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.

A mutated factor X activatable by thrombin corrects bleedings in vivo in a rabbit model of antibody-induced hemophilia A.

Rendering coagulation factor X sensitive to thrombin was proposed as a strategy that can bypass the need for factor VIII. In this paper, this non-replacement strategy was evaluated in vitro and in vivo in its ability to correct factor VIII but also factor IX, X and XI deficiencies. A novel modified factor X, named Actiten, was generated and produced in the HEK293F cell line. The molecule possesses the required post-translational modifications, partially keeps its ability to be activated by RVV-X, factor VIIa/tissue factor, factor VIIIa/factor IXa and acquires the ability to be activated by thrombin. The potency of the molecule was evaluated in respective deficient plasmas or hemophilia A plasmas, for some with inhibitors. Actiten corrects dose dependently all the assayed deficient plasmas. It is able to normalize the thrombin generation at 20 µg/mL showing however an increased lagtime. It was then assayed in a rabbit antibody-induced model of hemophilia A where, in contrast to recombinant factor X wild-type, it normalized the bleeding time and the loss of hemoglobin. No sign of thrombogenicity was observed and the generation of activated factor X was controlled by the anticoagulation pathway in all performed coagulation assays. This data indicates that Actiten may be considered as a possible non replacement factor to treat hemophilia's with the advantage of being a zymogen correcting bleedings only when needed.

Hyper-Enriched Anti-RSV Immunoglobulins Nasally Administered: A Promising Approach for Respiratory Syncytial Virus Prophylaxis.

Respiratory syncytial virus (RSV) is a public health concern that causes acute lower respiratory tract infection. So far, no vaccine candidate under development has reached the market and the only licensed product to prevent RSV infection in at-risk infants and young children is a monoclonal antibody (Synagis®). Polyclonal human anti-RSV hyper-immune immunoglobulins (Igs) have also been used but were superseded by Synagis® owing to their low titer and large infused volume. Here we report a new drug class of immunoglobulins, derived from human non hyper-immune plasma that was generated by an innovative bioprocess, called Ig cracking, combining expertises in plasma-derived products and affinity chromatography. By using the RSV fusion protein (F protein) as ligand, the Ig cracking process provided a purified and concentrated product, designated hyper-enriched anti-RSV IgG, composed of at least 15-20% target-specific-antibodies from normal plasma. These anti-RSV Ig displayed a strong in vitro neutralization effect on RSV replication. Moreover, we described a novel prophylactic strategy based on local nasal administration of this unique hyper-enriched anti-RSV IgG solution using a mouse model of infection with bioluminescent RSV. Our results demonstrated that very low doses of hyper-enriched anti-RSV IgG can be administered locally to ensure rapid and efficient inhibition of virus infection. Thus, the general hyper-enriched Ig concept appeared a promising approach and might provide solutions to prevent and treat other infectious diseases. IMPORTANCE: Respiratory Syncytial Virus (RSV) is the major cause of acute lower respiratory infections in children, and is also recognized as a cause of morbidity in the elderly. There are still no vaccines and no efficient antiviral therapy against this virus. Here, we described an approach of passive immunization with a new class of hyper-enriched anti-RSV immunoglobulins (Ig) manufactured from human normal plasma. This new class of immunoglobulin plasma derived product is generated by an innovative bioprocess, called Ig cracking, which requires a combination of expertise in both plasma derived products and affinity chromatography. The strong efficacy in a small volume of these hyper-enriched anti-RSV IgG to inhibit the viral infection was demonstrated using a mouse model. This new class of immunoglobulin plasma-derived products could be applied to other pathogens to address specific therapeutic needs in the field of infectious diseases or even pandemics, such as COVID-19.

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.

Plasmacytoid dendritic cells respond to Epstein-Barr virus infection with a distinct type I interferon subtype profile.

Infectious mononucleosis, caused by infection with the human gamma-herpesvirus Epstein-Barr virus (EBV), manifests with one of the strongest CD8+ T-cell responses described in humans. The resulting T-cell memory response controls EBV infection asymptomatically in the vast majority of persistently infected individuals. Whether and how dendritic cells (DCs) contribute to the priming of this near-perfect immune control remains unclear. Here we show that of all the human DC subsets, plasmacytoid DCs (pDCs) play a central role in the detection of EBV infection in vitro and in mice with reconstituted human immune system components. pDCs respond to EBV by producing the interferon (IFN) subtypes α1, α2, α5, α7, α14, and α17. However, the virus curtails this type I IFN production with its latent EBV gene products EBNA3A and EBNA3C. The induced type I IFNs inhibit EBV entry and the proliferation of latently EBV-transformed B cells but do not influence lytic reactivation of the virus in vitro. In vivo, exogenous IFN-α14 and IFN-α17, as well as pDC expansion, delay EBV infection and the resulting CD8+ T-cell expansion, but pDC depletion does not significantly influence EBV infection. Thus, consistent with the observation that primary immunodeficiencies compromising type I IFN responses affect only alpha- and beta-herpesvirus infections, we found that EBV elicits pDC responses that transiently suppress viral replication and attenuate CD8+ T-cell expansion but are not required to control primary infection.

[The alternative NF-kB activation pathway and cancer: friend or foe?]. / Voie alternative d'activation de NF-kB et cancer: amis ou ennemis?

The family of NF-kappaB transcription factors plays a key role in diverse biological processes, such as inflammatory and immune responses, cell survival and tumor development. A new NF-kappaB activation pathway has been recently uncovered, the so-called alternative NF-kappaB activation pathway. It has been shown that this pathway mainly controls the activity of RelB, a member of the NF-kappaB family, and its activation requires IKKalpha, one of the two catalytic subunits of the IKK complex. Recent studies suggest that constitutive activation of the alternative pathway may be involved in tumor development, but most surprisingly it is also reported that IKKalpha exerts a protective function against uncontrolled cell proliferation and cancer development. This review discusses recent progresses in the understanding on how the alternative pathway may participate in cancer development, focusing more specifically on RelB and IKKalpha.

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.

BAFF activation of the ERK5 MAP kinase pathway regulates B cell survival.

B cell activating factor (BAFF) stimulation of the BAFF receptor (BAFF-R) is essential for the homeostatic survival of mature B cells. Earlier in vitro experiments with inhibitors that block MEK 1 and 2 suggested that activation of ERK 1 and 2 MAP kinases is required for BAFF-R to promote B cell survival. However, these inhibitors are now known to also inhibit MEK5, which activates the related MAP kinase ERK5. In the present study, we demonstrated that BAFF-induced B cell survival was actually independent of ERK1/2 activation but required ERK5 activation. Consistent with this, we showed that conditional deletion of ERK5 in B cells led to a pronounced global reduction in mature B2 B cell numbers, which correlated with impaired survival of ERK5-deficient B cells after BAFF stimulation. ERK5 was required for optimal BAFF up-regulation of Mcl1 and Bcl2a1, which are prosurvival members of the Bcl-2 family. However, ERK5 deficiency did not alter BAFF activation of the PI3-kinase-Akt or NF-κB signaling pathways, which are also important for BAFF to promote mature B cell survival. Our study reveals a critical role for the MEK5-ERK5 MAP kinase signaling pathway in BAFF-induced mature B cell survival and homeostatic maintenance of B2 cell numbers.

IKK-induced NF-κB1 p105 proteolysis is critical for B cell antibody responses to T cell-dependent antigen.

The importance of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in B cells was investigated using Nfkb1(SSAA/SSAA) mice, in which this NF-κB signaling pathway is blocked. Nfkb1(SSAA) mutation had no effect on the development and homeostasis of follicular mature (FM) B cells. However, analysis of mixed bone marrow chimeras revealed that Nfkb1(SSAA/SSAA) FM B cells were completely unable to mediate T cell-dependent antibody responses. Nfkb1(SSAA) mutation decreased B cell antigen receptor (BCR) activation of NF-κB in FM B cells, which selectively blocked BCR stimulation of cell survival and antigen-induced differentiation into plasmablasts and germinal center B cells due to reduced expression of Bcl-2 family proteins and IRF4, respectively. In contrast, the antigen-presenting function of FM B cells and their BCR-induced migration to the follicle T cell zone border, as well as their growth and proliferation after BCR stimulation, were not affected. All of the inhibitory effects of Nfkb1(SSAA) mutation on B cell functions were rescued by normalizing NF-κB activation genetically. Our study identifies critical B cell-intrinsic functions for IKK-induced NF-κB1 p105 proteolysis in the antigen-induced survival and differentiation of FM B cells, which are essential for T-dependent antibody responses.

RelA repression of RelB activity induces selective gene activation downstream of TNF receptors.

TNF-alpha is a potent proinflammatory cytokine that regulates immune and inflammatory responses and programmed cell death. TNF-alpha stimulation causes nuclear translocation of several NF-kappaB dimers, including RelA/p50 and RelB/p50. However, contrary to RelA, RelB entering the nucleus in response to TNF-alpha cannot bind to DNA in mouse embryonic fibroblasts, strongly suggesting that RelB DNA-binding activity is modulated by additional nuclear mechanisms. Here, we demonstrate that TNF-alpha promotes the association of RelA with RelB in the nucleus and that TNF-alpha-induced RelA/RelB heterodimers do not bind to kappaB sites. Remarkably, we show that RelA serine-276, the phosphorylation of which is induced by TNF receptor ligation, is crucial for RelA/RelB complex formation and subsequent inhibition of RelB DNA binding. In the absence of RelA phosphorylation on serine-276, TNF-alpha stimulation leads to a strong increase in the expression of endogenous NF-kappaB-responsive genes, such as Bcl-xL, whose transcriptional up-regulation is mainly controlled by RelB. Our findings demonstrate that RelA has a major regulatory role serving to dampen RelB activity in response to TNF-alpha and define a previously unrecognized mechanism that represents an essential step leading to selective NF-kappaB target gene expression.