Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire.

Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition

Exploring the Dynamic Range of the Kinetic Exclusion Assay in Characterizing Antigen-Antibody Interactions

Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA) by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (KD values) spanning six orders of magnitude, from approximately 100 fM to 100 nM. Using a previously calibrated antibody concentration and working in a suitable concentration range, we show that a single experiment can yield accurate and precise values for both the apparent KD and the apparent active concentration of the antigen, thereby increasing the information content of an assay and decreasing sample consumption. Orthogonal measurements obtained on Biacore and Octet label-free biosensor platforms further validated our KinExA-derived affinity and active concentration determinations. We obtained excellent agreement in the apparent affinities obtained across platforms and within the KinExA method irrespective of the assay orientation employed or the purity of the recombinant or native antigens

Appetite Enhancement and Weight Gain by Peripheral Administration of TrkB Agonists in Non-Human Primates

Loss of function mutations in the receptor tyrosine kinase TrkB pathway resulted in hyperphagia and morbid obesity in human and rodents. Conversely, peripheral or central stimulation of TrkB by its natural ligands BDNF or NT4 reduced body weight and food intake in mice, supporting the idea that TrkB is a key anorexigenic signal downstream of the melanocortin-4 receptor (Mc4r) system. Here we show that in non-human primates TrkB agonists were anorexigenic when applied centrally, but surprisingly orexigenic, leading to gain in appetite, body weight, fat deposits and serum leptin levels, when given peripherally. The orexigenic and pro-obesity effects of peripherally administered TrkB agonists appear to be dose dependent, not associated with fluid retention nor with evidence of receptor down regulation. Our findings revealed that TrkB signaling exerts dual control on energy homeostasis in the primates that could be targeted for the treatment of either wasting disorders or obesity

Determining the Binding Affinity of Therapeutic Monoclonal Antibodies towards Their Native Unpurified Antigens in Human Serum

<div><p>Monoclonal antibodies (mAbs) are a growing segment of therapeutics, yet their <i>in vitro</i> characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen’s endogenous concentration, by combining the kinetic exclusion assay and Biacore’s calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9), progranulin (PGRN), and fatty acid binding protein (FABP4). We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling. </p> </div

Influence of pH on the apparent affinity (top) and apparent activity (bottom) of different mAbs towards their purified recombinant antigens.

<p>The K_D values and mAb activities for each interaction were obtained from a single curve KinExA analysis performed at different pH values that spanned the pH range encountered during serum experiments. The bars represent the best fit values and the error bars represent the 95% confidence interval. The arrows indicate the trend observed with increasing pH and the range of best fit values for K_D and activity. Only sweet spot experiments enabled a determination of both the K_D and the mAb activity (no mAb activity is reported for 19F7 and 33B12 because those curves were mostly K_D-controlled). The antigen concentrations used were 128 pM rhPCSK9, 42 pM rhPGRN, 21 pM rhPGRN, 100 pM rhFABP4, and 1nM rhFABP4 (from left to right).</p

KinExA-determined affinities of three mAbs (J17, 2B2, and 33B12) towards their purified recombinant antigens (left) and native antigens in human serum (right).

<p>The data are presented in the same way as in Figure 2.</p

KinExA-determined apparent affinities for four mAbs (J16, J17, 19F7, and 21B8) towards their purified recombinant antigens (left) and their serum antigens (right) at stable, non-neutral pH values.

<p>Titration curves are presented as described in Figure 2.</p

Kinetic analysis of anti-PGRN mAbs 2B2 (left) and 19F7 (right) in TBST buffers at different pH values.

<p>The data were collected on a ProteOn XPR36 biosensor by injecting a dilution series of rhPGRN (0.8, 2.4, 7.1, 21.3, and 64 nM) over amine-coupled mAbs. Double-referenced sensorgrams (colored lines) obtained from two ligand channels per mAb were fit globally to a 1:1 binding model with mass transport limitation (fit shown in black); the results from one channel per mAb is shown along with the global best fit K_D.</p