Extension

Programme Discovery Lead Optimisation Pre-clinical Clinical

• Research collaboration with Moderna Therapeutics developing Affimers for messenger RNA therapies. (Multiple undisclosed IO targets)

Proof of concept study • Solvent stability of Affimers AVA-006 (Model Systems) • Control of Affimer-toxin ratio Drug Conjugates

Memorial Sloan Kettering Proof of concept study Cancer Centre • CD19 binding Affimer • Expression on a CAR-T cell • Cell killing potency AVA-008 CD19 CAR-T

The Leeds • Modulation of blood clotting by targeting Teaching Hospitals fibrinogen NHS Trust • Multiple Affimer modulators AVA-005 Fibrinogen

The Leeds • Modulation of blood clotting by targeting a-2- Teaching Hospitals antiplasmin NHS Trust • Multiple Affimer modulators AVA-016 a-2-antiplasmin

Human PD-L1 Affimer Antagonist Biacore (1) (2) (3) (4) kDa Apparent KD 2 2 180 Protein ka (1/Ms) kd (1/s) Chi (RU ) Tetramer G4S3 G4S3 G4S3 130 (M) 100 70 Monomer His 2.81E+06 2.51E-02 8.91E-09 0.0740

55 Dimer His 1.12E+06 5.35E-04 4.79E-10 0.0251 Trimer G4S3 G4S3 40 Trimer His 1.13E+06 4.73E-04 4.18E-10 0.0153

35 Tetramer His 1.01E+06 3.48E-04 3.44E-10 0.0292 25

Dimer G4S3 15 Competitive ELISA

10 PDL1_141 Monomer His Monomer PDL1_141 Dimer His PDL1_141 Trimer His PDL1_141 Tetramer His hPD-1 Fc

Yield after one- Yield after two- Format of the Expected step purification step purification Affimer protein MW (kDa) (mg/L) (mg/L)

Monomer His 14 270 56 IC50 (nM) Dimer His 25 278 116

Trimer His 42 212 37 hPD-1 Fc Monomer His Dimer His Trimer His Tetramer His

Tetramer His 56 205 47 0.17 2.5 0.28 0.13 0.04

• PoC to demonstrate AVA04-236-6(EAAAK) hIgG1 Fc 1 that Affimers can be formatted at various Purity = 96% sites on an Fc, and so M 1 2 3 KDa should translate to 140 IgG-Affimer fusions 115 AVA04-236 hIgG1 Fc C 80 terminal Affimer 2 70 • Constructs were 50 expressed in HEK239 40 30 Purity = 97% cells and purified using 25

standard Pr A- 15

sepharose affinity AVA04-236 dimer on 10 chromatography hIgG1 Fc 3 • Analytical SEC-HPLC Reducing SDS-PAGE used to assess purity Purity = 92%

AVA04-236-6(EAAAK) hIgG1 Fc AVA04-236 hIgG1 Fc C-terminal Affimer

• Determined the KD’s of the KD=0.89 nM KD=2.01 nM Affimer formats using Biacore Response (RUs) • PD-L1 Fc antigen was Response (RUs) immobilised onto the chip

surface and so the antigen Time (sec) Time (sec) is dimeric • With dimeric antigen avidity AVA04-236 dimer on hIgG1 Fc AVA04-236 hIgG1 Fc N-terminal Affimer effects can be observed KD=0.19 nM KD=0.63 nM Response (RUs) Response (RUs)

Time (sec) Time (sec)

AVA04-251 hFc1 SEC-HPLC AVA04-251hFc1 Biacore Kinetics

Affimer Monomer FACs Binding to CHO PD-L1 Cells PD-1/PD-L1 Competition ELISA

Affimer EC50 (nM) AVA04-141 6.2 Clone IC50 (nM) AVA04-236 1.6 AVA04-251 Fc1 0.4 AVA04-228 0.9 29E.2A3 mAb 0.5 AVA04-261 1.0 Atezolizumab” 1.2

From Initial Affimer Generation to Animal Efficacy Data in Nine Months

• Mouse CT26 syngeneic tumour model • Anti-tumour effect seen with anti-mPDL1 Affimer comparable to mAb • Repeat high dosing of anti-mPDL1 Affimer was well tolerated

Reduction in Tumour Growth Reduction in Tumour Growth Rate: Affimer Fc-182 Rate: 10F9.G2 mAb

From Initial Affimer Discovery to Animal Efficacy Data in Nine Months

• Significant increase of T-reg cell population (CD4+, FoxP3+) compared to controls in the tumour micro-environment for Affimer and 10F9.G2 control Ab • No neutralising ADAs detected

Tumour T-reg Cell Population

• Co-crystalised Affimer with PD-L1/PD- 1 binding domain. Affimer • No non-antibody scaffold/PD-L1 complex yet published. • Structure solved to 2.1 Å (detailed Loop 4 interactions not shown in figure) • Confirms main binding interactions are via loop 2. • Confirms epitope binding site similar to Atezolizumab. • Will support the affinity maturation of lead Affimer.

Human PD-L1 Loop 2 Domain

• Cryopreserved PBMC samples from 50 healthy donors selected to represent the different HLA- allotypes in the human population • Test products: – Type 1 Affimer scaffold (human Affimer scaffold) – A Type 2 Affimer protein (plant consensus sequence Affimer protein) – Type 3 Affimer scaffold (next generation human Affimer scaffold) – Avastin (Bevacizumab) benchmark – Keyhole Limpet Hemocyanin (positive control; raw data not shown for clarity) – Buffer (negative control) • Incubate PBMC for 7 days in the presence of test products at 50 µg/ml (8 replicates) – Note: Affimer test products are at 5x the effective molar concentration of the Avastin sample (which has a ten- fold greater molecular weight and two binding regions per molecule) • Assess proliferation of CD4+ T cells using flow cytometry by: – Cell surface marker staining (CD3+, CD4+ and CD8+) – Measuring EdU incorporation as an indicator of T-cell proliferation • Analysis: – Normalized count of CD3+CD4+EdU+ cells per well – Calculation of Stimulation Index (SI) (ratio mean of stimulated over mean non-stimulated wells (Blank or Buffer) – Responders defined by industry standard SI>2 and p value < 0.05 using an unpaired, two sample student’s t- test

Commentary Responder Rate • High responder rate to KLH positive control as expected 100% 84% • 90% No responders in this set of 50 donors 80% to T1, T2 Affimers or Avastin 70% • One responder (2%) to T3 Affimer 60% scaffold 50% • The Avastin test product is a marketed 40% monoclonal therapeutic antibody 30% manufactured to GMP standards % Responders 20% 2% • 10% The Affimer test products were 0% produced in-house under non-GMP -ve Type 1 Type 2 Type 3 Avastin KLH +ve conditions Control Affimer Affimer Affimer Control • The Affimer test products have been tested at five times the concentration of Avastin

Published PBMC data for a range of monoclonal antibody therapies for comparison1 • Responder rates for these mAbs for these donors are in the range 3 – 15% • Scale bar shows % incidence of clinical (in vivo) immunogenicity for these products showing good correlation with PBMC results

-S-

Fc Fusions Serum Albumin PEGylation

Utilising IgG FcRn recycling to Affimer biotherapeutic binds Increased hydrodynamic size maintain high serum levels to SA in the circulation of the protein to prevent clearance via the kidneys Direct genetic fusion to SA

• Repeated mouse PK to confirm long in vivo half-life • AVA04-141 hFc4 does not bind mouse PD-L1 and has a human IgG4 Fc • Dosed animals via the IV route (10 mg/kg) • Serum half-life of Affimer Fc fusion ~126 hrs • Serum half-life similar to other scaffold- Fc fusions • Serum half-life of isotype IgG4 mAb ~220 hrs • Next steps: PK of other Affimer Fc fusions (e.g. IgG1 which has ADCC) and also biodistribution.

• Identified a range of human serum albumin binders following Affimer selections • Affimers bind to mouse, dog, cyno and human serum albumin with differing affinities

Lane Sample M Molecular weight marker >97% purity by SEC-HPLC 1 AVA03-42 1 μg 2 AVA03-42 5 μg

• AVA03-42 (human serum albumin (HSA) binding Affimer) was expressed from E. coli and purified using IMAC, IEX and SEC. • Expression of the AVA03-42 was >200mg/L

Affimer HSA binders t1/2 AUC 0-t Clone (hrs) h*µg/mL

AVA03-42 38.2 5,670 AVA03-37 37.7 3,435 AVA03-21 30.6 1,401 AVA03-19 24.3 1,059 AVA03-32 29.0 112 Affimer monomer Non-binder 1.6 18.1

• Demonstrated half-life extension in mouse compared • AVA03 Affimers significantly extend the

to non-binding Affimer monomer. serum half-life (t1/2) in mouse. • Affimer labelled with I125 and dosed at 10 mg/kg (IV). • The half-life can be tuned by the affinity • Next steps: generate Affimer fusions (e.g. dimers etc) to albumin. and plan for further mouse and cyno PK studies • Albumin binding Affimers give the option using AVA03 monomers. for manufacturing in E. coli if the partner biomolecule can be produced in bacteria as well.

• Licensing Business Model • Key Benefits of Affimers • Case Studies: 1. Generating Robust Critical Reagents - Anti-idiotypic Trastuzumab Affimers 2. Increasing Assay Specificity of an Existing Clinical ELISA Kit Using Affimers as a Capture Surface – C. Difficile ToxinB 3. Rapid Generation and Formatting of Affimer Pairs to a Clinically Important Target - CRP

27 Affimer Reagents Licensing Model Paid for evaluations of Affimer technology leading to licensing

Custom Affimer Service Commercial Licensing • £25-50K per project • Research tools, lab assays, diagnostic immunoassays, separation systems • R&D license only • Royalty bearing commercial licenses • Prioritise commercial licenses and high volume repeat users

Affimer Licensing for £ Customer Commercial Generation product Royalties (9-12 weeks) evaluation development License

£ Repeat business Licensing for in-house research use

• 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-

3 nm batch variation. • Easily engineered 11 nm • Fusions to other functional proteins are simple to generate and manufacture. • Generation of multimers is comparatively straightforward. • Easy to orientate • Site-specific chemistries allow production of high-capacity surfaces for target protein capture or Affimer labelling. • Highly stable 29 Avacta Life Sciences © 2017 • pH tolerant over a broad range (pH2-12). • Thermally stable. • Broad tolerance to organic solvents.

• Availability of capture reagents for Assay format therapeutic antibodies PK assays can be rate limiting in product development: – Anti-idiotype antibodies development can be a long and difficult process. Fluorescence Signal / Detection – Ligand capture reagents not always reliably available and can be expensive. AP - Conjugate – Specificity of capture reagents can sometimes be problematic (matrix Anti-Human IgG effect). Trastuzumab /Test mAb

Immobilised Her2 • Affimer binders can provide a fast, reliable 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.

Western Blot with Strep-HRP detection 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

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 )

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: Inter-assay precision • • <16.3 %CV across LLOQ of 60ng/ml <4.6 %CV across LLOQ of 40ng/ml - to ULOQ of 1600 ng/ml (LQC outlier, ULOQ of 3000ng/ml (n=9). remainder <6%). • Broader dynamic range than existing • Intra-assay precision bioanalysis assay (60-3000ng/ml • <12.4 %CV across LLOQ of 60ng/ml - compared to 40-750ng/ml with anti-ID ULOQ of 2000ng/ml (n=6). 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 2 reverse is not the case . https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4885049/ • Annual case load/annual cost estimated $1b annually US alone, $3,600 per patient. 1. Magill et al, 2014. N.Eng.J.Med, 370:1198-1208 2. Bella et al, 2016. Toxins, 8(5):134 • A growing proportion of cases are not treatable by antibiotic therapy alone.

• 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 using Optim Aggregation testing of Clone B-45 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 surface: n.d.

• 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.

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.

Target Protein Primary Screen ELISA Phage Display QC & Sequencing Validation

12 – 14 weeks

• Clone 2237 performed well as a capture surface with 2240 as a 2 2 3 7 - h C R P - 2 2 4 0 - B 0 1 ( b u ffe r ) h C R P s a n d w ic h E L IS A detection reagent. 2 2 3 7 - h C R P - 2 2 4 0 - B 0 1 ( s e r u m ) 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 0 5 4 ( 2 2 5 4 - h C R P - 2 2 4 8 - B 0 1 ( b u ffe r ) 2 2248 as a detection reagent. D

O 2 2 5 4 - h C R P - 2 2 4 8 - B 0 1 ( s e r u m )

1 • 2237/2240 chosen as 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 ) development pair. 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

4 )

m • Assay was optimised and validated n

3 using 1:10 dilution of serum, therefore 6 3 - 0

5 the range of the assay is 20 - 500ng/mL 4 (

e 2 CRP in undiluted serum samples c n a b r 1 o s b A 0 0 5 0 1 0 0 1 5 0 C R P (n g /m L )

Intra- LOQ Accuracy Inter-assay Precision assay (ng/mL) 50ng/mL 25ng/mL 125ng/mL 0.5ng/mL 0ng/mL Precision

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

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 ) ) ) m m m n n n

0 3 0 0 3 3 3 3 3 6 6 6 - - - 0 0 0 5 5 5 4 4 4 ( 2 ( 2 ( 2 D D D O 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)

Affimers can be engineered to Affimers have been used as: specifically recognise: • Immunoassay reagents. • Ligand-receptor complexes. • Immuno-precipitation reagents. • • Antibody-antigen complexes. Imaging reagents. • Crystallisation chaperones. • Very homologous protein sequences. • Biosensor capture reagents. • Very specific motifs/epitope. • Affinity purification reagents. • Antibody idiotypes. • Protein-protein inhibitors.

We have functionalised Affimers with: • Biotin. • Terminal cysteines to enable malemide chemistries. • Various protein fusion (GFP…). • Fluorophore labelling. • Resins and beads.

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