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Summary: Background: Merkel cell carcinoma (MCC) is an aggressive skin cancer, whose tumour cells often express CD56. While immune checkpoint inhibitors constitute a major advance for treating patients with MCC with advanced disease, new therapeutic options are still urgently required. Objectives: To produce and evaluate the therapeutic performance of a new antibody–drug conjugate (Adcitmer®) targeting CD56 in preclinical models of MCC. Methods: CD56 expression was evaluated in a MCC cohort (immunohistochemistry on a tissue microarray of 90 tumour samples) and MCC cell lines. Interaction of an unconjugated CD56‐targeting antibody with CD56+ MCC cell lines was investigated by immunohistochemistry and imaging flow cytometry. Adcitmer® product was generated by the bioconjugation of CD56‐targeting antibody to a cytotoxic drug (monomethyl auristatin E) using the McSAF Inside® bioconjugation process. The chemical properties and homogeneity of Adcitmer® were characterized by hydrophobic interaction chromatography. Adcitmer® cytotoxicity was evaluated in vitro and in an MCC xenograft mice model. Results: Similar to previous reports, CD56 was expressed by 66% of MCC tumours in our cohort, confirming its relevance as a therapeutic target. Specific binding and internalization of the unconjugated CD56‐targeting antibody was validated in MCC cell lines. The high homogeneity of the newly generated Adcitmer® was confirmed by hydrophobic interaction chromatography. The CD56‐mediated cytotoxicity of Adcitmer® was demonstrated in vitro in MCC cell lines. Moreover, Adcitmer® significantly reduced tumour growth in a MCC mouse model. Conclusions: Our study suggests that Adcitmer® should be further assessed as a therapeutic option in patients with MCC, as an alternative therapy or combined with immune checkpoint inhibitors. What is already known about this topic? Merkel cell carcinoma (MCC) is an aggressive skin cancer.While immune checkpoint inhibitors constitute a major advance in treating patients with MCC with advanced disease, new therapeutic options are still urgently required.CD56, which is expressed by most MCC tumours, might constitute a therapeutic target for the development of antibody–drug conjugate therapies. What does this study add? We generated a CD56‐targeting monomethyl auristatin E (MMAE) bioconjugate antibody (Adcitmer®) and demonstrated its optimal biochemical properties, specific binding and cytotoxicity in vitro in MCC cell lines, and its capacity to reduce tumour growth in vivo in a MCC mouse model. What is the translational message? Adcitmer®, a new CD56‐targeting MMAE bioconjugate antibody, may represent a potential therapeutic option to be investigated as an alternative approach or in combination with immune checkpoint inhibitors in patients with inoperable MCC. Linked Comment: L. Leiendecker et al. Br J Dermatol 2022; 186:209–210. Plain language summary available online [ABSTRACT FROM AUTHOR]

Activation of the Jak/STAT pathway by cytokines has been shown to regulate differentiation, proliferation or apoptosis in hematopoeitic cells. Among the Stat proteins, STAT5 is activated by a broad range of cytokines. In order to study the role of STAT5 in hematopoietic cells, we stably expressed a dominant negative form of STAT5 (STAT5AΔ749) in the IL-3 dependent bone marrow derived Ba/F3 cell line. Ba/F3 cells expressing STAT5AΔ749 were found to be more sensitive to apoptosis than parental or control Ba/F3 cells after IL-3 withdrawal. Analysis of the expression of the cell death regulators, Bcl-2 and Bcl-x, revealed that the level of Bcl-x was lower in Ba/F3 cells expressing STAT5AΔ749 than in control cells. IL-3 regulation of Bcl-x expression at protein and mRNA levels was impaired in these cells while that of Bcl-2 expression was unaffected. We further demonstrated that the Bcl-x gene promoter contained a proximal STAT consensus sequence that bound STAT5. Transactivation of a Bcl-x gene promoter reporter construct by STAT5 was observed in Ba/F3 cells. Introduction of a mutation in the STAT binding site abolished this transactivation. These data indicate that Bcl-x is probably a STAT5 target gene. They also support the involvement of STAT5 in hematopoietic cell survival. [ABSTRACT FROM AUTHOR]

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology.)

The concept of grafting mannose 6-phosphonate derivatives (M6Pn), named AMFA, on therapeutic proteins was first developed for the improvement of enzyme delivery in lysosomal storage disorders. This glycoengineering increases the cellular uptake of the protein via the cation-independent mannose 6-phosphate receptor (M6PR) which further allows their targeting to the lysosomes. In the present study, we investigated the extent to which the direct grafting of AMFA onto a drug, here a monoclonal antibody (mAb), affects the cell uptake and recycling of the antibody. The antibodies infliximab (IFX) and adalimumab (ADA), directed against the tumor necrosis factor α (TNFα), grafted with AMFA acquired an affinity for the M6PR, resulting in a >3-fold increase in drug release in cells. Subsequently, the impact of AMFA grafting to the Fc portion of mAb on its affinity for the neonatal Fc receptor (FcRn), which is the key receptor for antibody recycling, was evaluated. Whether one to three AMFA moieties were grafted, FcRn-mediated recycling of mAb was not affected. AMFA grafting did not impair the pharmacokinetics of both ADA and IFX and presented a high stability since AMFA were still bound to mAb in the plasma of mice 21 days after the treatment. In conclusion, this type of antibody engineering with a reduced number of AMFA confers M6PR targeting property and increases endocytosis, and yet appears fully compatible with FcRn binding and with antibody recycling and transcytosis., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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(K D ) 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.

Recent technological developments in ultrasound (US) imaging and ultrasound contrast agents (UCAs) have improved diagnostic confidence in echography. In the clinical management of melanoma, contrast-enhanced ultrasound (CEUS) imaging complements conventional US imaging (i.e., high-resolution US and Doppler imaging) for clinical examination and therapeutic follow-up. These developments have set into motion the combined use of ultrasound and UCAs as a new modality for drug delivery. This modality, called sonoporation, has emerged as a non-invasive, targeted and safe method for the delivery of therapeutic drugs into melanoma. This review focuses on the results and prospects of using US and UCAs as dual modalities for CEUS imaging and melanoma treatment., Competing Interests: Conflict of interest disclosure The authors have no conflicts of interest to disclose., (Copyright © 2022 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

The neonatal Fc receptor (FcRn) plays a central role in recycling and biodistributing immunoglobulin G. FcRn is also involved in many physiological immune functions as well as pathological immune responses in cancer or autoimmune diseases. Low levels of FcRn in tumor cells and the microenvironment is associated with poor prognosis in non-small cell lung cancers. Among cells that are present in the tumor microenvironment, macrophages express high levels of FcRn. Macrophages are involved in these pathophysiological contexts by their dual differentiation states of pro- or anti-inflammatory macrophages. However, variations in FcRn protein expression have not been described in macrophage subtypes. In this work, we studied FcRn expression in an in vitro model of pro- and anti-inflammatory macrophage differentiation. We demonstrated an inverse relation between FcRn protein and mRNA expression in macrophage populations. Autophagy, which is involved in protein degradation and acquisition of phagocytic function in macrophages, participated in regulating FcRn levels. Intravenous immunoglobulin protected FcRn against autophagosome degradation in anti-inflammatory macrophages. Our data demonstrate that autophagy participates in regulating FcRn expression in pro- and anti-inflammatory macrophages. This finding raises new questions concerning the regulation of FcRn in immune functions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lamamy, Larue, Mariot, Dhommée, Demattei, Delneste and Gouilleux-Gruart.)

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.

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., Competing Interests: The authors declare no conflict of interest.

Bispecific antibodies (BsAbs) represent an important advance in innovative therapeutic strategies. Among the countless formats of BsAbs, fusion with molecules such as anticalins linked to a monoclonal antibody (mAb), represents an easy and low-cost way to obtain innovative molecules. We fused an anticalin against human fibronectin to a molecule biosimilar to trastuzumab (H0) or rituximab (R0), in four different positions, two on the N terminal region of heavy or light chains and two on the C terminal region. The eight BsAbs (H family (HF) 1 to 4 and R family (RF) 1 to 4) were produced and their affinity parameters and functional properties evaluated. The presence of anticalin did not change the glycosylation of the BsAb, shape or yield. The antigenic recognition of each BsAb family, Her2 for HF1 to 4 and CD20 for RF1 to 4, was slightly decreased (HF) or absent (RF) for the anticalin N-terminal in the light chain position. The anticalin recognition of FN was slightly decreased for the HF family, but a dramatic decrease was observed for RF members with lowest affinity for RF1. Moreover, functional properties of Abs, such as CD16 activation of NK, CD32-dependent phagocytosis and FcRn transcytosis, confirmed that this anticalin position leads to less efficient BsAbs, more so for RF than HF molecules. Nevertheless, all BsAbs demonstrated affinities for CD16, CD32 and FcRn, which suggests that more than affinity for FcRs is needed for a functioning antibody. Our strategy using anticalin and Abs allows for rapid generation of BsAbs, but as suggested by our results, some positions of anticalins on Abs result in less functionality.

Antibody-based therapeutics have become a major class of therapeutics with over 120 recombinant antibodies approved or under review in the EU or US. This therapeutic class has experienced a remarkable expansion with an expected acceleration in 2021-2022 due to the extraordinary global response to SARS-CoV2 pandemic and the public disclosure of over a hundred anti-SARS-CoV2 antibodies. Mainly delivered intravenously, alternative delivery routes have emerged to improve antibody therapeutic index and patient comfort. A major hurdle for antibody delivery and efficacy as well as the development of alternative administration routes, is to understand the different natural and pathological barriers that antibodies face as soon as they enter the body up to the moment they bind to their target antigen. In this review, we discuss the well-known and more under-investigated extracellular and cellular barriers faced by antibodies. We also discuss some of the strategies developed in the recent years to overcome these barriers and increase antibody delivery to its site of action. A better understanding of the biological barriers that antibodies have to face will allow the optimization of antibody delivery near its target. This opens the way to the development of improved therapy with less systemic side effects and increased patients' adherence to the treatment., Competing Interests: Declaration of Competing Interest YA, TB, JL, MG, AP, CDS, NJ, JPP, VGG, DL, SP, TS, have nothing to declare. NHV is co-founder and scientific expert for Cynbiose Respiratory. In the past two years, she received consultancy fees from Eli Lilly, Argenx, Novartis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Merkel cell carcinoma (MCC) is an aggressive skin cancer, whose tumour cells often express CD56. While immune checkpoint inhibitors constitute a major advance for treating patients with MCC with advanced disease, new therapeutic options are still urgently required., Objectives: To produce and evaluate the therapeutic performance of a new antibody-drug conjugate (Adcitmer ® ) targeting CD56 in preclinical models of MCC., Methods: CD56 expression was evaluated in a MCC cohort (immunohistochemistry on a tissue microarray of 90 tumour samples) and MCC cell lines. Interaction of an unconjugated CD56-targeting antibody with CD56 + MCC cell lines was investigated by immunohistochemistry and imaging flow cytometry. Adcitmer ® product was generated by the bioconjugation of CD56-targeting antibody to a cytotoxic drug (monomethyl auristatin E) using the McSAF Inside ® bioconjugation process. The chemical properties and homogeneity of Adcitmer ® were characterized by hydrophobic interaction chromatography. Adcitmer ® cytotoxicity was evaluated in vitro and in an MCC xenograft mice model., Results: Similar to previous reports, CD56 was expressed by 66% of MCC tumours in our cohort, confirming its relevance as a therapeutic target. Specific binding and internalization of the unconjugated CD56-targeting antibody was validated in MCC cell lines. The high homogeneity of the newly generated Adcitmer ® was confirmed by hydrophobic interaction chromatography. The CD56-mediated cytotoxicity of Adcitmer ® was demonstrated in vitro in MCC cell lines. Moreover, Adcitmer ® significantly reduced tumour growth in a MCC mouse model., Conclusions: Our study suggests that Adcitmer ® should be further assessed as a therapeutic option in patients with MCC, as an alternative therapy or combined with immune checkpoint inhibitors., (© 2021 British Association of Dermatologists.)

Background: Rabbit antithymocyte globulins (rATGs) are polyclonal antibodies used to prevent acute cellular rejection in kidney transplantation. Their dosing remains largely empirical and the question of an individualized dose is still unresolved., Methods: Data from a prospective study in 17 kidney transplant patients were used to develop a model describing the dose-concentration-response relationship of rATG with T-lymphocyte subpopulation counts over time. The model was validated using an independent cohort of kidney transplant patients treated by rATG in the same center., Results: Pharmacokinetics of rATG was described using a two-compartment model integrating a third compartment and a target-mediated elimination for active rATG. The kinetics of CD3 + , CD4 + , CD8 + , and CD3 - CD56 + cell counts over time were described by a pharmacokinetic-pharmacodynamic model with transit compartments, integrating both CD3 - CD56 + -independent and CD3 - CD56 + -dependent rATG-mediated lymphocyte depletion, and a positive feedback. Elimination of rATG was influenced by age and body surface area, while its distribution was also influenced by body surface area. CD3 + proliferation rate decreased with age and CD3 - CD56 + -mediated elimination was influenced by the V158F-FCGR3A polymorphism. Binary efficacy and tolerance endpoints were defined as a CD3 + count < 20 mm -3 for at least 7 days and a CD4 + count > 200 mm -3 at 1 year, respectively. Simulations showed that increasing or decreasing the standard 6-mg/kg dose will impact both tolerance and efficacy, while a dose decrease may be beneficial in elderly patients., Conclusions: Our results can be used to design prospective clinical trials testing dose individualization based on patients' characteristics., Clinical Trial Registration: Eudract No. 2009-012673-35., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Angiogenesis is in a constant balance between pro and anti-angiogenic factors. Neoangiogenesis, implicated in metastatic spreading is characterized in solid cancers, but fairly new in chronic lymphocytic leukemia (CLL). We hypothesize that secretion of angiogenic factors could be correlated to the pathogenesis of CLL, and therefore predict the outcome of patients. We investigated concentrations of 22 cytokines and chemokines in 137 non-del 17p B-CLL patients, treated with a fludarabine-cyclophosphamide-rituximab (FCR)-based regimen. We constructed a biomarker index defining different risk groups based on lymphocyte count, the intensity of CD20 antigen on CD19 + cells, Ang-2, and PDGF-BB plasma concentrations at diagnosis. Four groups were defined, exhibiting specific molecular signatures and correlated with progression-free survival of patients. Our results suggest that we can determine at diagnosis of non-del 17p B-CLL patients, those with a very high probability of progression-free survival, independently of IGVH mutational status and residual disease at the end of treatment.

The neonatal Fc receptor (FcRn) is a ubiquitously expressed protein historically involved in IgG and albumin recycling. Recent data suggest an involvement in the pathophysiology of antibody-mediated autoimmune diseases. Among them, systemic lupus erythematosus (SLE) implies clinical and biological abnormalities of innate and adaptive circulating immune cells, potentially involving newly described functions of FcRn. In this study, FcRn expression was assessed by flow cytometry in peripheral blood leukocytes of 41 SLE patients with either active or inactive disease and 32 healthy donors. FcRn expression in B cells, natural killer cells, and T cells of SLE patients was statistically lower as compared to healthy donors. Conversely, FcRn level was statistically higher in non-classical monocyte subpopulations (CD14+CD16+ monocytes) of SLE patients versus healthy donors providing an interesting perspective to further explore its role in SLE pathophysiology.

Since the neonatal IgG Fc receptor (FcRn) was discovered, its role has evolved from immunoglobulin recycling and biodistribution to antigen presentation and immune complex routing, bringing it to the center of both humoral and cellular immune responses. FcRn is thus involved in the pathophysiology of immune-related diseases such as cancer, infection, and autoimmune disorders. This review focuses on the role of FcRn in autoimmunity, based on the available data from both animal models and human studies. The knowledge concerning ways in which FcRn is involved in autoimmune response has led to the development of inhibitors for the treatment of autoimmune diseases, also described here. Up to date, the literature remains scarce, shedding light on the need for further studies to fully understand the various pathophysiological roles of this unique receptor., (© 2021 Published by Elsevier B.V.)

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 × 10 9 /L, respectively, to ≈13 days (both HR and RR) at 350 × 10 9 /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.

Granulomatosis with polyangiitis (GPA) is a severe autoimmune vasculitis associated with the presence of anti-neutrophil cytoplasmic antibodies (ANCA) mainly targeting proteinase 3 (PR3), a neutrophilic serine proteinase. PR3-ANCA binding to membrane-bound PR3 on neutrophils induce their auto-immune activation responsible for vascular lesions. However, the correlation between PR3-ANCA level and disease activity remains inconsistent, suggesting the existence of non-pathogenic PR3-ANCA. In order to prove their existence, we immortalized B lymphocytes from blood samples of GPA patients in remission having persistent PR3-ANCA to isolate non-activating PR3-ANCA. We obtained for the first time a non-activating human IgG1κ anti-PR3 monoclonal antibody (mAb) named 4C3. This new mAb binds soluble PR3 with a high affinity and membrane-bound PR3 on an epitope close to the PR3 hydrophobic patch and in the vicinity of the active site. 4C3 is able to bind FcγRIIA and FcγRIIIB and has a G2F glycosylation profile on asparagine 297. 4C3 did not induce activation of neutrophils and could inhibit human polyclonal PR3-ANCA-induced activation suggesting that 4C3 is non-pathogenic. This characteristic relies on the recognized epitope on PR3 rather than to the Fc portion properties. The existence of non-pathogenic PR3-ANCA, which do not activate neutrophils, could explain the persistence of high PR3-ANCA levels in some GPA patients in remission and why PR3-ANCA would not predict relapse. Finally, these results offer promising perspectives particularly regarding the understanding of PR3-ANCA pathogenicity and the development of new diagnostic and therapeutic strategies in GPA., (Copyright © 2020 Granel, Lemoine, Morello, Gallais, Mariot, Drapeau, Musnier, Poupon, Pugnière, Seren, Nouar, Gouilleux-Gruart, Watier, Korkmaz and Hoarau.)

Post-transplantation lymphoproliferative disorder (PTLD) is a severe complication in organ transplant recipients. The use of T lymphocyte-depleting antibodies (TLDAb), especially rabbit TLDAb, contributes to PTLD, and the V158F polymorphism of Fc gamma receptor IIIA (FcγRIIIA) also named CD16A could affect the concentration-effect relationship of TLDAb. We therefore investigated the association of this polymorphism with PTLD in kidney transplant recipients. We characterized the V158F polymorphism in two case-control cohorts (discovery, n = 196; validation, n = 222). Then, we evaluated the binding of rabbit IgG to human FcγRIIIA-158V and FcγRIIIA-158F. The V158F polymorphism was not linked to PTLD in the overall cohorts, but risk of PTLD was increased in VV homozygous recipients receiving TLDAb compared with F carriers in both cohorts, especially in recipients receiving TLDAb without muromonab (discovery: HR = 2.22 [1.03-4.76], P = 0.043, validation: HR = 1.75 [1.01-3.13], P = 0.049). In vitro, we found that the binding of rabbit IgG to human NK-cell FcγRIIIA was increased when cells expressed the 158-V versus the 158-F allotype. While the 158-V allotype of human FcγRIIIA binds rabbit immunoglobulin-G with higher affinity, the risk of PTLD was increased in homozygous VV kidney transplant recipients receiving polyclonal TLDAb., (© 2020 Steunstichting ESOT. Published by John Wiley & Sons Ltd.)

Dendritic cells (DCs) play a key role in immunity and are highly potent at presenting antigens and orienting the immune response. Depending on the environmental signals, DCs could turn the immune response toward immunity or immune tolerance. Several subsets of DCs have been described, with each expressing various surface receptors and all participating in DC-associated immune functions according to their specific skills. DC subsets could also contribute to the vicious circle of inflammation in immune diseases and establishment of immune tolerance in cancer. They appear to be appropriate targets in the control of inflammatory diseases or regulation of autoimmune responses. For all these reasons, in situ DC targeting with therapeutic antibodies seems to be a suitable way of modulating the entire immune system. At present, the field of antibody-based therapies has mainly been developed in oncology, but it is undergoing remarkable expansion thanks to a wide variety of antibody formats and their related functions. Moreover, current knowledge of DC biology may open new avenues for targeting and modulating the different DC subsets. Based on an update of pathogen recognition receptor expression profiles in human DC subsets, this review evaluates the possibility of inducing tolerant DCs using antibody-based therapeutic agents.

Background:  Heparin-induced thrombocytopenia (HIT) is a rare complication of heparin treatments, and only a few large patient cohorts have been reported. In this study, biological and clinical data from 144 French patients with HIT were analyzed in comparison with the literature., Methods:  The diagnosis of HIT was confirmed in all patients by an immunoassay combined with serotonin release assay. In the literature, only cohorts of at least 20 HIT patients published from 1992 were selected for a comparative analysis., Results:  Two-thirds of patients were hospitalized in surgery and most were treated with unfractionated heparin (83.2% vs. 16.8% with low molecular weight heparin only). Thrombotic events in 54 patients (39.7%) were mainly venous (41/54). However, arterial thrombosis was more frequent after cardiac surgery (13.2% vs. 2.4% in other surgeries, p  = 0.042) with a shorter recovery time (median = 3 vs. 5 days, p  < 0.001). The mortality rate was lower in our series than in the 22 selected published studies (median = 6.3% vs. 15.9%). Three genetic polymorphisms were also studied and homozygous subjects FcγRIIA RR were more frequent in patients with thrombosis (37.8 vs. 18.2% in those without thrombosis, p  = 0.03)., Conclusion:  This study shows that the mortality rate due to HIT has recently decreased in France, possibly due to earlier diagnosis and improved medical care. It also confirms the strong association between polymorphism FcγRIIA H131R and thrombosis in HIT., Competing Interests: Y.G.: Consulting and/or travel fees from Octapharma, Shire, CSL Behring, Novo Nordisk, Bayer, LFB, and Léo Pharma. J.R.: Travel fees from Octapharma, Shire, and CSL Behring. C.V.: Travel fees from Sobi, Roche, Shire, and CSL Behring. D.F.: Consulting and/or travel fees from Aspen, Boehringer, Werfen, Stago, and Léo Pharma. A.B.: Travel fees from Aspen, Boehringer, Werfen, Pfizer, and LFB. A.L.: Consulting and/or travel fees from Bayer, LFB, Pfizer, Sobi, and Octapharma. E.D.M.: Travel fees from Sobi, Bayer, Novo Nordisk, and Pfizer. B.T.P.: Consulting and/or travel fees from Sobi, Bayer, Shire, CSL Behring, and Pfizer. I.E.: Consulting and/or travel fees from BMS, Aspen, Léo Pharma, Daiichi Sankyo, Pfizer, Sanofi-Aventis, and Shire. C.L.B.: Travel fees from Sobi, Octapharma, CSL Berhing, and Novo Nordisk. C.M.: Consulting and/or travel fees from BMS, Aspen, Pfizer, and Bayer. C.B.: Consulting and/or travel fees from CSL, Sobi, Bayer, and Novo Nordisk. C.T.: Consulting and travel fees from Sobi, Roche, Octapharma. F.N.G.: Travel fees from Sobi, Bayer, BMS, and Boehringer. J.D.: Travel fees from CSL and Novo Nordisk. T.B.A.: Travel fees from Servier and MSD. E.D.R.: Consulting and/or travel fees from Shire, Alexion, Sobi, Bayer, and Novo Nordisk. I.G.T.: Consulting and/or travel fees from Bayer, MSD, Pfizer, Sanofi-Aventis, and BMS. L.R.: Consulting and/or travel fees from CSL, LFB, Novo Nordisk, Sobi, and Bayer. B.T.: Consulting and/or travel fees from Sobi, Bayer, Novo Nordisk Aspen, CSL Behring, and Pfizer. C.P.: Consulting and/or travel fees from Sobi, Roche, Novo Nordisk, and CSL Behring. P.W., M.A.G., S.V., V.G.G., J.P., and B.G. have no conflict to disclose. All the authors did not receive any personal honorarium or funds related to the study reported in this manuscript. Y.G. reports grants from Ministère de la Santé (Government), during the conduct of the study; personal fees and nonfinancial support from CSL Behring, personal fees and nonfinancial support from Octapharma, nonfinancial support from Shire, personal fees from Léo Pharma, personal fees and nonfinancial support from LFB, nonfinancial support from Bayer, nonfinancial support from Novo Nordisk, outside the submitted work., (Georg Thieme Verlag KG Stuttgart · New York.)

Since the neonatal IgG Fc receptor (FcRn) was discovered, it was found to be involved in immunoglobulin recycling and biodistribution, immune complexes routing, antigen presentation, humoral immune response, and cancer immunosurveillance. The latest data show that FcRn plays a part in cancer pathophysiology. In various types of cancers, such as lung and colorectal cancer, FcRn has been described as an early marker for prognosis. Dysregulation of FcRn expression by cancer cells allows them to increase their metabolism, and this process could be exploited for passive targeting of cytotoxic drugs. However, the roles of this receptor depend on whether the studied cell population is the tumor tissue or the infiltrating cells, bringing forward the need for further studies., (© 2020 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background and Objectives: Rituximab is approved in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and leads to a decrease of ANCA levels. The objectives of this study were to investigate the non-linear pharmacokinetics of rituximab and the relationship between its concentrations and ANCA levels in AAV patients., Methods: Ninety-two AAV patients from the RAVE (Rituximab in ANCA-Associated Vasculitis) trial were assessed. Both ANCA anti-myeloperoxidase (MPO-ANCA) and anti-proteinase 3 (PR3-ANCA) levels were used as biomarkers. The pharmacokinetics of rituximab were described using a semi-mechanistic two-compartment model that included a latent target antigen turnover and allowed the estimation of specific target-mediated elimination in addition to its non-specific elimination of rituximab. The effect of rituximab on the ANCA level was described using a semi-mechanistic compartment model with a negative feedback (Friberg) model with no transit compartment. A population modeling approach was used., Results: Our pharmacokinetic and pharmacokinetic-pharmacodynamic (PK-PD) models satisfactorily described both concentration-time and concentration-effect relationship data. The mean (inter-individual standard deviation) estimated non-specific clearance was 0.15 L/day (0.30%) and the target-mediated elimination rate constant was 2.4 × 10 -5 nmol/day. The elimination half-lives for MPO-ANCA and PR3-ANCA were 24 and 18 days, respectively., Conclusions: A non-linear target-mediated elimination of rituximab was detected in AAV patients. Our PK-PD model allowed quantification of the association between rituximab concentrations and ANCA levels. This decrease was deep but delayed, and more sustained in patients with MPO-ANCA than in those with PR3-ANCA. Our results suggest that repeating courses of rituximab might improve the clinical response to rituximab.

Antibodies directed against CD22 have been used in radioimmunotherapy (RIT) clinical trials to treat patients with diffuse large B-cell lymphoma (DLBCL) with promising results. However, relevant preclinical models are needed to facilitate the evaluation and optimization of new protocols. Spontaneous DLBCL in dogs is a tumor model that may help accelerate the development of new methodologies and therapeutic strategies for RIT targeting CD22. Seven murine monoclonal antibodies specific for canine CD22 were produced by the hybridoma method and characterized. The antibodies' affinity and epitopic maps, their internalization capability and usefulness for diagnosis in immunohistochemistry were determined. Biodistribution and PET imaging on a mouse xenogeneic model of dog DLBCL was used to choose the most promising antibody for our purposes. PET-CT results confirmed biodistribution study observations and allowed tumor localization. The selected antibody, 10C6, was successfully used on a dog with spontaneous DLBCL for SPECT-CT imaging in the context of disease staging, validating its efficacy for diagnosis and the feasibility of future RIT assays. This first attempt at phenotypic imaging on dogs paves the way to implementing quantitative imaging methodologies that would be transposable to humans in a theranostic approach. Taking into account the feedback of existing human radioimmunotherapy clinical trials targeting CD22, animal trials are planned to investigate protocol improvements that are difficult to consider in humans due to ethical concerns., (Copyright © 2020 Etienne, Berthaud, Nguyen, Bernardeau, Maurel, Bodet-Milin, Diab, Abadie, Gouilleux-Gruart, Vidal, Bourgeois, Chouin, Ibisch and Davodeau.)

Fluorescent labelling of monoclonal antibodies (mAbs) is classically performed by chemical bioconjugation methods. The most frequent labelling technique to generate antibody-fluorophore conjugates (AFCs) involves the bioconjugation onto the mAb lysines of a dye bearing an N -hydroxysuccinimide ester or an isothiocyanate group. However, discrepancies between labelling experiments or kits can be observed, related to reproducibility issues, alteration of antigen binding, or mAb properties. The lack of information on labelling kits and the incomplete characterization of the obtained labelled mAbs largely contribute to these issues. In this work, we generated eight AFCs through either lysine or interchain cysteine cross-linking bioconjugation of green-emitting fluorophores (fluorescein or BODIPY) onto either trastuzumab or rituximab. This strategy allowed us to study the influence of fluorophore solubility, bioconjugation technology, and antibody nature on two known labelling procedures. The structures of these AFCs were thoroughly analyzed by mass spectroscopy, and their antigen binding properties were studied. We then compared these AFCs in vitro by studying their respective spectral properties and stabilities. The shelf stability profiles and sensibility to pH variation of these AFCs prove to be dye-, antibody- and labelling-technology-dependent. Fluorescence emission in AFCs was higher when lysine labelling was used, but cross-linked AFCs were revealed to be more stable. This must be taken into account for the design of any biological study involving antibody labelling.

The hinge region is a short sequence of the heavy chains (H) of antibodies linking the Fab (Fragment antigen binding) region to the Fc (Fragment crystallisable) region. The functional properties of the four IgG subclasses partly result from the sequence differences of their hinge regions as some amino acids of the lower hinge region are located within or in the close vicinity of the C1q and FcγR binding sites on the IgG H chains. In addition, the hinge is susceptible to proteolytic cleavage by many proteases present in tumor and/or inflammatory microenvironment capable of affecting functional responses. Thus, an optimal format of the hinge region remains a major challenge for the development of new therapeutic antibodies., (© 2019 médecine/sciences – Inserm.)

Despite being the least abundant immunoglobulin G in human plasma, IgG4 are used therapeutically when weak effector functions are needed. The increase in engineered IgG4-based antibodies on the market led us to study the patent landscape of IgG4 Fc engineering, i.e ., patents claiming modifications in the heavy chain. Thirty-seven relevant patent families were identified, comprising hundreds of IgG4 Fc variants focusing on removal of residual effector functions (since IgG4s bind to FcγRI and weakly to other FcγRs), half-life enhancement and IgG4 stability. Given the number of expired or soon to expire major patents in those 3 areas, companies developing blocking antibodies now have, or will in the near future, access to free tools to design silenced, half-life extended and stable IgG4 antibodies.