A novel, label-free, pre-equilibrium assay to determine the association and dissociation rate constants of therapeutic antibodies on living cells.

BACKGROUND AND PURPOSE: Monoclonal antibodies (Ab) represent the fastest growing drug class. Knowledge of the biophysical parameters (kon , koff and KD ) that dictate Ab:receptor interaction is critical during the drug discovery process. However, with the increasing complexity of Ab formats and their targets, it became apparent that existing technologies present limitations and are not always suitable to determine these parameters. Therefore, novel affinity determination methods represent an unmet assay need. EXPERIMENTAL APPROACH: We developed a pre-equilibrium kinetic exclusion assay using recent mathematical advances to determine the kon , koff and KD of monoclonal Ab:receptor interactions on living cells. The assay is amenable to all human IgG1 and rabbit Abs. KEY RESULTS: Using our novel assay, we demonstrated for several monoclonal Ab:receptor pairs that the calculated kinetic rate constants were comparable with orthogonal methods that were lower throughput or more resource consuming. We ran simulations to predict the critical conditions to improve the performance of the assays. We further showed that this method could successfully be applied to both suspension and adherent cells. Finally, we demonstrated that kon and koff , but not KD , correlate with in vitro potency for a panel of monoclonal Abs. CONCLUSIONS AND IMPLICATIONS: Our novel assay has the potential to systematically probe binding kinetics of monoclonal Abs to cells and can be incorporated in a screening cascade to identify new therapeutic candidates. Wide-spread adoption of pre-equilibrium assays using physiologically relevant systems will lead to a more holistic understanding of how Ab binding kinetics influence their potency.

Dysregulation of the kallikrein-kinin system in bronchoalveolar lavage fluid of patients with severe COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to the angiotensin-converting enzyme 2 (ACE2) receptor, a critical component of the kallikrein-kinin system. Its dysregulation may lead to increased vascular permeability and release of inflammatory chemokines. Interactions between the kallikrein-kinin and the coagulation system might further contribute to thromboembolic complications in COVID-19. METHODS: In this observational study, we measured plasma and tissue kallikrein hydrolytic activity, levels of kinin peptides, and myeloperoxidase (MPO)-DNA complexes as a biomarker for neutrophil extracellular traps (NETs), in bronchoalveolar lavage (BAL) fluid from patients with and without COVID-19. FINDINGS: In BAL fluid from patients with severe COVID-19 (n = 21, of which 19 were mechanically ventilated), we observed higher tissue kallikrein activity (18·2 pM [1·2-1535·0], median [range], n = 9 vs 3·8 [0·0-22·0], n = 11; p = 0·030), higher levels of the kinin peptide bradykinin-(1-5) (89·6 [0·0-2425·0], n = 21 vs 0·0 [0·0-374·0], n = 19, p = 0·001), and higher levels of MPO-DNA complexes (699·0 ng/mL [66·0-142621·0], n = 21 vs 70·5 [9·9-960·0], n = 19, p < 0·001) compared to patients without COVID-19. INTERPRETATION: Our observations support the hypothesis that dysregulation of the kallikrein-kinin system might occur in mechanically ventilated patients with severe pulmonary disease, which might help to explain the clinical presentation of patients with severe COVID-19 developing pulmonary oedema and thromboembolic complications. Therefore, targeting the kallikrein-kinin system should be further explored as a potential treatment option for patients with severe COVID-19. FUNDING: Research Foundation-Flanders (G0G4720N, 1843418N), KU Leuven COVID research fund.

KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements.

Design of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

Systemic exposure following intravitreal administration of therapeutic agents: an integrated pharmacokinetic approach. 1. THR-149.

Intravitreal (IVT) injection of pharmacological agents is an established and widely used procedure for the treatment of many posterior segment of the eye diseases. IVT injections permit drugs to reach high concentrations in the retina whilst limiting systemic exposure. Beyond the risk of secondary complications such as intraocular infection, the potential of systemic adverse events cannot be neglected. Therefore, a detailed understanding of the rules governing systemic exposure following IVT drug administration remains a prerequisite for the evaluation and development of new pharmacological agents intended for eye delivery. We present here a novel mathematical model to describe and predict circulating drug levels following IVT in the rabbit eye, a species which is widely used for drug delivery, pharmacokinetic, and pharmacodynamic studies. The mathematical expression was derived from a pharmacokinetic model that assumes the existence of a compartment between the vitreous humor compartment itself and the systemic compartment. We show that the model accurately describes circulating levels of THR-149, a plasma kallikrein inhibitor in development for the treatment of diabetic macular edema. We hypothesize that the model based on the rabbit eye has broader relevance to the human eye and can be used to analyze systemic exposure of a variety of drugs delivered in the eye.

Systemic exposure following intravitreal administration of therapeutic agents: an integrated pharmacokinetic approach. 2. THR-687.

Intravitreal (IVT) injection remains the preferred administration route of pharmacological agents intended for the treatment of back of the eye diseases such as diabetic macular edema (DME) and neovascular age-related macular degeneration (nvAMD). The procedure enables drugs to be delivered locally at high concentrations whilst limiting whole body exposure and associated risk of systemic adverse events. Nevertheless, intravitreally-delivered drugs do enter the general circulation and achieving an accurate understanding of systemic exposure is pivotal for the evaluation and development of drugs administered in the eye. We report here the full pharmacokinetic properties of THR-687, a pan RGD integrin antagonist currently in clinical development for the treatment of DME, in both rabbit and minipig. Pharmacokinetic characterization included description of vitreal elimination, of systemic pharmacokinetics, and of systemic exposure following IVT administration. For the latter, we present a novel pharmacokinetic model that assumes clear partition between the vitreous humor compartment itself where the drug is administered and the central systemic compartment. We also propose an analytical solution to the system of differential equations that represent the pharmacokinetic model, thereby allowing data analysis with standard nonlinear regression analysis. The model accurately describes circulating levels of THR-687 following IVT administration in relevant animal models, and we suggest that this approach is relevant to a range of drugs and analysis of subsequent systemic exposure.

A method to assess the robustness of complex mathematical models used for quantitative interpretation of experimental data by nonlinear regression analysis: application to high-affinity binding models.

Nonlinear regression is widely used to fit experimental data to a specific mathematical model to extract numerical values for parameters representing the studied process. However, assessing the degree of precision that can be expected from such an analysis for a given parameter can be quite challenging for complex mathematical models. To address this issue, we propose here a method based on the analysis of a large number of data sets generated in such a way to mimic specific experimental conditions. Applying this methodology to high-affinity binding models, we report here a quantitative analysis of the robustness of such models, and how the precision on the fitting parameters can be expected to vary based on, e.g., the initial experimental conditions, the number or distribution of experimental points, or the experimental variability. We also show that these models, although widely used, are intrinsically limited by the fact that the two main fitting parameters, one representing the concentration of the studied species and the other its affinity, are inversely correlated, but demonstrate that this limitation can be overcome by global analysis of multiple data series generated at different concentrations of the titrated species.

A novel assay based on pre-equilibrium titration curves for the determination of enzyme inhibitor binding kinetics.

Selection of pharmacological agents based on potency measurements performed at equilibrium fail to incorporate the kinetic aspects of the drug-target interaction. Here we describe a method for screening or characterization of enzyme inhibitors that allows the concomitant determination of the equilibrium inhibition constant in unison with rates of complex formation and dissociation. The assay is distinct from conventional enzymatic assays and is based on the analysis of inhibition curves recorded prior to full equilibration of the system. The methodology is illustrated using bicyclic peptide inhibitors of the serine protease plasma kallikrein.

The potent small molecule integrin antagonist THR-687 is a promising next-generation therapy for retinal vascular disorders.

Integrins are associated with various eye diseases such as diabetic retinopathy (DR) and wet age-related macular degeneration (AMD) and implicated in main pathologic disease hallmarks like neovascularization, inflammation, fibrosis and vascular leakage. Targeting integrins has the potential to attenuate these vision-threatening processes, independent of anti-vascular endothelial growth factor (VEGF) responsiveness. The current investigation characterized THR-687 as a novel pan RGD (arginylglycylaspartic acid) integrin receptor antagonist able to compete for binding with the natural ligand with nanomolar potency (e.g. αvß3 (IC50 of 4.4 ±â€¯2.7 nM), αvß5 (IC50 of 1.3 ±â€¯0.5 nM) and α5ß1 (IC50 of 6.8 ±â€¯3.2 nM)). THR-687 prevented the migration of human umbilical vein endothelial cells (HUVECs) into a cell-free area (IC50 of 258 ±â€¯113 nM) as well as vessel sprouting in an ex vivo mouse choroidal explant model (IC50 of 236 ±â€¯173 nM), and was able to induce the regression of pre-existing vascular sprouts. Moreover, combined intravitreal and intraperitoneal administration of THR-687 potently inhibited VEGF-induced leakage in the mouse retina. In addition, THR-687 injected intravitreally at 3 different dose levels (0.45 mg, 2.25 mg or 4.5 mg/eye) potently inhibited neovascularization-induced leakage in the cynomolgus laser-induced choroidal neovascularization (CNV) model. These data suggest that THR-687 is a promising drug candidate for the treatment of vision-threatening retinal vascular eye diseases such as DR and wet AMD.

Affinity determination of biomolecules: a kinetic model for the analysis of pre-equilibrium titration curves.

Equilibrium titration curves have been used to determine the affinity of biomolecules using a variety of platforms and technologies. Such measurements, however, are experimentally limited by the time that is needed for the system to reach the equilibrium, which may exceed the time during which the studied biomolecules are stable. We propose here a kinetic model to analyze titration curves obtained prior to full equilibration of the system.

Stable and Long-Lasting, Novel Bicyclic Peptide Plasma Kallikrein Inhibitors for the Treatment of Diabetic Macular Edema.

Plasma kallikrein, a member of the kallikrein-kinin system, catalyzes the release of the bioactive peptide bradykinin, which induces inflammation, vasodilation, vessel permeability, and pain. Preclinical evidence implicates the activity of plasma kallikrein in diabetic retinopathy, which is a leading cause of visual loss in patients suffering from diabetes mellitus. Employing a technology based on phage-display combined with chemical cyclization, we have identified highly selective bicyclic peptide inhibitors with nano- and picomolar potencies toward plasma kallikrein. Stability in biological matrices was either intrinsic to the peptide or engineered via the introduction of non-natural amino acids and nonpeptidic bonds. The peptides prevented bradykinin release in vitro, and in vivo efficacy was demonstrated in both a rat paw edema model and in rodent models of diabetes-induced retinal permeability. With a highly extended half-life of ∼40 h in rabbit eyes following intravitreal administration, the bicyclic peptides are promising novel agents for the treatment of diabetic retinopathy and diabetic macular edema.

Intravitreally Injected Fluid Dispersion: Importance of Injection Technique.

Purpose: The purpose of this study was to evaluate the dispersion of intravitreally injected solutions and investigate the influence of varying injection techniques. Methods: This was a prospective study using enucleated porcine eyes and ultra-high-resolution computed tomography (UHRCT) scanning to visualize iomeprol intravitreal dispersion. Sixty eyes were divided over 12 different groups according to the injection procedure: fast (2 seconds) or slow (10 seconds) injection speed and needle tip location (6- and 12-mm needle shaft insertion or premacular tip placement verified by indirect ophthalmoscopy). For each of these combinations, eyes were either injected with the combination of V20I (which is an analogue of ocriplasmin) and iomeprol or iomeprol alone. Distance to the macula and volume measurements were performed at 1, 2, 3, and 5 hours after injection. Results: The measured contrast bolus volume increases slowly over time to an average of 0.70 (P = 0.03), 1.04 (P = 0.006), and 0.79 (P = 0.0001) cm3 5 hours after the injection for the 6-mm needle shaft insertion, 12-mm needle shaft insertion, and premacular needle tip placement, respectively. The distance to the macular marker was significantly lower for premacular needle tip placement injections compared with 6- and 12-mm needle shaft insertion depths. Conclusions: Ultra-high-resolution computed tomography with three-dimensional reconstruction offers the possibility to study the dispersion of intravitreally injected solutions in a noninvasive manner. Intravitreal premacular solution delivery is possible with an indirect ophthalmoscope-guided injection technique and significantly reduces the time to reach the posterior pole in respect to 6- and 12-mm needle insertion depths. The speed of injection does not influence dispersion significantly.

Pharmacokinetics of ocriplasmin in vitreous.

PURPOSE: Ocriplasmin contains the active moiety of plasmin enzyme. At a physiologic pH, ocriplasmin is highly proteolytic and autolytic, limiting its duration of activity. Specific inhibitors of plasmin are present in the vitreous under normal and disease conditions and could affect its activity. Each may contribute to its mode of action. METHODS: Degradation characteristics were determined in porcine, human vitreous, and PBS under reducing conditions with different incubation periods between 0 and 24 hours on SDS-PAGE Tris-glycine gels. Residual activity was determined by spectrophotometry of p-nitroaniline release through hydrolysis of L-pyroglutamyl-L-phenylalanyl-L-lysine-p-nitroaniline hydrochloride. The presence of endogenous inactivators of ocriplasmin in human vitreous was determined in a series of vitreous samples using an ELISA specific for alpha(2)-antiplasmin, antithrombin, and antitrypsin. RESULTS: Degradation productions from autolysis are similar between vitreous and PBS with a significant prolongation of the effect in vitreous. Both follow a nonlinear pattern over time. The degradation corresponds best to a second-order kinetic process. The resulting rate constants were 207 ± 60 M(-1) s(-1) in PBS, 81 ± 15 M(-1) s(-1) in porcine vitreous, and 195 M(-1) s(-1) in human vitreous natural inhibitors were identified in samples of donor vitreous. Amounts differed significantly between samples, which may help explain the observed variability in human subjects. CONCLUSIONS: Ocriplasmin is autolytic in vitreous. Biologic activity extends to several days following injection. The exact duration will vary based on the presence and concentration of serine protease inhibitors.

Mechanism of inactivation of ocriplasmin in porcine vitreous.

Ocriplasmin, a 249-amino acid recombinant C-terminal fragment of human plasmin, has the potential to degrade, within the eye, the protein scaffold that links the vitreous to the retina. This may be beneficial to the treatment of a number of important ophthalmic indications, such as symptomatic vitreomacular adhesion. We demonstrate here that ocriplasmin used at therapeutically-relevant concentrations is inactivated in porcine vitreous through autolytic degradation. Autolytic cleavage occurs at a limited number of sites, primarily K156-E157, K166-V167 and R177-V178, which, as predicted, contain a positively-charged arginine or lysine residue at the P1 position. Our data also suggest that autolytic degradation requires at least local or partial unfolding of the protein.

Characterization of a stabilized form of microplasmin for the induction of posterior vitreous detachment.

PURPOSE: To investigate the stability and safety of a diluted acidified form of microplasmin and its ability to induce a posterior vitreous detachment (PVD) following intravitreal injection in post-mortem porcine eyes. METHODS: Microplasmin diluted in normal saline (NS) and balanced salt solution (BSS+) was assayed for residual activity by hydrolysis of the chromogenic substrate Glu-Phe-Lys-pNA. Residual activity on vitreous was determined by injecting aliquots of microplasmin reconstituted in balanced salt solution (BSS+) or normal saline (NS) kept at room temperature (RT) for up to 1 hr, then injected in aliquots of porcine vitreous and incubated for 2 hr at 37°C. The breakdown products were submitted to SDS Page electrophoresis and compared to determine the level of enzymatic activity. Pig eyes were incubated with graded concentrations of microplasmin 0.625, 1.25, or 2.50 mg/mL reconstituted in BBS+ or NS. Morphologic alterations and the ability to induce a PVD was assessed by light and electron microscopy. RESULTS: Microplasmin's enzymatic activity in an in vitro assay in BSS+ was 70% of its baseline value after 30 min, and about 50% after 60 min at RT. The corresponding effect on degradation of vitreous gel was 60 and 40% baseline at 30 and 60 min. There was no loss of activity in the microplasmin diluted in normal saline over this time period. Dilution of acidified microplasmin in normal saline did not lead to structural changes within the retina. A dose dependent PVD was observed in eyes treated with microplasmin diluted in NS. CONCLUSIONS: Acidified (stabilized) microplasmin has the same intraocular activity profile as microplasmin at a neutral pH. Better retention of activity at room temperature makes it a better candidate for use in clinical practice.

Selective inhibition of matrix metalloproteinase-14 blocks tumor growth, invasion, and angiogenesis.

Inhibition of specific matrix metalloproteinases (MMP) is an attractive noncytotoxic approach to cancer therapy. MMP-14, a membrane-bound zinc endopeptidase, has been proposed to play a central role in tumor growth, invasion, and neovascularization. Besides cleaving matrix proteins, MMP-14 activates proMMP-2 leading to an amplification of pericellular proteolytic activity. To examine the contribution of MMP-14 to tumor growth and angiogenesis, we used DX-2400, a highly selective fully human MMP-14 inhibitory antibody discovered using phage display technology. DX-2400 blocked proMMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions. The combination of potency, selectivity, and robust in vivo activity shows the potential of a selective MMP-14 inhibitor for the treatment of solid tumors.

Rapid kinetic-based screening of human Fab fragments.

Human antibodies able to bind with high affinity and specificity to numerous targets have been successfully identified from Fab phage display libraries. A key step in the library selection screening process is the early characterization of library isolates in order to determine which of these isolates to pursue further. Here we describe a Biacore assay that allows isolated clones expressed as soluble Fab fragments in E. coli to be screened and ranked based on their affinity against the target. The assay takes advantage of our ability to measure Fab concentrations in crude bacterial extracts in Biacore using very high density Protein A chips. The procedure allows up to 100 clones per week to be screened and permits the identification of a small number of high-affinity Fabs from a large batch obtained following library selection or affinity maturation.

Rapid generation of functional human IgG antibodies derived from Fab-on-phage display libraries.

We introduce a procedure for the rapid generation of fully human antibodies derived from "Fab-on-phage" display libraries. The technology is based on the compatibility of display vectors and IgG expression constructs, and allows reformatting of individual Fab clones to IgG, as well as reformatting of antibody repertoires. Examples of batch reformatting of an uncharacterized Fab repertoire and of a pool of Fabs, previously analyzed at the phage level, are presented. The average transient expression levels of the IgG constructs in HEK293T cells are above 10 microg/ml, allowing the use of conditioned media in functional assays without antibody purification. Furthermore, we describe a high-throughput purification method yielding IgG amounts sufficient for initial antibody characterization. Our technology allows the generation and production of antigen-specific complete human antibodies as fast or even faster than raising monoclonal antibodies by conventional hybridoma techniques.

Crystal structures of the Bacillus licheniformis BS3 class A beta-lactamase and of the acyl-enzyme adduct formed with cefoxitin.

The Bacillus licheniformis BS3 beta-lactamase catalyzes the hydrolysis of the beta-lactam ring of penicillins, cephalosporins, and related compounds. The production of beta-lactamases is the most common and thoroughly studied cause of antibiotic resistance. Although they escape the hydrolytic activity of the prototypical Staphylococcus aureus beta-lactamase, many cephems are good substrates for a large number of beta-lactamases. However, the introduction of a 7alpha-methoxy substituent, as in cefoxitin, extends their antibacterial spectrum to many cephalosporin-resistant Gram-negative bacteria. The 7alpha-methoxy group selectively reduces the hydrolytic action of many beta-lactamases without having a significant effect on the affinity for the target enzymes, the membrane penicillin-binding proteins. We report here the crystallographic structures of the BS3 enzyme and its acyl-enzyme adduct with cefoxitin at 1.7 A resolution. The comparison of the two structures reveals a covalent acyl-enzyme adduct with perturbed active site geometry, involving a different conformation of the omega-loop that bears the essential catalytic Glu166 residue. This deformation is induced by the cefoxitin side chain whose position is constrained by the presence of the alpha-methoxy group. The hydrolytic water molecule is also removed from the active site by the 7beta-carbonyl of the acyl intermediate. In light of the interactions and steric hindrances in the active site of the structure of the BS3-cefoxitin acyl-enzyme adduct, the crucial role of the conserved Asn132 residue is confirmed and a better understanding of the kinetic results emerges.