Albumin influences leucocyte FcRn expression in the early days of kidney transplantation.

FcRn, a receptor originally known for its involvement in IgG and albumin transcytosis and recycling, is also important in the establishment of the innate and adaptive immune response. Dysregulation of the immune response has been associated with variations in FcRn expression, as observed in cancer. Recently, a link between autophagy and FcRn expression has been demonstrated. Knowing that autophagy is strongly involved in the development of reperfusion injury in kidney transplantation and that albuminemia is transiently decreased in the first 2 weeks after transplantation, we investigated variations in FcRn expression after kidney transplantation. We monitored FcRn levels by flow cytometry in leukocytes from 25 renal transplant patients and considered parameters such as albumin concentrations, estimated glomerular filtration rate, serum creatinine, serum IgG levels, and ischaemia/reperfusion time. Two groups of patients could be distinguished according to their increased or non-increased FcRn expression levels between days 2 and 6 (d2-d6) post-transplantation. Leukocyte FcRn expression at d2-d6 was correlated with albumin concentrations at d0-d2. These results suggest that albumin concentrations at d0-d2 influence FcRn expression at d2-d6, raising new questions about the mechanisms underlying these original observations.

Harnessing Fc/FcRn Affinity Data from Patents with Different Machine Learning Methods.

Monoclonal antibodies are biopharmaceuticals with a very long half-life due to the binding of their Fc portion to the neonatal receptor (FcRn), a pharmacokinetic property that can be further improved through engineering of the Fc portion, as demonstrated by the approval of several new drugs. Many Fc variants with increased binding to FcRn have been found using different methods, such as structure-guided design, random mutagenesis, or a combination of both, and are described in the literature as well as in patents. Our hypothesis is that this material could be subjected to a machine learning approach in order to generate new variants with similar properties. We therefore compiled 1323 Fc variants affecting the affinity for FcRn, which were disclosed in twenty patents. These data were used to train several algorithms, with two different models, in order to predict the affinity for FcRn of new randomly generated Fc variants. To determine which algorithm was the most robust, we first assessed the correlation between measured and predicted affinity in a 10-fold cross-validation test. We then generated variants by in silico random mutagenesis and compared the prediction made by the different algorithms. As a final validation, we produced variants, not described in any patents, and compared the predicted affinity with the experimental binding affinities measured by surface plasmon resonance (SPR). The best mean absolute error (MAE) between predicted and experimental values was obtained with a support vector regressor (SVR) using six features and trained on 1251 examples. With this setting, the error on the log(KD) was less than 0.17. The obtained results show that such an approach could be used to find new variants with better half-life properties that are different from those already extensively used in therapeutic antibody development.

Finding the Right Heavy Chains for Immunostimulatory Antibodies.

For twelve years, the oncology field has been revolutionized by antibodies targeting immune checkpoints. They must be considered as a heterogenous family of immunostimulatory antibodies displaying very different mechanisms of action, not only depending on the target or on the cells expressing it, but also on the IgG subclass or IgG variant that has been chosen. To dissect this complex landscape, the clinical experience has been confronted with a precise analysis of the heavy chain isotypes, referred as new Ge nomenclature. For antibodies targeting inhibitory receptors, anti-CTLA-4 antibodies (whose main effect is to kill regulatory T cells) will be distinguished from anti-PD-1 antibodies and other true antagonistic antibodies. Antibodies targeting ligands of inhibitory receptors (PD-L1, CD47) represent another different category, due to the antigen expression on tumors and a possible beneficial killing effect. The case of agonistic antibodies targeting lymphocyte activatory receptors, such as CD40 or 4-1BB, is still another "under construction" category because these products are less advanced in their clinical development. Altogether, it appears that choosing the right heavy chain is crucial to obtain the desired pharmacological effect in patients.

Monoclonal Antibody Engineering and Design to Modulate FcRn Activities: A Comprehensive Review.

Understanding the biological mechanisms underlying the pH-dependent nature of FcRn binding, as well as the various factors influencing the affinity to FcRn, was concurrent with the arrival of the first recombinant IgG monoclonal antibodies (mAbs) and IgG Fc-fusion proteins in clinical practice. IgG Fc-FcRn became a central subject of interest for the development of these drugs for the comfort of patients and good clinical responses. In this review, we describe (i) mAb mutations close to and outside the FcRn binding site, increasing the affinity for FcRn at acidic pH and leading to enhanced mAb half-life and biodistribution, and (ii) mAb mutations increasing the affinity for FcRn at acidic and neutral pH, blocking FcRn binding and resulting, in vivo, in endogenous IgG degradation. Mutations modifying FcRn binding are discussed in association with pH-dependent modulation of antigen binding and (iii) anti-FcRn mAbs, two of the latest innovations in anti-FcRn mAbs leading to endogenous IgG depletion. We discuss the pharmacological effects, the biological consequences, and advantages of targeting IgG-FcRn interactions and their application in human therapeutics.

A New in Silico Antibody Similarity Measure Both Identifies Large Sets of Epitope Binders with Distinct CDRs and Accurately Predicts Off-Target Reactivity.

Developing a therapeutic antibody is a long, tedious, and expensive process. Many obstacles need to be overcome, such as biophysical properties (issues of solubility, stability, weak production yields, etc.), as well as cross-reactivity and subsequent toxicity, which are major issues. No in silico method exists today to solve such issues. We hypothesized that if we were able to properly measure the similarity between the CDRs of antibodies (Ab) by considering not only their evolutionary proximity (sequence identity) but also their structural features, we would be able to identify families of Ab recognizing similar epitopes. As a consequence, Ab within the family would share the property to recognize their targets, which would allow (i) to identify off-targets and forecast the cross-reactions, and (ii) to identify new Ab specific for a given target. Testing our method on 238D2, an antagonistic anti-CXCR4 nanobody, we were able to find new nanobodies against CXCR4 and to identify influenza hemagglutinin as an off-target of 238D2.

Association of IgG1 Antibody Clearance with FcγRIIA Polymorphism and Platelet Count in Infliximab-Treated Patients.

The FcγRIIA/CD32A is mainly expressed on platelets, myeloid and several endothelial cells. Its affinity is considered insufficient for allowing significant binding of monomeric IgG, while its H131R polymorphism (histidine > arginine at position 131) influences affinity for multimeric IgG2. Platelet FcγRIIA has been reported to contribute to IgG-containing immune-complexe clearance. Given our finding that platelet FcγRIIA actually binds monomeric IgG, we investigated the role of platelets and FcγRIIA in IgG antibody elimination. We used pharmacokinetics analysis of infliximab (IgG1) in individuals with controlled Crohn's disease. The influence of platelet count and FcγRIIA polymorphism was quantified by multivariate linear modelling. The infliximab half-life increased with R allele number (13.2, 14.4 and 15.6 days for HH, HR and RR patients, respectively). It decreased with increasing platelet count in R carriers: from ≈20 days (RR) and ≈17 days (HR) at 150 × 109/L, respectively, to ≈13 days (both HR and RR) at 350 × 109/L. Moreover, a flow cytometry assay showed that infliximab and monomeric IgG1 bound efficiently to platelet FcγRIIA H and R allotypes, whereas panitumumab and IgG2 bound poorly to the latter. We propose that infliximab (and presumably any IgG1 antibody) elimination is partly due to an unappreciated mechanism dependent on binding to platelet FcγRIIA, which is probably tuned by its affinity for IgG2.

MAbTope: A Method for Improved Epitope Mapping.

Abs are very efficient drugs, ∼70 of them are already approved for medical use, over 500 are in clinical development, and many more are in preclinical development. One important step in the characterization and protection of a therapeutic Ab is the determination of its cognate epitope. The gold standard is the three-dimensional structure of the Ab/Ag complex by crystallography or nuclear magnetic resonance spectroscopy. However, it remains a tedious task, and its outcome is uncertain. We have developed MAbTope, a docking-based prediction method of the epitope associated with straightforward experimental validation procedures. We show that MAbTope predicts the correct epitope for each of 129 tested examples of Ab/Ag complexes of known structure. We further validated this method through the successful determination, and experimental validation (using human embryonic kidney cells 293), of the epitopes recognized by two therapeutic Abs targeting TNF-α: certolizumab and golimumab.

Obinutuzumab: what is there to learn from clinical trials?

Obinutuzumab (OBZ) is a recombinant type II anti-CD20 and immunoglobulin G1 Fc-optimized monoclonal antibody (mAb), recently approved in chronic lymphocytic leukemia (CLL; B-cell CLL) and follicular lymphoma (FL). Rituximab (RTX) is frequently considered as its "ancestor" and OBZ clinical development was justified by the importance of FcγRIIIA-mediated mechanisms in RTX clinical activity. However, RTX differs from OBZ in 2 critical independent properties: being a type I anti-CD20 mAb and not being Fc-optimized. Moreover, the use of a different dosing regimen for RTX and OBZ further complicates any interpretation of clinical results. The results obtained for OBZ in CLL provide new arguments for FcγRIIIA-mediated mechanisms when the target antigen is expressed at a low density. Results of OBZ in FL confirm the interest for FcγRIIIA-mediated mechanisms, with some limitations, some of them being possibly due to lack of OBZ-induced complement activation. The situation in diffuse large B-cell lymphoma is deceiving, as the possible gains of activity of OBZ appear to be annihilated by the lack of complement activation. Although RTX was by chance an anti-CD20 mAb with equilibrated pharmacodynamic properties, the reinforcement of some of these properties, which has been done at the expense of complement activation, has conferred an advantage in some B-cell disorders while restricting OBZ indications. The OBZ story nicely demonstrates that the future of naked mAbs is to design agents with optimized and tailored properties, and that this must be done step by step, with a full clinical validation.

Crucial Role for Immune Complexes but Not FcRn in Immunization against Anti-TNF-α Antibodies after a Single Injection in Mice.

The immunogenicity of infliximab and adalimumab is a major concern because patients may develop Abs also called antidrug Abs (ADA), directed against these anti-TNF-α Abs after just a few weeks of treatment. These ADAs can lead to a decrease in biologic concentration, which is associated with lower treatment efficacy. Our aim was to study the involvement of immune complexes and neonatal Fc receptor (FcRn) in the emergence of ADAs in the case of anti-TNF-α Abs. Wild type and FcRn knockout mice were injected once with either infliximab or adalimumab, alone or preincubated with TNF-α. Adalimumab cross-reacts with murine TNF-α whereas infliximab is species specific. When injected alone, only adalimumab elicited a humoral response. By preforming immune complexes with TNF-α, an anti-infliximab response was elicited. Surprisingly, both wild type and FcRn knockout mice were able to mount an immune response against anti-TNF-α Abs, suggesting that immune complexes are a major determinant of this immunization.

Inhibitors and Antibody Fragments as Potential Anti-Inflammatory Therapeutics Targeting Neutrophil Proteinase 3 in Human Disease.

Proteinase 3 (PR3) has received great scientific attention after its identification as the essential antigenic target of antineutrophil cytoplasm antibodies in Wegener's granulomatosis (now called granulomatosis with polyangiitis). Despite many structural and functional similarities between neutrophil elastase (NE) and PR3 during biosynthesis, storage, and extracellular release, unique properties and pathobiological functions have emerged from detailed studies in recent years. The development of highly sensitive substrates and inhibitors of human PR3 and the creation of PR3-selective single knockout mice led to the identification of nonredundant roles of PR3 in cell death induction via procaspase-3 activation in cell cultures and in mouse models. According to a study in knockout mice, PR3 shortens the lifespan of infiltrating neutrophils in tissues and accelerates the clearance of aged neutrophils in mice. Membrane exposure of active human PR3 on apoptotic neutrophils reprograms the response of macrophages to phagocytosed neutrophils, triggers secretion of proinflammatory cytokines, and undermines immune silencing and tissue regeneration. PR3-induced disruption of the anti-inflammatory effect of efferocytosis may be relevant for not only granulomatosis with polyangiitis but also for other autoimmune diseases with high neutrophil turnover. Inhibition of membrane-bound PR3 by endogenous inhibitors such as the α-1-protease inhibitor is comparatively weaker than that of NE, suggesting that the adverse effects of unopposed PR3 activity resurface earlier than those of NE in individuals with α-1-protease inhibitor deficiency. Effective coverage of PR3 by anti-inflammatory tools and simultaneous inhibition of both PR3 and NE should be most promising in the future.

IgG1 Allotypes Influence the Pharmacokinetics of Therapeutic Monoclonal Antibodies through FcRn Binding.

Because IgG1 allotypes might have different half-lives, their influence on infliximab (G1m17,1 allotype) pharmacokinetics was investigated in a group of spondyloarthritis patients. Infliximab was found to have a shorter half-life in patients homozygous for the G1m17,1 allotypes than in those carrying the G1m3 with no G1m1 (G1m3,-1) allotype. Because the neonatal FcR (FcRn) is involved in the pharmacokinetics of mAbs, the interaction of different IgG1 allotypes with FcRn was examined using cellular assays and surface plasmon resonance. G1m17,1 mAbs, such as infliximab and rituximab, were shown to bind more efficiently to FcRn and to be transcytosed better than the G1m3,-1 mAb cetuximab, which explains why infliximab is a better competitor for endogenous IgG1 in G1m3,-1 allotype-bearing patients. A set of four allotype variants of adalimumab (G1m17,1; G1m17,-1; G1m3,1; and G1m3,-1) was also tested for its binding to FcRn, revealing that the G1m3,1 variant, not present in commercial mAbs, binds more efficiently to FcRn and is transcytosed better than the other three variants, all of which are found in therapeutic mAbs.

Immunoassays for Measuring Serum Concentrations of Monoclonal Antibodies and Anti-biopharmaceutical Antibodies in Patients.

Monoclonal antibodies (mAbs) may be used as biopharmaceuticals to treat various diseases, ranging from oncology to inflammatory and cardiovascular affections. Trustworthy analytical methods are necessary to study their pharmacokinetics, both during their development and in post-marketing studies. Because biopharmaceuticals are macromolecules, ligand-binding assays (both immunoassays and bioassays) are methods of choice to measure their concentrations. Immunoassays are based on the capture of biopharmaceuticals by their target, which may be a circulating or membrane antigen or by an antibody recognizing their structure. Bioassays measure the activity of the biopharmaceutical in a specific in vitro test. A number of techniques have been reported, but their limits of detection and quantification vary widely. Anti-drug antibodies (ADA) against biopharmaceuticals are often formed and sometimes interfere with clinical efficacy. Accurate and reliable detection of ADA is therefore necessary. Binding of ADA is dependent on affinity and avidity, which makes quantification challenging. In this review, we discuss the benefits and limitations of each method to determine mAb levels and carefully compare ADA assays.

Pharmacokinetics and concentration-effect relationship of adalimumab in rheumatoid arthritis.

AIMS: This study aimed at describing adalimumab pharmacokinetics (PK) and the concentration-effect relationship of adalimumab using pharmacokinetic-pharmacodynamic (PK-PD) modelling in patients with rheumatoid arthritis (RA). METHODS: Adalimumab PK and PK-PD data were obtained from a multicentric observational study. Adalimumab (40 mg) was administered subcutaneously every other week, and its pharmacokinetics was described using a one-compartment model. The relationship between adalimumab concentration and C-reactive protein (CRP) concentration was described using an indirect response model with inhibition of CRP input, whereas the relationship between adalimumab concentration and disease activity score in 28 joints (DAS28) was described using a direct inhibition model. Dose regimens that included a loading dose of adalimumab were simulated. RESULTS: Thirty patients treated for RA were analysed. The following pharmacokinetic and PK-PD parameters were estimated (interidividual coefficient of variation): apparent volume of distribution (Vd /F) = 10.8 l (92%); apparent clearance (CL/F) = 0.32 l day(-1) (17%); first-order absorption rate (ka ) = 0.28 day(-1) ; CRP input (kin ) = 22.0 mg l(-1) day(-1) (65%); adalimumab concentration leading to a 50% decrease in kin (C50 ) = 3.6 mg l(-1) (88%); baseline DAS28 (DAS0 ) = 5.5 mg l(-1) (11%); and adalimumab concentration leading to 50% decrease of DAS0 (IC50 ) = 11.0 mg l(-1) (71%). Simulations showed that a 160 mg loading dose should reduce the time to reach efficacy in terms of both CRP and DAS28 after the first injection. CONCLUSIONS: This is the first study to describe adalimumab pharmacokinetics and the concentration-effect relationship in RA. A 160 mg loading dose may lead to an increased benefit from treatment in RA patients.

Influence of FcγRIIIA genetic polymorphism on T-lymphocyte depletion induced by rabbit antithymocyte globulins in kidney transplant patients.

INTRODUCTION: Polyclonal antithymocyte globulins (ATG) have been used in transplantation for several decades, but the sources of the interindividual variability of their effect are poorly understood. An influence of the FCGR3A-158V/F genetic polymorphism on the horse ATG concentration-effect relationship was reported in kidney transplant patients. The objective of the present study was to confirm the influence of the FCGR3A polymorphism on the extent of lymphocyte depletion in kidney transplant patients treated with rabbit antithymocyte globulin (r-ATG). MATERIALS AND METHODS: Of the 194 transplant patients treated with r-ATG between 1998 and 2002 in our institution, 69 patients were eligible and included in this retrospective study. Biomarkers of response were CD3 and CD4 counts. Dose-effect data were analyzed using a population approach, and a two-compartment turnover model with stimulation of lymphocyte 'output'. Since r-ATG concentrations were not available, a K-PD model was used. The influence of FCGR3A genotype on estimated parameters was investigated. RESULTS: The r-ATG infusion rate leading to a 50% stimulation of CD3+ output (EDK(50)), which is inversely related to patient sensitivity to r-ATG treatment, decreased with the number of V alleles (P=0.0016). CONCLUSION: The genetic polymorphism of FCGR3A influences r-ATG effect on CD3 count in kidney transplant patients, those with the V allele being more sensitive to antilymphocyte serum. These results also suggest that r-ATG act, at least in part, by antibody-dependent cellular cytotoxicity.

Relationship between inflammation and infliximab pharmacokinetics in rheumatoid arthritis.

AIMS: Infliximab, an anti-tumour necrosis factor-α monoclonal antibody, is indicated in rheumatoid arthritis (RA). Our objective was to evaluate the influence of the sources of infliximab pharmacokinetic variability in RA. METHODS: Eighty-four patients treated with infliximab for RA were included in a prospective noncomparative study. They were analysed between two consecutive infliximab infusions. Infliximab concentrations were measured before the infusion, 2 h, 1 and 4 weeks after the infusion and immediately before the next infusion. Infliximab concentrations were described using a two-compartment population pharmacokinetic model. RESULTS: The mean (interindividual standard deviation) estimated central volume of distribution was 2.3 l (36%) and systemic clearance was 0.019 l h(-1) (37%). The central volume of distribution increased with bodyweight; it was doubled between 50 and 90 kg. Systemic clearance increased with pre-infusion C-reactive protein concentration by 20%, varying from 3 to 14 mg l(-) 1, and was decreased by 30% when methotrexate was coadministered. CONCLUSIONS: The influence of methotrexate and inflammation on infliximab clearance suggests that individual adjustment of infliximab doses according to disease activity may be useful in RA.

Detection of antiranibizumab antibodies among patients with exudative age-related macular degeneration.

PURPOSE: The aim of this study was to detect immune responses induced by intravitreal injection (IVT) of ranibizumab in patients with exudative age-related macular degeneration (AMD) in real life conditions. METHODS: An ELISA protocol from blood samples, following 2 different steps, was used to detect antibodies directed against the variable regions of ranibizumab. RESULTS: Among 91 patients included, 46 received more than 10 IVTs, 36 had received 10 IVTs or fewer, and 9 were treatment naïve. Specific antiranibizumab immunoglobulins G were detected in 14/82 treated patients (17.1%). No immunization was detected among naïve patients. For patients with 10 or fewer previous IVTs, immunization against ranibizumab was detected in 4/36 patients (11.1%) whereas immunization was observed in 10/46 patients (21.7%) with more IVTs (p = 0.20). CONCLUSIONS: Immunization against ranibizumab can be detected in 17% of treated patients. Further clinical studies are needed to investigate the relationship between specific immunization to anti-vascular endothelial growth factor antibodies and response or resistance to ranibizumab treatments.

[Biotherapies, immunotherapies, targeted therapies, biopharmaceuticals which word should be used?]. / Biothérapies, immunothérapies, thérapies ciblées, biomédicaments - De quoi faut-il parler ?

The ability to accurately describe and name medical advances is a prerequisite to foster public debates with scientists and physicians, and favour faith over fear among patients and citizens. Therapeutic antibodies are a good example of a medical breakthrough which has met with considerable clinical success, and which terminology has changed over the years. If the appellation serotherapy was appropriate a century ago, it has become obsolete. Recent names such as biotherapy, immunotherapy, targeted therapy, biopharmaceuticals have been introduced and are now commonly used, each of those can apply to therapeutic antibodies. It is thus interesting to question the real meaning of these different appellations. Our goal in this manuscript is to analyse the genesis of these terms but also to suggest how to simplify the terminology: biotherapy or targeted therapy need to be eliminated, as well as immunotherapy when communicating with non scientific public. It is recommended to favour the term biopharmaceuticals (biomédicaments in French), which clearly indicates the origin of these molecules, intermediate between chemical drugs and living biologics, whose borders need to be accurately defined also.

Bispecific antibodies tethering innate receptors induce human tolerant-dendritic cells and regulatory T cells.

There is an urgent need for alternative therapies targeting human dendritic cells (DCs) that could reverse inflammatory syndromes in many autoimmune and inflammatory diseases and organ transplantations. Here, we describe a bispecific antibody (bsAb) strategy tethering two pathogen-recognition receptors at the surface of human DCs. This cross-linking switches DCs into a tolerant profile able to induce regulatory T-cell differentiation. The bsAbs, not parental Abs, induced interleukin 10 and transforming growth factor ß1 secretion in monocyte-derived DCs and human peripheral blood mononuclear cells. In addition, they induced interleukin 10 secretion by synovial fluid cells in rheumatoid arthritis and gout patients. This concept of bsAb-induced tethering of surface pathogen-recognition receptors switching cell properties opens a new therapeutic avenue for controlling inflammation and restoring immune tolerance.

Bacteria and HIT: a close connection?

HIT is caused by antibodies specific to PF4/heparin complexes. In this issue of Blood, Krauel et al report novel findings supporting the hypothesis that primary synthesis of these antibodies results from bacterial infections. HIT, therefore, appears to be a misdirected antibacterial host defense response.