The Dual Targeting of FcRn and FcγRs via Monomeric Fc Fragments Results in Strong Inhibition of IgG-Dependent Autoimmune Pathologies.

Novel molecules that directly target the neonatal Fc receptor (FcRn) and/or Fc gamma receptors (FcγRs) are emerging as promising treatments for immunoglobulin G (IgG)-dependent autoimmune pathologies. Mutated Fc regions and monoclonal antibodies that target FcRn are currently in clinical development and hold promise for reducing the levels of circulating IgG. Additionally, engineered structures containing multimeric Fc regions allow the dual targeting of FcRn and FcγRs; however, their tolerance needs to first be validated in phase I clinical studies. Here, for the first time, we have developed a modified monomeric recombinant Fc optimized for binding to all FcRns and FcγRs without the drawback of possible tolerance associated with FcγR cross-linking. A rational approach using Fc engineering allowed the selection of LFBD192, an Fc with a combination of six mutations that exhibits improved binding to human FcRn and FcγR as well as mouse FcRn and FcγRIV. The potency of LFBD192 was compared with that of intravenous immunoglobulin (IVIg), an FcRn blocker (Fc-MST-HN), and a trimeric Fc that blocks FcRn and/or immune complex-mediated cell activation through FcγR without triggering an immune reaction in several in vitro tests and validated in three mouse models of autoimmune disease.

Fc Sialylation Prolongs Serum Half-Life of Therapeutic Antibodies.

The long serum t 1/2 of IgGs is ensured by their interaction with the neonatal Fc receptor (FcRn), which salvages IgG from intracellular degradation. Fc glycosylation is thought not to influence FcRn binding and IgG longevity in vivo. In this article, we demonstrate that hypersialylation of asparagine 297 (N297) enhances IgG serum persistence. This polarized glycosylation is achieved using a novel Fc mutation, a glutamate residue deletion at position 294 (Del) that endows IgGs with an up to 9-fold increase in serum lifespan. The strongest impact was observed when the Del was combined with Fc mutations improving FcRn binding (Del-FcRn+). Enzymatic desialylation of a Del-FcRn+ mutant or its production in a cell line unable to hypersialylate reduced the in vivo serum t 1/2 of the desialylated mutants to that of native FcRn+ mutants. Consequently, our study proves that sialylation of the N297 sugar moiety has a direct impact on human IgG serum persistence.

Improved in vitro and in vivo activity against CD303-expressing targets of the chimeric 122A2 antibody selected for specific glycosylation pattern.

Plasmacytoid dendritic cells (pDCs) play a central role for both innate and adaptive antiviral responses, as they direct immune responses through their unique ability to produce substantial concentrations of type I interferon (IFNs) upon viral encounter while also activating multiple immune cells, including macrophages, DCs, B, natural killer and T cells. Recent evidence clearly indicates that pDCs also play a crucial role in some cancers and several auto-immune diseases. Although treatments are currently available to patients with such pathologies, many are not fully efficient. We are proposing here, as a new targeted-based therapy, a novel chimeric monoclonal antibody (mAb) that mediates a strong cellular cytotoxicity directed against a specific human pDC marker, CD303. This antibody, ch122A2 mAb, is characterized by low fucose content in its human IgG1 constant (Fc) region, which induces strong in vitro and in vivo activity against human pDCs. We demonstrated that this effect relates in part to its specific Fc region glycosylation pattern, which increased affinity for CD16/FcγRIIIa. Importantly, ch122A2 mAb induces the down-modulation of CpG-induced IFN-α secretion by pDCs. Additionally, ch122A2 mAb shows in vitro high pDC depletion mediated by antibody-dependent cell-mediated cytotoxicity and antibody-dependent cellular phagocytosis. Remarkably, in vivo ch122A2 mAb efficacy is also demonstrated in humanized mice, resulting in significant pDC depletion in bloodstream and secondary lymphoid organs such as spleen. Together, our data indicates that ch122A2 mAb could represent a promising cytotoxic mAb candidate for pathologies in which decreasing type I IFNs or pDCs depleting may improve patient prognosis.

The anti-tumor efficacy of 3C23K, a glyco-engineered humanized anti-MISRII antibody, in an ovarian cancer model is mainly mediated by engagement of immune effector cells.

Ovarian cancer is the leading cause of death in women with gynecological cancers and despite recent advances, new and more efficient therapies are crucially needed. Müllerian Inhibiting Substance type II Receptor (MISRII, also named AMHRII) is expressed in most ovarian cancer subtypes and is a novel potential target for ovarian cancer immunotherapy. We previously developed and tested 12G4, the first murine monoclonal antibody (MAb) against human MISRII. Here, we report the humanization, affinity maturation and glyco-engineering steps of 12G4 to generate the Fc-optimized 3C23K MAb, and the evaluation of its in vivo anti-tumor activity. The epitopes of 3C23K and 12G4 were strictly identical and 3C23K affinity for MISRII was enhanced by a factor of about 14 (KD = 5.5 × 10-11 M vs 7.9 × 10-10 M), while the use of the EMABling® platform allowed the production of a low-fucosylated 3C23K antibody with a 30-fold KD improvement of its affinity to FcγRIIIa. In COV434-MISRII tumor-bearing mice, 3C23K reduced tumor growth more efficiently than 12G4 and its combination with carboplatin was more efficient than each monotherapy with a mean tumor size of 500, 1100 and 100 mm3 at the end of treatment with 3C23K (10 mg/kg, Q3-4D12), carboplatin (60 mg/kg, Q7D4) and 3C23K+carboplatin, respectively. Conversely, 3C23K-FcKO, a 3C23K form without affinity for the FcγRIIIa receptor, did not display any anti-tumor effect in vivo. These results strongly suggested that 3C23K mechanisms of action are mainly Fc-related. In vitro, antibody-dependent cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP) were induced by 3C23K, as demonstrated with human effector cells. Using human NK cells, 50% of the maximal lysis was obtained with a 46-fold lower concentration of low-fucosylated 3C23K (2.9 ng/ml) than of 3C23K expressed in CHO cells (133.35 ng/ml). As 3C23K induced strong ADCC with human PBMC but almost none with murine PBMC, antibody-dependent cell phagocytosis (ADCP) was then investigated. 3C23K-dependent (100 ng/ml) ADCP was more active with murine than human macrophages (only 10% of living target cells vs. about 25%). These in vitro results suggest that the reduced ADCC with murine effectors could be partially balanced by ADCP activity in in vivo experiments. Taken together, these preclinical data indicate that 3C23K is a new promising therapeutic candidate for ovarian cancer immunotherapy and justify its recent introduction in a phase I clinical trial.

Characterization of monoclonal antibodies by a fast and easy liquid chromatography-mass spectrometry time-of-flight analysis on culture supernatant.

Rapid and efficient structural analysis is key to the development of new monoclonal antibodies. We have developed a fast and easy process to obtain mass spectrometry profiles of antibodies from culture supernatant. Treatment of the supernatant with IdeS generates three fragments of 25 kDa that can be analyzed by liquid chromatography-mass spectrometry time-of-flight (LC-MS TOF) in one run: LC, Fd, and Fc/2. This process gives rapid access to isoform and glycoform profiles. To specifically measure the fucosylation yield, we included a one-pot treatment with EndoS that removes the distal glycan heterogeneity. Our process was successfully compared with high-performance capillary electrophoresis with laser-induced fluorescence detection (HPCE-LIF), currently considered as the "gold standard" method.

Vegetation limits the impact of a warm climate on boreal wildfires.

Strategic introduction of less flammable broadleaf vegetation into landscapes was suggested as a management strategy for decreasing the risk of boreal wildfires projected under climatic change. However, the realization and strength of this offsetting effect in an actual environment remain to be demonstrated. Here we combined paleoecological data, global climate models and wildfire modelling to assess regional fire frequency (RegFF, i.e. the number of fires through time) in boreal forests as it relates to tree species composition and climate over millennial time-scales. Lacustrine charcoals from northern landscapes of eastern boreal Canada indicate that RegFF during the mid-Holocene (6000-3000 yr ago) was significantly higher than pre-industrial RegFF (AD c. 1750). In southern landscapes, RegFF was not significantly higher than the pre-industrial RegFF in spite of the declining drought severity. The modelling experiment indicates that the high fire risk brought about by a warmer and drier climate in the south during the mid-Holocene was offset by a higher broadleaf component. Our data highlight an important function for broadleaf vegetation in determining boreal RegFF in a warmer climate. We estimate that its feedback may be large enough to offset the projected climate change impacts on drought conditions.

Potential changes in forest composition could reduce impacts of climate change on boreal wildfires.

There is general consensus that wildfires in boreal forests will increase throughout this century in response to more severe and frequent drought conditions induced by climate change. However, prediction models generally assume that the vegetation component will remain static over the next few decades. As deciduous species are less flammable than conifer species, it is reasonable to believe that a potential expansion of deciduous species in boreal forests, either occurring naturally or through landscape management, could offset some of the impacts of climate change on the occurrence of boreal wildfires. The objective of this study was to determine the potential of this offsetting effect through a simulation experiment conducted in eastern boreal North America. Predictions of future fire activity were made using multivariate adaptive regression splines (MARS) with fire behavior indices and ecological niche models as predictor variables so as to take into account the effects of changing climate and tree distribution on fire activity. A regional climate model (RCM) was used for predictions of future fire risk conditions. The experiment was conducted under two tree dispersal scenarios: the status quo scenario, in which the distribution of forest types does not differ from the present one, and the unlimited dispersal scenario, which allows forest types to expand their range to fully occupy their climatic niche. Our results show that future warming will create climate conditions that are more prone to fire occurrence. However, unlimited dispersal of southern restricted deciduous species could reduce the impact of climate change on future fire occurrence. Hence, the use of deciduous species could be a good option for an efficient strategic fire mitigation strategy aimed at reducing fire Propagation in coniferous landscapes and increasing public safety in remote populated areas of eastern boreal Canada under climate change.

Control of the multimillennial wildfire size in boreal North America by spring climatic conditions.

Wildfire activity in North American boreal forests increased during the last decades of the 20th century, partly owing to ongoing human-caused climatic changes. How these changes affect regional fire regimes (annual area burned, seasonality, and number, size, and severity of fires) remains uncertain as data available to explore fire-climate-vegetation interactions have limited temporal depth. Here we present a Holocene reconstruction of fire regime, combining lacustrine charcoal analyses with past drought and fire-season length simulations to elucidate the mechanisms linking long-term fire regime and climatic changes. We decomposed fire regime into fire frequency (FF) and biomass burned (BB) and recombined these into a new index to assess fire size (FS) fluctuations. Results indicated that an earlier termination of the fire season, due to decreasing summer radiative insolation and increasing precipitation over the last 7.0 ky, induced a sharp decrease in FF and BB ca. 3.0 kyBP toward the present. In contrast, a progressive increase of FS was recorded, which is most likely related to a gradual increase in temperatures during the spring fire season. Continuing climatic warming could lead to a change in the fire regime toward larger spring wildfires in eastern boreal North America.

Priority wetland invertebrates as conservation surrogates.

Invertebrates are important functionally in most ecosystems, but seldom appraised as surrogate indicators of biological diversity. Priority species might be good candidates; thus, here we evaluated whether three freshwater invertebrates listed in the U.K. Biodiversity Action Plan indicated the richness, composition, and conservation importance of associated wetland organisms as defined respectively by their alpha diversity, beta diversity, and threat status. Sites occupied by each of the gastropods Segmentina nitida, Anisus vorticulus, and Valvata macrostoma had greater species richness of gastropods and greater conservation importance than other sites. Each also characterized species assemblages associated with significant variations between locations in alpha or beta diversity among other mollusks and aquatic macrophytes. Because of their distinct resource requirements, conserving the three priority species extended the range of wetland types under management for nature conservation by 18% and the associated gastropod niche-space by around 33%. Although nonpriority species indicated variations in richness, composition, and conservation importance among other organisms as effectively as priority species, none characterized such a wide range of high-quality wetland types. We conclude that priority invertebrates are no more effective than nonpriority species as indicators of alpha and beta diversity or conservation importance among associated organisms. Nevertheless, conserving priority species can extend the array of distinct environments that are protected for their specialized biodiversity and environmental quality. We suggest that this is a key role for priority species and conservation surrogates more generally, and, on our evidence, can best be delivered through multiple species with contrasting habitat requirements.