Biophysical characterization of PVR family interactions and therapeutic antibody recognition to TIGIT.

The clinical successes of immune checkpoint blockade have invigorated efforts to activate T cell-mediated responses against cancer. Targeting members of the PVR family, consisting of inhibitory receptors TIGIT, CD96, and CD112R, has been an active area of clinical investigation. In this study, the binding interactions and molecular assemblies of the PVR family receptors and ligands have been assessed in vitro. Furthermore, the anti-TIGIT monoclonal antibody BMS-986207 crystal structure in complex with TIGIT was determined and shows that the antibody binds an epitope that is commonly targeted by the CD155 ligand as well as other clinical anti-TIGIT antibodies. In contrast to previously proposed models, where TIGIT outcompetes costimulatory receptor CD226 for binding to CD155 due to much higher affinity (nanomolar range), our data rather suggest that PVR family members all engage in interactions with relatively weak affinity (micromolar range), including TIGIT and CD155 interactions. Thus, TIGIT and other PVR inhibitory receptors likely elicit immune suppression via increased surface expression rather than inherent differences in affinity. This work provides an improved foundational understanding of the PVR family network and mechanistic insight into therapeutic antibody intervention.

Biacore surface matrix effects on the binding kinetics and affinity of an antigen/antibody complex.

To characterize a proprietary therapeutic monoclonal antibody (mAb) candidate, a rigorous biophysical study consisting of 53 Biacore and kinetic exclusion assay (KinExA) experiments was undertaken on the therapeutic mAb complexing with its target antigen. Unexpectedly, the observed binding kinetics depended on the chip used, suggesting that the negatively charged carboxyl groups on CM5, CM4, and C1 chips were adversely affecting the Biacore kinetic results. To study this hypothesis, Biacore solution-phase and KinExA equilibrium titrations, as well as KinExA kinetic measurements, were performed to establish accurate values for the affinity and kinetic rate constants of the binding reaction between antigen and mAb. The results revealed that as the negative charge on the biosensor surface decreased, the binding kinetics and K(D) approached the accurate binding parameters more closely when measured in solution. Two potential causes for the artifactual Biacore surface-based measurements are (i) steric hindrance of antigen binding arising from an interaction of the negatively charged carboxymethyldextran matrix with the mAb, which is a highly basic protein with a pI of 9.4, and (ii) an electrostatic repulsion between the negatively charged antigen and the carboxymethyldextran matrix. Importantly, simple diagnostic tests can be performed early in the measurement process to identify these types of matrix-mediated artifacts.

Miscalculating Equilibrium Constants for Multivalent Protein Complexes: Site-Binding Concentration or Protein Molecular Concentration?

Because the authors continue to note instances in the scientific literature of failure to use the correct receptor binding site concentration for determining binding constants, herein we discuss the fundamental concepts that need to be considered to determine correct binding constants or conversely calculate accurate reactant concentrations with known equilibrium constants. We also show the derivation and analytical solutions of the cubic and quartic equations that give the exact free ligand concentration in bivalent and trivalent receptor systems at equilibrium as a function of the macroscopic equilibrium dissociation constants and the total concentrations of ligand and multivalent protein. These equations and solutions strongly reemphasize the critical dependency of deriving the correct concentrations of bound or free ligand and multivalent protein on the choice of the correct concentration basis for the multivalent protein, which is in turn dependent upon the type of equilibrium constant used. These results demonstrate the importance of choosing the proper multivalent protein concentration for the determination of either valid microscopic or valid macroscopic equilibrium dissociation constants from binding isotherms of ligand-multivalent protein complexes.

A rigorous multiple independent binding site model for determining cell-based equilibrium dissociation constants.

A new 4-parameter nonlinear equation based on the standard multiple independent binding site model (MIBS) is presented for fitting cell-based ligand titration data in order to calculate the ligand/cell receptor equilibrium dissociation constant and the number of receptors/cell. The most commonly used linear (Scatchard Plot) or nonlinear 2-parameter model (a single binding site model found in commercial programs like Prism(R)) used for analysis of ligand/receptor binding data assumes only the K(D) influences the shape of the titration curve. We demonstrate using simulated data sets that, depending upon the cell surface receptor expression level, the number of cells titrated, and the magnitude of the K(D) being measured, this assumption of always being under K(D)-controlled conditions can be erroneous and can lead to unreliable estimates for the binding parameters. We also compare and contrast the fitting of simulated data sets to the commonly used cell-based binding equation versus our more rigorous 4-parameter nonlinear MIBS model. It is shown through these simulations that the new 4-parameter MIBS model, when used for cell-based titrations under optimal conditions, yields highly accurate estimates of all binding parameters and hence should be the preferred model to fit cell-based experimental nonlinear titration data.

Detection of high- and low-affinity antibodies against a human monoclonal antibody using various technology platforms.

The effect of monoclonal antibody (mAb) affinity on the detection limit of enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and electrochemiluminescence (ECL) methods was evaluated using a panel of murine mAbs with affinities ranging from 0.057 to 340 nM. M1 and M7 are anti-idiotypic mAbs against a human mAb, ABX10, with dissociation equilibrium constant (KD) values of 0.057 and 7.2 nM, respectively. HP6030 and HP6002 are anti human IgG mAbs with KD values of 30 and 340 nM, respectively. The limit of detection (LOD) for these mAbs was determined using ELISA, SPR, and ECL technologies and was generally correlated with the rank order of their affinities. The LODs for M1, M7, HP6030, and HP6002 by ELISA were 17 +/- 13, 26,000 +/- 9,020, 344,000 +/- 271,000, and 792,000 +/- 1,050,000 ng/ml, respectively. According to an industry-suggested detection limit of 500 ng/ml, the ELISA was not sensitive enough for detecting M7, HP6030, and HP6002, demonstrating its limitation for detection of low- affinity mAbs. The SPR method lowered the LOD for M7 to 3,900 ng/ml, which was above the industry requirement. The ECL method lowered the LOD for all antibodies tested. Importantly, the ECL method lowered the LOD for M7 to 570 +/- 370 ng/ml, which is close to the industry requirement. Since the ECL method had demonstrated a high serum tolerance, its detection capability may be improved by using a higher percentage of serum in the assay matrix. Although a hook effect was observed with ECL methods, the methods could still detect anti-drug antibody (ADA) concentrations greater than 1 mg/ml, which minimizes concerns that high-titer ADA responses could be missed. The results demonstrated the superiority of an ECL method in detecting high- and low- affinity antibodies when compared to the ELISA and SPR methods.

A potent anti-HB-EGF monoclonal antibody inhibits cancer cell proliferation and multiple angiogenic activities of HB-EGF.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family and has a variety of physiological and pathological functions. Modulation of HB-EGF activity might have a therapeutic potential in the oncology area. We explored the therapeutic possibilities by characterizing the in vitro biological activity of anti-HB-EGF monoclonal antibody Y-142. EGF receptor (EGFR) ligand and species specificities of Y-142 were tested. Neutralizing activities of Y-142 against HB-EGF were evaluated in EGFR and ERBB4 signaling. Biological activities of Y-142 were assessed in cancer cell proliferation and angiogenesis assays and compared with the anti-EGFR antibody cetuximab, the HB-EGF inhibitor CRM197, and the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab. The binding epitope was determined with alanine scanning. Y-142 recognized HB-EGF as well as the EGFR ligand amphiregulin, and bound specifically to human HB-EGF, but not to rodent HB-EGF. In addition, Y-142 neutralized HB-EGF-induced phosphorylation of EGFR and ERBB4, and blocked their downstream ERK1/2 and AKT signaling. We also found that Y-142 inhibited HB-EGF-induced cancer cell proliferation, endothelial cell proliferation, tube formation, and VEGF production more effectively than cetuximab and CRM197 and that Y-142 was superior to bevacizumab in the inhibition of HB-EGF-induced tube formation. Six amino acids in the EGF-like domain were identified as the Y-142 binding epitope. Among the six amino acids, the combination of F115 and Y123 determined the amphiregulin cross-reactivity and that F115 accounted for the species selectivity. Furthermore, it was suggested that the potent neutralizing activity of Y-142 was derived from its recognition of R142 and Y123 and its high affinity to HB-EGF. Y-142 has a potent HB-EGF neutralizing activity that modulates multiple biological activities of HB-EGF including cancer cell proliferation and angiogenic activities. Y-142 may have a potential to be developed into a therapeutic agent for the treatment of HB-EGF-dependent cancers.

A strategic and systematic approach for the determination of biosensor regeneration conditions.

Determining the optimal conditions for surface regeneration is fundamental for performance of efficient and robust protein-protein interaction kinetic studies. We devised a systematic methodology comprised of an automated seven-cycle analyte and buffer injection Biacore scheme and data interpretation algorithm. The efficiency and utility is illustrated using an antigen/monoclonal antibody interaction that required ultimately six pulses of acid for regeneration. This technique has broad applicability to any biosensor assay that requires regeneration of a surface.

Screening antibody-antigen interactions in parallel using Biacore A100.

Label-free optical biosensor technology has become a standard tool for characterizing monoclonal antibodies for therapeutic and diagnostic applications. The availability of high-quality binding data at an early stage greatly improves the ability to select antibodies for further development. This article shows how Biacore A100, a protein interaction array system, is capable of providing high-quality data with increased throughput. In a 12-h automated run, we analyzed 386 crude hybridoma samples to identify those with the desired kinetic profiles. Selected antibodies were further characterized by higher resolution analysis, and binding interactions were studied under a range of buffer conditions. We demonstrate how this new parallel processing system significantly expands the throughput of protein interaction analysis while maintaining data quality.

Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods.

Two biophysical methods, Biacore and KinExA, were used to kinetically and thermodynamically characterize high-affinity antigen/antibody complexes. Three to five independent experiments were performed on each platform with three different antigen/antibody complexes possessing nanomolar to picomolar equilibrium dissociation constants. By monitoring the dissociation phase on Biacore for 4 h, we were able to measure dissociation rate constants (kd) on the order of 1 x 10(-5)s(-1). To characterize high-affinity interactions by KinExA, samples needed to be equilibrated for up to 35 h to reach equilibrium. In the end, we show that similar kinetic rate constants and affinities were determined with both solution-phase and solid-phase methodologies. These results help further validate both interaction technologies and illustrate their suitability for characterizing extremely high-affinity interactions.