Therapeutic drug monitoring of immunotherapies with novel Affimer-NanoBiT sensor construct.

Concentration-therapeutic efficacy relationships have been observed for several therapeutic monoclonal antibodies (TmAb), where low circulating levels can result in ineffective treatment and high concentrations can cause adverse reactions. Rapid therapeutic drug monitoring (TDM) of TmAb drugs would provide the opportunity to adjust an individual patient's dosing regimen to improve treatment results. However, TDM for immunotherapies is currently limited to centralised testing methods with long sample-collection to result timeframes. Here, we show four point-of-care (PoC) TmAb biosensors by combining anti-idiotypic Affimer proteins and NanoBiT split luciferase technology at a molecular level to provide a platform for rapid quantification (<10 minutes) for four clinically relevant TmAb (rituximab, adalimumab, ipilimumab and trastuzumab). The rituximab sensor performed best with 4 pM limit of detection (LoD) and a quantifiable range between 8 pM-2 nM with neglectable matrix effects in serum up to 1%. After dilution of serum samples, the resulting quantifiable range for all four sensors falls within the clinically relevant range and compares favourably with the sensitivity and/or time-to-result of current ELISA standards. Further development of these sensors into a PoC test may improve treatment outcome and quality of life for patients receiving immunotherapy.

"Affimer" synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation.

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol, and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidized low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low-density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified five synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently labeled oxLDL by the LOX-1-expressing cell model was inhibited with subnanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p < 0.01) by preincubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease.

Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition.

Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible.

Enzyme - Switch sensors for therapeutic drug monitoring of immunotherapies.

Therapeutic monoclonal antibodies (TmAb) have emerged as effective treatments for a number of cancers and autoimmune diseases. However, large interpatient disparities in the pharmacokinetics of TmAb treatment requires close therapeutic drug monitoring (TDM) to optimise dosage for individual patients. Here we demonstrate an approach for achieving rapid, sensitive quantification of two monoclonal antibody therapies using a previously described enzyme switch sensor platform. The enzyme switch sensor consists of a ß-lactamase - ß-lactamase inhibitor protein (BLA-BLIP) complex with two anti-idiotype binding proteins (Affimer proteins) as recognition elements. The BLA-BLIP sensor was engineered to detect two TmAbs (trastuzumab and ipilimumab) by developing constructs incorporating novel synthetic binding reagents to each of these mAbs. Trastuzumab and ipilimumab were successfully monitored with sub nM sensitivity in up to 1% serum, thus covering the relevant therapeutic range. Despite the modular design, the BLA-BLIP sensor was unsuccessful in detecting two further TmAbs (rituximab and adalimumab), an explanation for which was explored. In conclusion, the BLA-BLIP sensors provide a rapid biosensor for TDM of trastuzumab and ipilimumab with the potential to improve therapy. The sensitivity of this platform alongside its rapid action would be suitable for bedside monitoring in a point-of-care (PoC) setting.

Fibrin-glycoprotein VI interaction increases platelet procoagulant activity and impacts clot structure.

BACKGROUND: The glycoprotein VI (GPVI) signaling pathway was previously reported to direct procoagulant platelet activity through collagen binding. However, the impact of GPVI-fibrin interaction on procoagulant platelet development and how it modulates the clot structure are unknown. OBJECTIVES: To determine the effect of GPVI-fibrin interaction on the platelet phenotype and its impact on the clot structure. METHODS: Procoagulant platelets in platelet-rich plasma clots were determined by scanning electron microscopy (wild-type and GPVI-deficient murine samples) and confocal microscopy. Procoagulant platelet number, clot density, clot porosity, and clot retraction were determined in platelet-rich plasma or whole blood clots of healthy volunteers in the presence of tyrosine kinase inhibitors (PRT-060318, ibrutinib, and dasatinib) and eptifibatide. RESULTS: GPVI-deficient clots showed a higher nonprocoagulant vs procoagulant platelet ratio than wild-type clots. The fiber density and the procoagulant platelet number decreased in the presence of Affimer proteins, inhibiting GPVI-fibrin(ogen) interaction and the tyrosine kinase inhibitors. The effect of GPVI signaling inhibitors on the procoagulant platelet number was exacerbated by eptifibatide. The tyrosine kinase inhibitors led to an increase in clot porosity; however, no differences were observed in the final clot weight, following clot retraction with the tyrosine kinase inhibitors, except for ibrutinib. In the presence of eptifibatide, clot retraction was impaired. CONCLUSION: Our findings showed that GPVI-fibrin interaction significantly contributes to the development of procoagulant platelets and that inhibition of GPVI signaling increases clot porosity. Clot contractibility was impaired by the integrin αIIbß3 and Btk pathway inhibition. Thus, inhibition of GPVI-fibrin interactions can alleviate structural characteristics that contribute to a prothrombotic clot phenotype, having potential important implications for novel antithrombotic interventions.

Labeling of a mutant estrogen receptor with an Affimer in a breast cancer cell line.

Mutations of the intracellular estrogen receptor alpha (ERα) is implicated in 70% of breast cancers. Therefore, it is of considerable interest to image various mutants (L536S, Y537S, D538G) in living cancer cell lines, particularly as a function of various anticancer drugs. We therefore developed a small (13 kDa) Affimer, which, after fluorescent labeling, is able to efficiently label ERα by traveling through temporary pores in the cell membrane, created by the toxin streptolysin O. The Affimer, selected by a phage display, predominantly labels the Y537S mutant and can tell the difference between L536S and D538G mutants. The vast majority of Affimer-ERαY537S is in the nucleus and is capable of an efficient, unrestricted navigation to its target DNA sequence, as visualized by single-molecule fluorescence. The Affimer can also differentiate the effect of selective estrogen receptor modulators. More generally, this is an example of a small binding reagent-an Affimer protein-that can be inserted into living cells with minimal perturbation and high efficiency, to image an endogenous protein.

Protein-conjugated microbubbles for the selective targeting of S. aureus biofilms.

Staphylococcus aureus (S. aureus) is an important human pathogen and a common cause of bloodstream infection. The ability of S. aureus to form biofilms, particularly on medical devices, makes treatment difficult, as does its tendency to spread within the body and cause secondary foci of infection. Prolonged courses of intravenous antimicrobial treatment are usually required for serious S. aureus infections. This work investigates the in vitro attachment of microbubbles to S. aureus biofilms via a novel Affimer protein, AClfA1, which targets the clumping factor A (ClfA) virulence factor - a cell-wall anchored protein associated with surface attachment. Microbubbles (MBs) are micron-sized gas-filled bubbles encapsulated by a lipid, polymer, or protein monolayer or other surfactant-based material. Affimers are small (∼12 kDa) heat-stable binding proteins developed as replacements for antibodies. The binding kinetics of AClfA1 against S. aureus ClfA showed strong binding affinity (KD = 62 ± 3 nM). AClfA1 was then shown to bind S. aureus biofilms under flow conditions both as a free ligand and when bound to microparticles (polymer beads or microbubbles). Microbubbles functionalized with AClfA1 demonstrated an 8-fold increase in binding compared to microbubbles functionalized with an identical Affimer scaffold but lacking the recognition groups. Bound MBs were able to withstand flow rates of 250 µL/min. Finally, ultrasound was applied to burst the biofilm bound MBs to determine whether this would lead to biofilm biomass loss or cell death. Application of a 2.25 MHz ultrasound profile (with a peak negative pressure of 0.8 MPa and consisting of a 22-cycle sine wave, at a pulse repetition rate of 10 kHz) for 2 s to a biofilm decorated with targeted MBs, led to a 25% increase in biomass loss and a concomitant 8% increase in dead cell count. The results of this work show that Affimers can be developed to target S. aureus biofilms and that such Affimers can be attached to contrast agents such as microbubbles or polymer beads and offer potential, with some optimization, for drug-free biofilm treatment.

Affinity purification of fibrinogen using an Affimer column.

BACKGROUND: Fibrinogen is an abundant plasma protein with an essential role in blood coagulation and haemostasis thus receiving significant research interest. However, protein purification is time consuming and commercial preparations often have protein contaminants. The aim of this study was to develop a new method to purify high quality and functional fibrinogen. METHODS: Fibrinogen-specific Affimer protein, isolated using phage display systems, was immobilised to SulfoLink resin column and employed for fibrinogen purification from plasma samples. Fibrinogen was eluted using a high pH solution. Commercial human fibrinogen was also further purified using the Affimer column. Fibrinogen purity was determined by SDS-PAGE and mass spectrometry, while functionality was assessed using turbidimetric analysis. RESULTS: Affimer-purified fibrinogen from human plasma showed purity at least comparable to commercially available preparations and was able to form physiological fibrin networks. Further purification of commercially available fibrinogen using the Affimercolumn eliminated multiple contaminant proteins, a significant number of which are key elements of the coagulation cascade, including plasminogen and factor XIII. CONCLUSIONS: The Affimercolumn represents a proof of concept novel, rapid method for isolating functional fibrinogen from plasma and for further purification of commercially available fibrinogen preparations. GENERAL SIGNIFICANCE: Our methodology provides an efficient way of purifying functional fibrinogen with superior purity without the need of expensive pieces of equipment or the use of harsh conditions.

Piggybacking on the Cholera Toxin: Identification of a CTB-Binding Protein as an Approach for Targeted Delivery of Proteins to Motor Neurons.

A significant unmet need exists for the delivery of biologic drugs such as polypeptides or nucleic acids to the central nervous system for the treatment and understanding of neurodegenerative diseases. Naturally occurring bacterial toxins have been considered as tools to meet this need. However, due to the complexity of tethering macromolecular drugs to toxins and the inherent dangers of working with large quantities of recombinant toxins, no such route has been successfully exploited. Developing a method where a bacterial toxin's nontoxic targeting subunit can be assembled with a drug immediately prior to in vivo administration has the potential to circumvent some of these issues. Using a phage-display screen, we identified two antibody mimetics, anticholera toxin Affimer (ACTA)-A2 and ACTA-C6 that noncovalently associate with the nonbinding face of the cholera toxin B-subunit. In a first step toward the development of a nonviral motor neuron drug-delivery vehicle, we show that Affimers can be selectively delivered to motor neurons in vivo.

RAS-inhibiting biologics identify and probe druggable pockets including an SII-α3 allosteric site.

RAS mutations are the most common oncogenic drivers across human cancers, but there remains a paucity of clinically-validated pharmacological inhibitors of RAS, as druggable pockets have proven difficult to identify. Here, we identify two RAS-binding Affimer proteins, K3 and K6, that inhibit nucleotide exchange and downstream signaling pathways with distinct isoform and mutant profiles. Affimer K6 binds in the SI/SII pocket, whilst Affimer K3 is a non-covalent inhibitor of the SII region that reveals a conformer of wild-type RAS with a large, druggable SII/α3 pocket. Competitive NanoBRET between the RAS-binding Affimers and known RAS binding small-molecules demonstrates the potential to use Affimers as tools to identify pharmacophores. This work highlights the potential of using biologics with small interface surfaces to select unseen, druggable conformations in conjunction with pharmacophore identification for hard-to-drug proteins.

Selective Affimers Recognise the BCL-2 Family Proteins BCL-xL and MCL-1 through Noncanonical Structural Motifs*.

The BCL-2 family is a challenging group of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL-2 family regulators of apoptosis are useful as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non-antibody-binding proteins based on a conserved scaffold) to identify ligands for MCL-1, BCL-xL , BCL-2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective recognition of their target BCL-2 protein. For anti-apoptotic targets BCL-xL and MCL-1, competitive inhibition of their canonical protein-protein interactions is demonstrated. Co-crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug-bound-like conformation. These proof-of-concept studies indicate that Affimers could be used as alternative templates to inspire the design of selective BCL-2 family modulators and more generally other protein-protein interaction inhibitors.

Fibrinogen interaction with complement C3: a potential therapeutic target to reduce thrombosis risk.

Complement C3 binds fibrinogen and compromises fibrin clot lysis thereby enhancing thrombosis risk. We investigated the role of fibrinogen-C3 interaction as a novel therapeutic target to reduce thrombosis risk by analysing: i) consistency in the fibrinolytic properties of C3, ii) binding sites between fibrinogen and C3 and iii) modulation of fibrin clot lysis by manipulating fibrinogen-C3 interactions. Purified fibrinogen and C3 from the same individuals (n=24) were used to assess inter-individual variability in the anti-fibrinolytic effects of C3. Microarray screening and molecular modelling evaluated C3 and fibrinogen interaction sites. Novel synthetic conformational proteins, termed Affimers, were used to modulate C3-fibrinogen interaction and fibrinolysis. C3 purified from patients with type 1 diabetes showed enhanced prolongation of fibrinolysis compared with healthy control protein [195±105 and 522±166 seconds, respectively (p=0.04)], with consistent effects but a wider range (5-51% and 5-18% lysis prolongation, respectively). Peptide microarray screening identified 2 potential C3-fibrinogen interactions sites within fibrinogen ß chain (residues 424-433, 435-445). One fibrinogen-binding Affimer was isolated that displayed sequence identity with C3 in an exposed area of the protein. This Affimer abolished C3-induced prolongation of fibrinolysis (728±25.1 seconds to 632±23.7 seconds, p=0.005) and showed binding to fibrinogen in the same region that is involved in C3-fibrinogen interactions. Moreover, it shortened plasma clot lysis of patients with diabetes, cardiovascular disease or controls by 7-11%. C3 binds fibrinogen ß-chain and disruption of fibrinogen-C3 interaction using Affimer proteins enhances fibrinolysis, which represents a potential novel target tool to reduce thrombosis in high risk individuals.

Nanoscale Pattern Extraction from Relative Positions of Sparse 3D Localizations.

Inferring the organization of fluorescently labeled nanosized structures from single molecule localization microscopy (SMLM) data, typically obscured by stochastic noise and background, remains challenging. To overcome this, we developed a method to extract high-resolution ordered features from SMLM data that requires only a low fraction of targets to be localized with high precision. First, experimentally measured localizations are analyzed to produce relative position distributions (RPDs). Next, model RPDs are constructed using hypotheses of how the molecule is organized. Finally, a statistical comparison is used to select the most likely model. This approach allows pattern recognition at sub-1% detection efficiencies for target molecules, in large and heterogeneous samples and in 2D and 3D data sets. As a proof-of-concept, we infer ultrastructure of Nup107 within the nuclear pore, DNA origami structures, and α-actinin-2 within the cardiomyocyte Z-disc and assess the quality of images of centrioles to improve the averaged single-particle reconstruction.

Isolation of Artificial Binding Proteins (Affimer Reagents) for Use in Molecular and Cellular Biology.

Artificial binding proteins have been validated as alternatives to antibodies in their use as research reagents in molecular and cellular biology. For example, they have been used as inhibitors of protein-protein interactions to modulate activity, to facilitate crystallization, and as probes for cellular imaging.Phage display is a widely used approach for isolating target-specific binding reagents, and it has even been used to isolate isoform-specific binding proteins and binders that can distinguish between highly homologous protein domains.Here, we describe methods that have been employed in isolating highly specific artificial binding proteins against a wide range of target proteins.

Characterization and applications of a Crimean-Congo hemorrhagic fever virus nucleoprotein-specific Affimer: Inhibitory effects in viral replication and development of colorimetric diagnostic tests.

Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is one of the most widespread medically important arboviruses, causing human infections that result in mortality rates of up to 60%. We describe the selection of a high-affinity small protein (Affimer-NP) that binds specifically to the nucleoprotein (NP) of CCHFV. We demonstrate the interference of Affimer-NP in the RNA-binding function of CCHFV NP using fluorescence anisotropy, and its inhibitory effects on CCHFV gene expression in mammalian cells using a mini-genome system. Solution of the crystallographic structure of the complex formed by these two molecules at 2.84 Å resolution revealed the structural basis for this interference, with the Affimer-NP binding site positioned at the critical NP oligomerization interface. Finally, we validate the in vitro application of Affimer-NP for the development of enzyme-linked immunosorbent and lateral flow assays, presenting the first published point-of-care format test able to detect recombinant CCHFV NP in spiked human and animal sera.

Photon induced quantum yield regeneration of cap-exchanged CdSe/CdS quantum rods for ratiometric biosensing and cellular imaging.

Full water-dispersion of commercial hydrophobic CdSe/CdS core/shell quantum rods (QRs) was achieved by cap-exchange using a dihydrolipoic acid zwitterion ligand at a low ligand:QR molar ratio (LQMR) of 1000. However, this process almost completely quenched the QR fluorescence, greatly limiting its potential in downstream fluorescence based applications. Fortunately, we found that the QR fluorescence could be recovered by exposure to near ultra-violet to blue light radiation (e.g. 300-450 nm). These "reborn" QRs were found to be compact, bright, and stable, and were resistant to non-specific adsorption, which make them powerful fluorescent probes in broad biomedical applications. We demonstrated their potential in two model applications: first, the QRs were conjugated with His8-tagged small antibody mimetic proteins (also known as Affimers) for the sensitive detection of target proteins via a Förster resonance energy transfer (FRET) readout strategy and second, the QR surface was functionalized with biotins for targeted imaging of cancer cells.

Affimers as anti-idiotypic affinity reagents for pharmacokinetic analysis of biotherapeutics.

Therapeutic antibodies are the fastest growing class of drugs in the treatment of cancer, and autoimmune and inflammatory diseases that require the concomitant development of assays to monitor therapeutic antibody levels. Here, we demonstrate that the use of Affimer nonantibody binding proteins provides an advantage over current antibody-based detection systems. For four therapeutic antibodies, we used phage display to isolate highly specific anti-idiotypic Affimer reagents, which selectively bind to the therapeutic antibody idiotype. For each antibody target the calibration curves met US Food and Drug Administration criteria and the dynamic range compared favorably with commercially available reagents. Affimer proteins therefore represent promising anti-idiotypic reagents that are simple to select and manufacture, and that offer the sensitivity, specificity and consistency required for pharmacokinetic assays.

Affimer-Enzyme-Inhibitor Switch Sensor for Rapid Wash-free Assays of Multimeric Proteins.

Robust technology is required to underpin rapid point-of-care and in-field diagnostics to improve timely decision making across broad sectors. An attractive strategy combines target recognition and signal generating elements into an "active" enzyme-switch that directly transduces target-binding into a signal. However, approaches that are broadly applicable to diverse targets remain elusive. Here, an enzyme-inhibitor switch sensor was developed by insertion of non-immunoglobulin Affimer binding proteins, between TEM1-ß-lactamase and its inhibitor protein, such that target binding disrupts the enzyme-inhibitor complex. Design principles for a successful switch architecture are illustrated by the rapid (min), simple (wash-free), and sensitive (pM) quantification of multimeric target analytes in biological samples (serum, plasma, leaf extracts), across three application areas. A therapeutic antibody (Herceptin), protein biomarker (human C-reactive protein), and plant virus (cow pea mosaic virus) were targeted, demonstrating assays for therapeutic drug monitoring, health diagnostics, and plant pathogen detection, respectively. Batch-to-batch reproducibility, shelf-life stability, and consistency with validated enzyme-linked immunosorbent assay analysis confirm that the principle of an Affimer-enzyme-inhibitor switch provides a platform for point-of-care and in-field diagnostics.

Affimer reagents as tools in diagnosing plant virus diseases.

Plant viruses can cause devastating losses to agriculture and are therefore a major threat to food security. The rapid identification of virally-infected crops allowing containment is essential to limit such threats, but plant viral diseases can be extremely challenging to diagnose. An ideal method for plant virus diagnosis would be a device which can be implemented easily in the field. Such devices require a binding reagent that is specific for the virus of interest. We chose to investigate the use of Affimer reagents, artificial binding proteins and a model plant virus Cowpea Mosaic virus (CPMV) empty virus like particles (eVLPs). CPMV-eVLP mimic the morphology of wild-type (WT) CPMV but lack any infectious genomic material and so do not have biocontainment issues. We have produced and purified an Affimer reagent selected for its ability to bind to CPMV-eVLP and have shown that the selected Affimer also specifically binds to WT CPMV. We have produced a 3.4 Å structure of WT CPMV bound to the Affimer using cryo-electron microscopy. Finally, we have shown that this Affimer is capable of reliably detecting the virus in crude extracts of CPMV-infected leaves and can therefore form the basis for the future development of diagnostic tests.

Affimer proteins as a tool to modulate fibrinolysis, stabilize the blood clot, and reduce bleeding complications.

Bleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding. Two fibrinogen-specific Affimer proteins, F5 and G2, were identified and characterized for their effects on clot structure/fibrinolysis, using turbidimetric and permeation analyses and confocal and electron microscopy. Binding studies and molecular modeling identified interaction sites, whereas plasmin generation assays determined effects on plasminogen activation. In human plasma, F5 and G2 prolonged clot lysis time from 9.8 ± 1.1 minutes in the absence of Affimers to 172.6 ± 7.4 and more than 180 minutes (P < .0001), respectively, and from 7.6 ± 0.2 to 28.7 ± 5.8 (P < .05) and 149.3 ± 9.7 (P < .0001) minutes in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy control patients and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure and G2 profoundly altered fibrin fiber arrangement, whereas F5 maintained physiological clot structure. Affimer F5 reduced fibrin-dependent plasmin generation and was predicted to bind fibrinogen D fragment close to tissue plasminogen activator (tPA; residues γ312-324) and plasminogen (α148-160) binding sites, thus interfering with tPA-plasminogen interaction and representing 1 potential mechanism for modulation of fibrinolysis. Our Affimer proteins provide a novel methodology for stabilizing fibrin networks with potential future clinical implications to reduce bleeding risk.