Mannose 6-phosphate receptor-targeting antibodies preserve Fc receptor-mediated recycling.

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.

The neonatal Fc receptor expression during macrophage differentiation is related to autophagy.

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.

Anticalin N- or C-Terminal on a Monoclonal Antibody Affects Both Production and In Vitro Functionality.

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.

4C3 Human Monoclonal Antibody: A Proof of Concept for Non-pathogenic Proteinase 3 Anti-neutrophil Cytoplasmic Antibodies in Granulomatosis With Polyangiitis.

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.

Choice of protocol for the in vivo bioassay of equine Chorionic Gonadotropin (eCG / PMSG) in immature female rats.

Equine Chorionic Gonadotropin (eCG) previously known as Pregnant Mare Serum Gonadotropin (PMSG) has been used for decades in regulating reproduction in various domestic animal species. Its use necessitates a good measurement of its bioactivity in commercial preparations. The EUROPEAN PHARMACOPEIA (EP 7.0) recommends 5-6 subcutaneous injections in immature female rats for the in vivo bioassay for eCG as in the case for measurement of FSH bioactivity in the Steelman & Pohley assay (1953). This recommendation is in marked contrast with the classical and long-time used PMSG assay of Cole & Erway (1941) that includes only one subcutaneous injection, 3 days before measurement of ovarian weight. As this difference introduces much confusion in the determination of eCG/PMSG bioactivity in commercial sources, we have performed parallel assays of several PMSG preparations, with both protocols. The single-injection protocol takes into account the long half-life of eCG in bloodstream and provokes an ovarian stimulation at lower concentrations than the multiple-injection protocol. As the single-injection protocol also mimicks the protocol used in cattle, it is preferable to the multiple-injection protocol of the current EP.

Low reversibility of intracellular cAMP accumulation in mouse Leydig tumor cells (MLTC-1) stimulated by human Luteinizing Hormone (hLH) and Chorionic Gonadotropin (hCG).

In order to study the intracellular cAMP response kinetics of Leydig cells to hormones with LH activity, we used MLTC-1 cells transiently expressing a chimeric cAMP-responsive luciferase so that real-time variations of intracellular cAMP concentration could be followed using oxiluciferin luminescence produced from catalyzed luciferin oxidation. The potencies of the different LHs and CGs were evaluated using areas under the curves (AUC) of their kinetics over 60 min stimulation. All mammalian LHs and CGs tested were found to stimulate cAMP accumulation in these cells. The reversibility of this stimulation was studied by removing the hormone from the culture medium after 10 min of incubation. The ratios of kinetics AUC after removing or not the hormone were used to evaluate the stimulation reversibility of each hormone. Natural and recombinant hLHs and hCGs were found to exhibit slowly reversible activation compared to pituitary rat, ovine, porcine, camel and equine LHs, serum-derived eCG (PMSG) and recombinant eLH/CGs. Carbohydrate side chains are not involved in this phenomenon since natural and recombinant homologous hormones exhibit the same reversibility rates. It is still unknown whether only one human subunit, α or ß, is responsible for this behaviour or whether it is due to a particular feature of the hLH and hCG quaternary structure.

Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures.

Quaternary structure of human, bovine and ovine Follicle-Stimulating Hormones (hFSH, bFSH and oFSH) and Luteinizing Hormone was assessed in sandwich ELISAs using monoclonal anti-oFSHß or anti-oLHß antibodies, respectively, for capture and a biotinylated anti-hFSHα (α4 epitope) for detection. Neither free subunit gave any signal in this assay so that it was possible to measure the residual heterodimeric fraction after thermal treatment of the gonadotropins under study. The hormones were subjected to 5-min heating between 37 and 90 °C before rapid cooling in melting ice before ELISA. The data show half-dissociation of natural and recombinant human and ovine FSH preparations between 68 and 74 °C whereas bovine FSH preparations exhibited lower stability in these conditions with half-dissociation between 61 and 64 °C. Moreover, whereas all human and bovine as well as most ovine FSH preparations were fully dissociated at temperatures above 80 °C, one natural oFSH and one recombinant hLH preparations contained an important fraction that resisted dissociation even at 93 °C and retained in vitro bioactivity. This suggests the existence of gonadotropin αß heterodimer with covalently linked subunits. Similarly, about 20% of the recombinant hLH preparation was also found withstand heat denaturation and also probably to have cross-linked subunits. The origin and chemical nature of these inter-subunit bonds remain to be determined.