Antibody-induced Internalization Research Articles

Fc gamma receptors promote antibody-induced LILRB4 internalization and immune regulation of monocytic AML.

The immune checkpoint leukocyte immunoglobulin-like receptor B4 (LILRB4) is found specifically on the cell surface of acute monocytic leukemia (monocytic AML), an aggressive and common subtype of AML. We have developed a humanized monoclonal IgG1 LILRB4-blocking antibody (h128-3), which improved immune regulation but reduced cell surface expression of LILRB4 in monocytic AML models by 40-60%. Interestingly, most of this effect was neutralized by mutation of the Fc region of the antibody (h128-3/N297A), which prevents interaction with Fc gamma receptors (FcγRs). This suggested that there is FcγR-dependent antigenic modulation underlying h128-3's effects, a mechanism known to alter the function of antibodies targeting B-cell malignancies. We disrupted the Fc-FcγR interaction pharmacologically and with stable CRISPR-Cas9-mediated genetic knockout of FcγRs in monocytic AML cell lines to investigate the role of FcγR-dependent antigenic modulation in the regulation of LILRB4 by h128-3. When FcγRI is inhibited or removed from the surface of monocytic AML cells, h128-3 cannot optimally perform its blocking function, resulting in activation of the LILRB4 inhibitory receptor and leading to a 15-25% decrease in T-cell-mediated cytotoxicity in vitro. In the absence of FcγRI, scaffolding by FcγRIIa allows h128-3 to maintain LILRB4-blocking function. Here we define a FcγR-dependent antigenic modulation mechanism underlying the function of an immunoreceptor blocking antibody for the first time in myeloid malignancy. This research will facilitate the development of safe, precision-targeted antibody therapeutics in myeloid malignancies with greater potency and efficacy.

281 Fc gamma receptor crosslinking promotes antibody-induced LILRB4 internalization and immune regulation of acute monocytic leukemia

OBJECTIVES/GOALS: The aims of this study were to: 1) determine the involvement of each Fc gamma receptor isoform in antibody-mediated crosslinking and internalization of LILRB4 in acute monocytic leukemia and 2) elucidate the role of this crosslinking and internalization in antibody-mediated immune regulation of these malignant cells. METHODS/STUDY POPULATION: To determine the involvement of Fc gamma receptors in antibody-mediated complex internalization, we generated acute monocytic leukemia cell lines with CRISPR-Cas9 knockout of each Fc gamma receptor isoform. We tested the effects of each knockout on anti-LILRB4 antibody-mediated internalization by flow cytometry and confirmed our findings with confocal microscopy. To elucidate the role of this crosslinking and internalization in immune regulation of acute monocytic leukemia, we conducted preliminary ELISA-based studies of the inflammatory signaling and cytokine release profiles of wild-type and Fc gamma receptor knockout cells treated with the LILRB4-targeting antibody. RESULTS/ANTICIPATED RESULTS: We have concluded that Fc gamma receptor I (CD64) plays a role in LILRB4 crosslinking and internalization by our anti-LILRB4 antibody and there are also contributions from Fc gamma receptor IIA (CD32A) observed in the absence of CD64 on the cell surface. Preliminary signaling studies have demonstrated that Fc gamma receptor-mediated antibody crosslinking and internalization of LILRB4 decreases anti-inflammatory signaling downstream of LILRB4 as well as pro-inflammatory signaling downstream of Fc gamma receptors, particularly in the absence of CD64 on the cell surface. The immunomodulatory effect of antibody-mediated LILRB4 crosslinking and internalization is being confirmed in follow-up signaling, cytokine release and lymphocyte activation studies. DISCUSSION/SIGNIFICANCE: This study will improve the efficacy of LILRB4-targeting antibody therapeutics for patients suffering from acute monocytic leukemia and help characterize CD64 and CD32A as potential clinical biomarkers for patients undergoing LILRB4-targeting antibody immunotherapeutic treatment, currently in first-in-human clinical trials.

Abstract 2886: Antigen internalization and its prevention during treatment with bispecific antibodies

Abstract Antigen-mediated antibody internalization is an important factor influencing kinetics and efficacy of antibody-based therapeutics. Antibodies directed against HER2, CD20, FLT3, EGFR, CD10 and CD22 have been shown to undergo internalization upon binding to their target antigens. Although antibody-induced internalization may be desirable under some circumstances, for example in case of antibody-drug conjugates, it can subvert the therapeutic efficacy of ADCC-inducing antibodies and bispecific antibodies (bsAbs) aiming to mobilize T cells against cancer. Here we examined the turnover of PSMA, a target antigen widely used for diagnostic and therapeutic interventions in prostate cancer. As a model system, we used prostate cancer cell lines expressing varying levels of PSMA in functional analyses with our PSMAxCD3 bsAb CC-1 which is currently being evaluated in two clinical trials (NCT04104607 and NCT04496674). Antigen internalization kinetics was analyzed by flow cytometric-internalization assays and live-cell imaging of antibody internalization. These analyses revealed that surface PSMA expression is rapidly downregulated after binding of PSMAxCD3 bsAb. Internalization occured in a dose-dependent manner, with complete negativity observed at concentrations ≥0.1 µg/ml. After withdrawal of the bsAb, antigen surface levels were fully restored within 24 hours. PSMA internalization was found to be partially prevented by inhibiting clathrin-dependent endocytosis. Currently, we are screening various clinically approved clathrin-dependent endocytosis inhibitors (e.g. statins and chloroquine) to identify compounds for combinatorial approaches to prevent undesired PSMA internalization in patients receiving PSMAxCD3 bsAbs. Altogether, our results document the involvement of clathrin-dependent endocytosis in bsAbs-driven PSMA internalization and provide a basis for combinatorial approaches to increase the efficacy of PSMA-directed therapeutics. Citation Format: Maria Klimovich, Latifa Zekri, Gundram Jung, Helmut R. Salih. Antigen internalization and its prevention during treatment with bispecific antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2886.

HIV-1 Envelope Glycoprotein Cell Surface Localization Is Associated with Antibody-Induced Internalization.

To minimize immune responses against infected cells, HIV-1 has evolved different mechanisms to limit the surface expression of its envelope glycoproteins (Env). Recent observations suggest that the binding of certain broadly neutralizing antibodies (bNAbs) targeting the ‘closed’ conformation of Env induces its internalization. On the other hand, non-neutralizing antibodies (nNAbs) that preferentially target Env in its ‘open’ conformation, remain bound to Env on the cell surface for longer periods of time. In this study, we attempt to better understand the underlying mechanisms behind the differential rates of antibody-mediated Env internalization. We demonstrate that ‘forcing’ open Env using CD4 mimetics allows for nNAb binding and results in similar rates of Env internalization as those observed upon the bNAb binding. Moreover, we can identify distinct populations of Env that are differentially targeted by Abs that mediate faster rates of internalization, suggesting that the mechanism of antibody-induced Env internalization partially depends on the localization of Env on the cell surface.

Antibody-Induced Internalization of HIV-1 Env Proteins Limits Surface Expression of the Closed Conformation of Env.

To minimize immune responses against infected cells, HIV-1 limits the surface expression of its envelope glycoprotein (Env). Here, we demonstrate that this mechanism is specific for the Env conformation and affects the efficiency of antibody-dependent cellular cytotoxicity (ADCC). Using flow cytometry and confocal microscopy, we show that broadly neutralizing antibodies (bNAbs) targeting the "closed" conformation of Env induce its internalization from the surface. In contrast, non-neutralizing antibodies (nNAbs) are displayed on the cell surface for prolonged period of times. The bNAb-induced Env internalization can be decreased by blocking dynamin function, which translates into higher susceptibilities of infected cells to ADCC. Our results suggest that antibody-mediated Env internalization is a mechanism used by HIV-1 to evade immune responses against the "closed" conformation of Env expressed on HIV-1-infected cells.IMPORTANCE HIV-1 has evolved to acquire several strategies to limit the exposure of its envelope glycoproteins (Env) on the surface of infected cells. In this study, we show that antibody-induced Env internalization is conformation specific and reduces the susceptibility of infected cells to antibody-dependent cellular cytotoxicity (ADCC). Thus, a better understanding of this mechanism might help develop antibodies with improved capacities to mediate ADCC.

Anti-NMDA Receptor Encephalitis: Clinical Features and Basic Mechanisms.

In slightly more than 10 years, anti-NMDA receptor (NMDAR) encephalitis has changed from a rare paraneoplastic syndrome to the most common cause of nonviral encephalitis. It presents fulminantly with progressive psychosis, seizures, and autonomic dysfunction, leading to death if untreated. However, rapid recognition and treatment can lead to survival and a return to baseline levels of functioning in many patients. While initially associated with ovarian teratomas, it is now associated with other tumors and can reflect a postviral event. The antibodies to the NMDAR made in this syndrome are pathogenic and are directed at the extracellular domain of the GluN1 subunit. Such antibodies lead to internalization of NMDARs in model systems, leading to a physiological state characterized by NMDAR hypofunction. Analogous disorders, characterized by antibodies to other synaptic receptors, present with neurological and psychiatric dysfunction and also appear to reflect antibody-induced internalization of receptors. However, this simple pathophysiology may be too simplistic to reflect the complexity of events in anti-NMDAR encephalitis. Future scientific investigations may allow a more complete understanding of this disorder and improve treatment of anti-NMDAR encephalitis.

Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein.

Respiratory syncytial virus (RSV) infections remain a major cause of respiratory disease and hospitalizations among infants. Infection recurs frequently and establishes a weak and short-lived immunity. To date, RSV immunoprophylaxis and vaccine research is mainly focused on the RSV fusion (F) protein, but a vaccine remains elusive. The RSV F protein is a highly conserved surface glycoprotein and is the main target of neutralizing antibodies induced by natural infection. Here, we analyzed an internalization process of antigen-antibody complexes after binding of RSV-specific antibodies to RSV antigens expressed on the surface of infected cells. The RSV F protein and attachment (G) protein were found to be internalized in both infected and transfected cells after the addition of either RSV-specific polyclonal antibodies (PAbs) or RSV glycoprotein-specific monoclonal antibodies (MAbs), as determined by indirect immunofluorescence staining and flow-cytometric analysis. Internalization experiments with different cell lines, well-differentiated primary bronchial epithelial cells (WD-PBECs), and RSV isolates suggest that antibody internalization can be considered a general feature of RSV. More specifically for RSV F, the mechanism of internalization was shown to be clathrin dependent. All RSV F-targeted MAbs tested, regardless of their epitopes, induced internalization of RSV F. No differences could be observed between the different MAbs, indicating that RSV F internalization was epitope independent. Since this process can be either antiviral, by affecting virus assembly and production, or beneficial for the virus, by limiting the efficacy of antibodies and effector mechanism, further research is required to determine the extent to which this occurs in vivo and how this might impact RSV replication.IMPORTANCE Current research into the development of new immunoprophylaxis and vaccines is mainly focused on the RSV F protein since, among others, RSV F-specific antibodies are able to protect infants from severe disease, if administered prophylactically. However, antibody responses established after natural RSV infections are poorly protective against reinfection, and high levels of antibodies do not always correlate with protection. Therefore, RSV might be capable of interfering, at least partially, with antibody-induced neutralization. In this study, a process through which surface-expressed RSV F proteins are internalized after interaction with RSV-specific antibodies is described. One the one hand, this antigen-antibody complex internalization could result in an antiviral effect, since it may interfere with virus particle formation and virus production. On the other hand, this mechanism may also reduce the efficacy of antibody-mediated effector mechanisms toward infected cells.

Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies

Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to ∼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications.

Mechanisms of Disease: Pemphigus and Bullous Pemphigoid.

Pemphigus and bullous pemphigoid are autoantibody-mediated blistering skin diseases. In pemphigus, keratinocytes in epidermis and mucous membranes lose cell-cell adhesion, and in pemphigoid, the basal keratinocytes lose adhesion to the basement membrane. Pemphigus lesions are mediated directly by the autoantibodies, whereas the autoantibodies in pemphigoid fix complement and mediate inflammation. In both diseases, the autoantigens have been cloned and characterized; pemphigus antigens are desmogleins (cell adhesion molecules in desmosomes), and pemphigoid antigens are found in hemidesmosomes (which mediate adhesion to the basement membrane). This knowledge has enabled diagnostic testing for these diseases by enzyme-linked immunosorbent assays and dissection of various pathophysiological mechanisms, including direct inhibition of cell adhesion, antibody-induced internalization of antigen, and cell signaling. Understanding these mechanisms of disease has led to rational targeted therapeutic strategies.

Abstract 640: Probing the tumorigenic properties of two pore potassium channels using inhibitory anti-KCNK9 mAbs

Abstract Ion channels are important portals that facilitate ionic passage across membranes in all organisms. KCNK9, a member of the family of two-pore domain potassium K+ channels, mediates “leak” currents critical to maintaining cell resting membrane potentials. Since its identification, KCNK9 has been implicated in various human disorders including cancer. KCNK9 overexpression has been observed in melanoma, colorectal, breast and lung tumors and KCNK9 has been found to promote tumor survival and growth, by increasing resistance to hypoxia and apoptosis. However, KCNK9's underlying pathophysiologic mechanisms have been elusive due to the absence of specific inhibitors. Here, we describe the development of novel monoclonal antibodies directed against the KCNK9 extracellular domain and their molecular/cellular effects on KCNK9-expressing cells. Murine monoclonal IgG1 antibodies were raised against recombinant KCNK9 extracellular domains expressed in a mammalian system. 40 monoclonal antibodies were generated and demonstrated channel subtype-specific recognition of human KCNK9. Among them, 1 antibody designated Y4-mAb displayed sub-nanomolar binding affinity and inhibited KCNK9 channel conductance by up to 30% when tested in a fluorescent ion-influx assay. Through confocal microscopic analysis, channel inhibition was found to result from antibody-induced internalization and endocytosis of cell-surface channels. Y4-mAb induced up to 30∼40% reduction in cell viability and increased cell death in BEN non-small cell lung carcinoma cells and BT-549 and 410.4 breast cancer cells. In addition to “direct cell killing”, this antibody activated complement, resulting in complement-dependent cancer cell cytotoxicity in vitro. Initial experiments in vivo showed that systemic Y4-mAb inhibited the capacity of BEN non-small cell lung carcinoma cells to propagate subcutaneous tumor xenografts in nude mice (p<0.05). In conclusion, we have generated a high affinity KCNK9-specific monoclonal antibody that perturbs KCNK9 function and inhibits tumor cell survival and xenograft formation. Anti-KCNK9 antibodies have the potential to be valuable research tools and may lead to novel therapeutics. Moreover, our strategies should be generally applicable to studying the functions of other ion channels in cancer. Citation Format: Han Sun, Liqun Luo, Bachchu Lal, John Laterra, Min Li. Probing the tumorigenic properties of two pore potassium channels using inhibitory anti-KCNK9 mAbs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 640. doi:10.1158/1538-7445.AM2015-640

Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies.

Monoclonal antibody-induced ErbB3 receptor internalization and degradation inhibits growth and migration of human melanoma cells

Members of the ErbB receptor family are targets of a growing numbers of small molecules and monoclonal antibodies inhibitors currently under development for the treatment of cancer. Although historical efforts have been directed against ErbB1 (EGFR) and ErbB2 (HER2/neu), emerging evidences have pointed to ErbB3 as a key node in the activation of proliferation/survival pathways from the ErbB receptor family and have fueled enthusiasm toward the clinical development of anti-ErbB3 agents. In this study, we have evaluated the potential therapeutic efficacy of a set of three recently generated anti-human ErbB3 monoclonals, A2, A3 and A4, in human primary melanoma cells. We show that in melanoma cells expressing ErbB1, ErbB3 and ErbB4 but not ErbB2 receptor ligands activate the PI3K/AKT pathway, and this leads to increased cell proliferation and migration. While antibodies A3 and A4 are able to potently inhibit ligand-induced signaling, proliferation and migration, antibody A2 is unable to exert this effect. In attempt to understand the mechanism of action and the basis of this different behavior, we demonstrate, through a series of combined approaches, that antibody efficacy strongly correlates with antibody-induced receptor internalization, degradation and inhibition of receptor recycling to the cell surface. Finally, fine epitope mapping studies through a peptide array show that inhibiting vs. non-inhibiting antibodies have a dramatically different mode of binding to the to the receptor extracellular domain. Our study confirms the key role of ErbB3 and points to exploitation of novel combination therapies for treatment of malignant melanoma.

Polyubiquitination of Insulin-like Growth Factor I Receptor (IGF-IR) Activation Loop Promotes Antibody-induced Receptor Internalization and Down-regulation

Ubiquitination has been implicated in negatively regulating insulin-like growth factor I receptor (IGF-IR) activity. Because of the relative stability of IGF-IR in the presence of ligand stimulation, IGF-IR ubiquitination sites have yet to be mapped and characterized, thus preventing a direct demonstration of how the receptor ubiquitination contributes to downstream molecular cascades. We took advantage of an anti-IGF-IR antibody (h10H5) that induces more efficient receptor down-regulation to show that IGF-IR is promptly and robustly ubiquitinated. The ubiquitination sites were mapped to the two lysine residues in the IGF-IR activation loop (Lys-1138 and Lys-1141) and consisted of polyubiquitin chains formed through both Lys-48 and Lys-29 linkages. Mutation of these ubiquitinated lysine residues resulted in decreased h10H5-induced IGF-IR internalization and down-regulation as well as a reduced cellular response to h10H5 treatment. We have therefore demonstrated that IGF-IR ubiquitination contributes critically to the down-regulating and antiproliferative activity of h10H5. This finding is physiologically relevant because insulin-like growth factor I appears to mediate ubiquitination of the same major sites as h10H5 (albeit to a lesser extent), and ubiquitination is facilitated by pre-existing phosphorylation of the receptor in both cases. Furthermore, identification of a breast cancer cell line with a defect in IGF-IR ubiquitination suggests that this could be an important tumor resistance mechanism to evade down-regulation-mediated negative regulation of IGF-IR activity in cancer.

Deletion or insertion in the first immunoglobulin-plexin-transcription (IPT) domain differentially regulates expression and tumorigenic activities of RON receptor Tyrosine Kinase

BackgroundActivation of the RON receptor tyrosine kinase, a member of the c-MET family, regulates tumorigenic phenotypes. The RON extracellular domains are critical in regulating these activities. The objective of this study was to determine the role of the first IPT domain in regulating RON-mediated tumorigenic activities and the underlying mechanisms.ResultsTwo RON variants, RON160 and RONE5/6in with deletion and insertion in the first IPT domain, respectively, were molecularly cloned. RON160 was a splicing variant generated by deletion of 109 amino acids encoded by exons 5 and 6. In contrast, RONE5/6in was derived from a transcript with an insertion of 20 amino acids between exons 5 and 6. Both RON160 and RONE5/6in were proteolytically matured into two-chain receptor and expressed on the cell surface. RON160 was constitutively active with tyrosine phosphorylation. However, activation of RONE5/6in required ligand stimulation. Deletion resulted in the resistance of RON160 to proteolytic digestion by cell associated trypsin-like enzymes. RON160 also resisted anti-RON antibody-induced receptor internalization. These features contributed to sustained intracellular signaling cascades. On the other hand, RONE5/6in was highly susceptible to protease digestion, which led to formation of a truncated variant known as RONp110. RONE5/6in also underwent rapid internalization upon anti-RON antibody treatment, which led to signaling attenuation. Although ligand-induced activation of RONE5/6in partially caused epithelial to mesenchymal transition (EMT), it was RON160 that showed cell-transforming activities in cell focus formation and anchorage-independent growth. RON160-mediated EMT is also associated with increased motile/invasive activity.ConclusionsAlterations in the first IPT domain in extracellular region differentially regulate RON mediated tumorigenic activities. Deletion of the first IPT results in formation of oncogenic variant RON160. Enhanced degradation and internalization with attenuated signaling cascades could be the mechanisms underlying non-tumorigenic features of RONE5/6in.

Monoclonal antibody (mAb)-induced down-regulation of RON receptor tyrosine kinase diminishes tumorigenic activities of colon cancer cells

Overexpression of the RON receptor tyrosine kinase contributes to pathogenesis of epithelial cancers and disruption of RON signals has potential for therapeutic intervention. Here, we report the inhibitory effects of monoclonal antibodies (Zt/g4, Zt/f2 and Zt/c9) on RON expression and tumorigenic activities in colon cancer cells. Persistent treatment of colon SW620 or other cells with Zt/g4 dramatically down-regulated RON expression as evident by Western blot and cell surface fluorescent analyses. The effect was both concentration and time-dependent and specific to RON but not to structure-related MET or -unrelated EGFR. The cause of reduction was antibody-induced receptor internalization followed by protein degradation through lysosome and proteasome-mediated pathways. Down-regulation of RON impaired intracellular signaling events. Phosphorylation of Erk1/2 and AKT was dramatically reduced after Zt/g4 treatment. Zt/g4 treatment also affects activities of DVL and GSK-3beta, which results in diminished beta-catenin nuclear translocation. Functional studies revealed that Zt/g4 treatment changes cellular morphology and affects colony formation in soft agar. It also increased the sensitivity of SW620 cells in response to gemcitabine-induced cytotoxicity. In this case, the death of SW620 cells was significantly increased when Zt/g4 was used in combination with gemcitabine. We conclude that persistent treatment of cancer cells with antibodies specific to RON extracellular domains results in down-regulation of RON expression. The reduced RON expression is accompanied with impaired signaling events, diminished tumorigenic activities and enhanced sensitivity towards cytotoxic drugs. Thus, Zt/g4-directed targeting could have therapeutic implication for controlling tumorigenic phenotypes of cancer cells.

Internalization of MUC1 by anti-MUC1 antibody from cell membrane through the macropinocytotic pathway

MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells. It is thought to serve as a physical barrier from the extracellular environment and also as a receptor for various extracellular molecules. However, little is known about the fate of MUC1 during and after the interaction with these molecules. In the present study, we used anti-MUC1 antibody as an interacting molecule and investigated the cellular trafficking of MUC1. Our results showed that: (1) anti-MUC1 antibody was internalized only in MUC1 expressing cells and triggered internalization and down-regulation of MUC1; (2) the internalization of MUC1 by anti-MUC1 antibody required the cytoplasmic tail of MUC1 and was suppressed by inhibitors of Na +/H + exchanger, and caveola/raft-dependent internalization, but not by an inhibitor of clathrin-dependent internalization. We conclude that antibody-induced internalization of MUC1 involves the macropinocytotic pathway.

Intrathecally infused antibodies against Nogo-A penetrate the CNS and downregulate the endogenous neurite growth inhibitor Nogo-A

Neutralizing antibodies against the neurite growth inhibitory protein Nogo-A are known to induce regeneration, enhance compensatory growth, and enhance functional recovery. In intact adult rats and monkeys or spinal cord injured adult rats, antibodies reached the entire spinal cord and brain through the CSF circulation from intraventricular or intrathecal infusion sites. In the tissue, anti-Nogo antibodies were found inside Nogo-A expressing oligodendrocytes and neurons. Intracellularly, anti-Nogo-A antibodies were colocalized with endogenous Nogo-A in large organels, some of which containing the lysosomal marker cathepsin-D. This suggests antibody-induced internalization of cell surface Nogo-A. Total Nogo-A tissue levels in spinal cord were decreased in intact adult rats following 7 days of antibody infusion. This mechanism was confirmed in vitro; cultured oligodendrocytes and neurons had lower Nogo-A contents in the presence of anti-Nogo-A antibodies. These results demonstrate that antibodies against a CNS cell surface protein reach their antigen through the CSF and can induce its downregulation.

Antibody-induced internalization of viral glycoproteins and gE-gI Fc receptor activity protect pseudorabies virus-infected monocytes from efficient complement-mediated lysis.

Pseudorabies virus (PRV)-infected blood monocytes are able to transport virus throughout the body of vaccination-immune pigs. PRV-infected monocytes express viral glycoproteins in their plasma membrane that can be recognized by virus-specific antibodies. Recently, it has been shown that addition of PRV-specific polyclonal immunoglobulins to PRV-infected monocytes at 37 degrees C induces internalization of the majority of plasma membrane-expressed viral glycoproteins. This study investigated whether this process may interfere with efficient antibody-dependent complement-mediated lysis (ADCML) of infected monocytes. Therefore, an ADCML assay was set up in vitro. A significant decrease in the percentage of cells lysed by ADCML was observed when antibody-induced internalization of PRV glycoproteins occurred (P<0.005). Furthermore, it is shown (i) that the PRV gE-gI complex, which, like certain other alpha herpesvirus orthologues, possesses IgG-binding capacity, aids in avoiding efficient ADCML of PRV-infected monocytes and (ii) that the efficiency of PRV gE-gI-mediated evasion of ADCML can be decreased by the presence of gE-gI-specific antibodies.

A tyrosine-based motif in the cytoplasmic tail of pseudorabies virus glycoprotein B is important for both antibody-induced internalization of viral glycoproteins and efficient cell-to-cell spread.

Pseudorabies virus (PRV), a swine alphaherpesvirus, is capable of causing viremia in vaccinated animals. Two mechanisms that may help PRV avoid recognition by the host immune system during this viremia are direct cell-to-cell spread in tissue and antibody-induced internalization of viral cell surface glycoproteins in PRV-infected blood monocytes, the carrier cells of the virus in the blood. PRV glycoprotein B (gB) is crucial during both processes. Here we show that mutating a tyrosine residue located in a YXXPhi motif in the gB cytoplasmic tail results in decreased efficiency of cell-to-cell spread and a strong reduction in antibody-induced internalization of viral cell surface glycoproteins. Mutating the dileucine motif in the gB tail led to an increased cell-to-cell spread of the virus and the formation of large syncytia.

Antibody-induced internalization of viral glycoproteins in pseudorabies virus-infected monocytes and role of the cytoskeleton: a confocal study

Addition of pseudorabies virus (PrV)-specific polyclonal immunoglobulins to PrV-infected monocytes induces internalization of plasma membrane anchored viral glycoproteins. This process may interfere with antibody-dependent cell lysis and resembles the well-studied physiological endocytosis process. A confocal study was designed to investigate whether the major cellular components, involved in physiological endocytosis (clathrin, actin, dynein and microtubules), play a role in this virological internalization process. In order to visualize the interaction of endosomes, which contain the internalized viral glycoproteins, with clathrin, actin, dynein and microtubules, a double labeling of viral glycoproteins and different cellular proteins was performed. Porcine monocytes were inoculated with the PrV-strain 89V87 at a multiplicity of infection of 50 for 13 h. After the addition of FITC-labeled porcine polyclonal PrV-specific antibodies, cells were fixed with para-formaldehyde at different time points and afterwards permeabilized. The different cellular components were visualized with monoclonal antibodies and a Texas Red ®-conjugate, with the exception of actin, which was stained with phalloidin-Texas Red ®. The cells were analyzed by confocal microscopy. A clear co-localization was observed between the viral glycoproteins and clathrin and dynein during the internalization process. The microtubules were in close contact with the internalized vesicles. For actin no co-localization could be observed. It can be stated that clathrin, dynein and microtubules, important components during physiological endocytosis, are also of importance during the antibody-induced internalization of viral glycoproteins.