The regulatory effect of growth hormone (GH) on its target cells is mediated via the GH receptor (GHR). GH binding to the GHR resultsin the formation of a GH-(GHR) 2 complex and the initiation of signal transduction cascades via the activation of the tyrosine kinase JAK2. Subsequent endocytosis and transport to the lysosome of the ligand-receptor complex is regulated via the ubiquitin system and requires the presence of an intact ubiquitin-dependent endocytosis (UbE) motif in the cytosolic tail of the GHR. Recently, the model of ligand-induced receptor dimerization has been challenged. In this study, ligand-independent GHR dimerization is demonstrated in the endoplasmic reticulum and at the cell surface by coimmunoprecipitation of an epitope-tagged truncated GHR with wild-type GHR. In addition, evidence is provided that the extracellular domain of the GHR is not required to maintain this interaction. Internalization of a chimeric receptor, which fails to dimerize, is independent of an intact UbE-motif. Therefore, we postulate that dimerization of GHR molecules is required for ubiquitin system-dependent endocytosis.
The CD38 molecule, with its high expression on Multiple Myeloma (MM), is considered a suitable target for antibody therapy of MM. We developed daratumumab (DARA), a human CD38 monoclonal antibody (mAb) with direct and Fc-mediated cell killing activity. DARA induces killing of tumor cells, mainly via complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers M. J Immunol 2011), and antibody-dependent cellular phagocytosis (ADCP) by macrophages (both murine and human). In addition, DARA induces apoptosis upon secondary cross-linking and modulates CD38 enzymatic function. DARA is currently in phase I, II and III clinical evaluation in patients with MM. Besides DARA, several other anti-CD38 mAb are in development; SAR650894 (SAR; clone 38SB19; Sanofi-Aventis) for MM and other CD38+ hematological malignancies, MOR03087 (MOR; Morphosys) for relapsed/refractory MM and Ab79 (Millenium/Takeda) which is in preclinical development. Similar mechanisms of action (MoA) are described for these mAb; nevertheless direct comparison studies would be critical for differentiation among these antibodies. . In this study, the efficacy of these anti-CD38 mAb was directly compared to DARA with respect to binding, apoptosis, CD38 ectoenzyme activity, and the induction of ADCC, ADCP and CDC. Surrogate antibodies of MOR, SAR and Ab79 were generated on the basis of protein sequences, as published in their corresponding patents families, and were attached to the backbone of DARA. Binding to CD38 expressing Daudi tumor cells was assessed by flow cytomery. All CD38 antibodies showed similar EC50 (~0.1 µg/mL) and maximal binding, except MOR which showed a lower apparent affinity (~0.3 µg/mL). Previously, CD38 amino acid residues Q272 and S274 were reported as critical for DARA binding. ELISA analyses using CD38 point mutants revealed MOR, SAR and Ab79 not to be affected by mutation of these residues. All CD38 mAb were equally potent in inducing ADCC of Daudi cells (40-60% lysis, 0.02 µg/mL), in classic Cr51-release ADCC assay using human PBMC effector cells (E:T ratio 100:1). Important differences were observed with respect to induction of CDC. SAR was unable to induce CDC in Daudi cells at concentrations up to 30 µg/mL, while DARA induced more than 80% lysis at concentrations above 1 µg/mL. Ab79 and MOR induced CDC, yet maximum lysis was 50% and 20%, respectively. Evaluation of Annexin V/propidium iodide (AnnV/PI) staining and activation of caspase-3 showed that only SAR induced AnnV/PI+ in Ramos cells (~40%) after 48 h exposure without Fc crosslinking, but did not activate caspase-3. In the presence of Fc crosslinking antibodies, all anti-CD38 mAb induced AnnV/PI+, caspase-3 mediated apoptosis. In enzyme activity assays using purified CD38 protein, SAR inhibited generation of cGDPR (indicative of the combined CD38 cyclase activity generating fluorescent cGDPR and hydrolase activity converting cGDPR into GDPR)more effectively than DARA (~70% vs. ~20% inhibition at 30 µg/mL). Ab79 had a modest effect on CD38 enzyme activity (~10% inhibition). MOR did not affect CD38 enzyme activity at all. The capacity to induce ADCP was only tested for DARA, MOR and Ab79 using mouse macrophages (mφ) as effector cells and Daudi target cells. mφ, isolated and matured from bone marrow cells, and calcein-AM labelled target cells (E:T ratio 1:1) were cultured in the presence of 0.0006-5 µg/mL antibody for 4 h. Non-phagocytosed target cells and mφ were collected and ADCP was evaluated by flow cytometry. All CD38 mAb induced mφ-mediated phagocytosis, as observed by a concentration dependent increase in the number of double positive mφ and killing of target cells. Ab79 was as effective as DARA (EC50 ~0.01 µg/mL) in ADCP induction, whereas MOR was less effective (EC50 0.04 µg/mL). In summary, DARA and surrogate mAb of MOR, SAR and Ab79 showed similar binding to cells and induced similar amounts of ADCC. Differences between these mAb involved the ability to directly induce apoptosis, to inhibit the enzymatic activity of CD38 and to induce ADCP. The most striking difference was observed for the ability to induce CDC, the MoA which is currently believed the most important mechanism of MM cell killing in the clinic. DARA efficiently induced high levels of CDC at low concentrations, whereas the other CD38 mAb were unable or less capable to induce CDC. Disclosures Lammerts van Bueren: Genmab: Employment, warrents Other. Jakobs:Genmab: Employment, warrents Other. Kaldenhoven:Genmab: Employment, warrents Other. Roza:Genmab: Employment, warrents Other. Hiddingh:Genmab: Employment. Meesters:Genmab: Employment, warrents Other. Voorhorst:Genmab: Employment, warrents Other. Gresnigt:Genmab: Employment, warrents Other. Wiegman:Genmab: Employment, warrents Other. Ortiz Buijsse:Genmab: Employment, warrents Other. Andringa:Genmab: Employment, warrents Other. Overdijk:Genmab: Employment, warrents Other. Doshi:Janssen R&D: Employment. Sasser:Janssen R&D: Employment. de Weers:Genmab: Employment, warrents Other. Parren:Genmab: Employment, inventor on patents regarding daratumumab Patents & Royalties, stock and warrents Other.
Transient expression systems in mammalian cells have become the method of choice for producing research quantities of antibodies. Both the speed and yield of the available transient systems and the natural posttranslational modifications favor these systems above expression in lower eukaryotes, prokaryotes or stable cell lines. We describe an optimized mammalian transient expression system, capable of producing up to 400mg/L of native secreted antibodies in less than a week. The system is composed of commercially available components and is based on expression in the fast growing suspension cell line, FreeStyle™ 293-F (HEK-293F). The method depends on an optimal combination of a gene transfer method, an expression vector and cotransfection with expression enhancing plasmids, encoding the large T antigen of the SV40 virus and the cell cycle inhibitors p21 and p27. Optimization of all components of the expression system, by experimental design techniques, yielded maximal expression levels (including antibody isotypes IgG1, 2, 3, 4 and Fab fragments of various species). Expression volumes were scalable from 0.1 ml up to 1.2L in a simple shaker flask system in animal component free, low protein medium, enabling consistent production of relatively high amounts of a large number of native antibodies.
Multidrug-resistant Neisseria gonorrhoeae is a global health problem. Monoclonal antibody (mAb) 2C7 recognizes a gonococcal lipooligosaccharide epitope that is expressed by >95% of clinical isolates and hastens gonococcal vaginal clearance in mice. Chimeric mAb 2C7 (human immunoglobulin G1 [IgG1]) with an E430G Fc modification that enhances Fc:Fc interactions and hexamerization following surface-target binding and increases complement activation (HexaBody technology) showed significantly greater C1q engagement and C4 and C3 deposition compared to mAb 2C7 with wild-type Fc. Greater complement activation by 2C7-E430G Fc translated to increased bactericidal activity in vitro and, consequently, enhanced efficacy in mice, compared with “Fc-unmodified” chimeric 2C7. Gonococci bind the complement inhibitors factor H (FH) and C4b-binding protein (C4BP) in a human-specific manner, which dampens antibody (Ab)-mediated complement-dependent killing. The variant 2C7-E430G Fc overcame the barrier posed by these inhibitors in human FH/C4BP transgenic mice, for which a single 1 μg intravenous dose cleared established infection. Chlamydia frequently coexists with and exacerbates gonorrhea; 2C7-E430G Fc also proved effective against gonorrhea in gonorrhea/chlamydia-coinfected mice. Complement activation alone was necessary and sufficient for 2C7 function, evidenced by the fact that (1) “complement-inactive” Fc modifications that engaged Fc gamma receptor (FcγR) rendered 2C7 ineffective, nonetheless; (2) 2C7 was nonfunctional in C1q −/− mice, when C5 function was blocked, or in C9 −/− mice; and (3) 2C7 remained effective in neutrophil-depleted mice and in mice treated with PMX205, a C5a receptor (C5aR1) inhibitor. We highlight the importance of complement activation for antigonococcal Ab function in the genital tract. Elucidating the correlates of protection against gonorrhea will inform the development of Ab-based gonococcal vaccines and immunotherapeutics.
Endocytosis of the growth hormone receptor (GHR) is regulated by the ubiquitin-conjugating system. A cytosolic 10 amino acid motif, referred to as the ubiquitin-dependent endocytosis (UbE) motif, is involved in the ubiquitination as well as in the endocytosis of the receptor. Proteins that are implicated in one of these processes have not been identified so far. Using a glutathione S-transferase (GST)-pulldown assay with a GST fusion protein encompassing the UbE motif of the GHR, a 35 kDa protein was purified. The protein was identified by MS as small glutamine-rich tetratricopeptide repeat (TPR)-containing protein (SGT). We found that GHR interacts with SGT. In vivo, both the precursor and the mature form of the receptor interacted with SGT. Inactivation of the ubiquitin-conjugating system did not affect the GHR-SGT interaction. Binding studies showed that the first TPR motif of SGT interacts with the UbE motif of the GHR. Taken together, these data show that SGT is a GHR-interacting protein, which binds independent of the ubiquitin-conjugating system.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.