The 4C5 Cell-Impermeable Anti-HSP90 Antibody with Anti-Cancer Activity, Is Composed of a Single Light Chain Dimer

MAb 4C5 is a cell impermeable, anti-HSP90 murine monoclonal antibody, originally produced using hybridoma technology. We have previously shown that mAb 4C5 specifically recognizes both the α- and to a lesser extent the β-isoform of HSP90. Additionally, in vitro and in vivo studies revealed that by selectively inhibiting the function of cell-surface HSP90, mAb 4C5 significantly impairs cancer cell invasion and metastasis. Here we describe the reconstitution of mAb 4C5 into a mouse-human chimera. More importantly we report that mAb 4C5 and consequently its chimeric counterpart are completely devoid of heavy chain and consist only of a functional kappa light chain dimer. The chimeric antibody is shown to retain the original antibody's specificity and functional properties. Thus it is capable of inhibiting the function of surface HSP90, leading to reduced cancer cell invasion in vitro. Finally, we present in vivo evidence showing that the chimeric 4C5 significantly inhibits the metastatic deposit formation of MDA-MB-453 cells into the lungs of SCID mice. These data suggest that a chimeric kappa light chain antibody could be potentially used as an anti-cancer agent, thereby introducing a novel type of antibody fragment, with reduced possible adverse immunogenic effects, into cancer therapeutics

Identification of 4C5 antigen as thew heat shock protein 90. Study of the molecule in the nervous system and breast cancer cell lines

The development of the vertebrate nervous system depends on extensive cell migration, which allows different cell types to reach their final destination and establish the composite organization of the adult nervous system. Cell migration is a complex process, which requires the coordination of many molecular and cellular events such as cell-cell recognition, adhesion, transmembrane signaling, and cell motility (131-135). The mechanisms underlying these processes involve orchestrated interactions between large numbers of molecules. In order to understand the molecular events regulating neuronal migration and development, we had previously characterized a monoclonal antibody (mAb), named 4C5, which was produced after immunization of mice with a brain membrane fraction of 15-day-old rat embryos. As shown by immunoblotting and immunocytochemistry, this antibody recognizes a 94 kDa cell surface peripheral antigen (4C5 antigen) in the developing rat nervous system (107). In the central nervous system, the 4C5 antigen is present in neurons. Moreover, antibody perturbation experiments, performed in cerebellar explant cultures using mAb 4C5, have indicated that the 4C5 antigen contributes to both the horizontal and the vertical neuron-neuron mediated migrations of granule cells during cerebellar development (108, 110). In the peripheral nervous system and in particular in the rat sciatic nerve, the presence of the 4C5 antigen was detected not only in neurons but also in myelin-forming and nonmyelin forming Schwann cells (109). Moreover, following adult sciatic nerve crush, the molecule was intensely re-expressed in Schwann cells of the distal segment, a few days after injury and during regeneration of the nerve. Antibody perturbation experiments performed in various in vitro systems of the peripheral nervous system using mAb 4C5 indicated that the 4C5 antigen participates in Schwann cell migration events during development and regeneration of the peripheral nervous system (111). Finally, data have been presented suggesting the involvement of this protein in actin cytoskeletal dynamics of migrating Schwann cells (111). The goals of the present work, was to investigate the identity of the 4C5 antigen and to further elucidate the molecular mechanisms underlying its action. In order to identify the 4C5 antigen we used a proteomic approach. In this context we performed MS analysis, which revealed that the 4C5 antigen is identical to the HSP90α protein, a member of the heat shock protein family. The identity of the 4C5 antigen was confirmed with immunoprecipitation experiments using mAb 4C5 and polyclonal anti-HSP90 antibodies.Η ανάπτυξη του Νευρικού Συστήματος των θηλαστικών, εξαρτάται από εκτεταμένες κυτταρικές μεταναστεύσεις, οι οποίες επιτρέπουν σε διαφορετικούς κυτταρικούς τύπους να φτάσουν στην τελική τους θέση και να συνθέσουν την πολύπλοκη οργάνωση του ενήλικου Νευρικού Συστήματος. Η κυτταρική μετανάστευση είναι μια πολύπλοκη διαδικασία, η οποία προϋποθέτει την ενορχήστρωση διάφορων μοριακών και κυτταρικών γεγονότων, όπως κυτταρική αναγνώριση, συγκόλληση, διαμεμβρανική μεταγωγή σήματος, και κυτταρική κίνηση (131-135). Οι μηχανισμοί οι οποίοι χαρακτηρίζουν όλες αυτές τις διαδικασίες, περιλαμβάνουν μεταξύ άλλων, ένα δίκτυο αλληλεπιδράσεων μεγάλου αριθμού μορίων. Με σκοπό την κατανόηση των μοριακών μηχανισμών που ρυθμίζουν τη μετανάστευση των νευρικών κυττάρων κατά την ανάπτυξη του νευρικού συστήματος, πραγματοποιήθηκε στο παρελθόν η παραγωγή και ο χαρακτηρισμός ενός μονοκλωνικού αντισώματος, του mAb 4C5 (107). Το συγκεκριμένο αντίσωμα, αναγνωρίζει μία περιφερική πρωτεΐνη μοριακού βάρους 94 kDa (αντιγόνο 4C5). Το αντιγόνο 4C5 μελετήθηκε στο νευρικό σύστημα, και βρέθηκε ότι εντοπίζεται στους νευρώνες του ΚΝΣ, ενώ στο ΠΝΣ εντοπίζεται τόσο στους νευρώνες όσο και στα μυελινοποιητικά και μη μυελινοποιητικά κύτταρα Schwann (109). Επιπλέον, πειράματα λειτουργικής παρεμπόδισης με το mAb 4C5 σε ιστοκαλλιέργειες παρεγκεφαλίδας και ισχιακού νεύρου, έδειξαν ότι το συγκεκριμένο μόριο συμμετέχει τόσο στην οριζόντια όσο και στην κάθετη μετανάστευση των κοκκιωδών νευρώνων κατά την ανάπτυξη της παρεγκεφαλίδας (108, 110) καθώς και στη μετανάστευση των Schwann κυττάρων κατά την ανάπτυξη και αναγέννηση του ισχιακού νεύρου μετά από τραυματισμό, αντίστοιχα (111). Τέλος, προκαταρκτικά δεδομένα εισηγούνταν την πιθανή συμμετοχή του συγκεκριμένου μορίου σε διαδικασίες αναδιάταξης του κυτταροσκελετού (111). Σκοπός της παρούσας εργασίας ήταν ο προσδιορισμός της αμινοξικής αλληλουχίας του αντιγόνου 4C5 και η περαιτέρω διερεύνηση της λειτουργίας του. Προκειμένου να ταυτοποιηθεί το αντιγόνο 4C5, πραγματοποιήθηκε φασματοσκοπία μάζας και σύγκριση της αμινοξικής αλληλουχίας του μορίου με βάσεις δεδομένων γνωστών πρωτεΐνών, η οποία αποκάλυψε ότι το αντιγόνο 4C5 είναι ταυτόσημο με την πρωτεΐνη θερμικού σοκ HSP90α. Η ταυτότητα του αντιγόνου 4C5 επιβεβαιώθηκε με πειράματα ανοσοκατακρήμνισης χρησιμοποιώντας το mAb 4C5 και δύο πολυκλωνικά αντισώματα έναντι των δύο ισομορφών της HSP90 (HSP90α και HSP90β). Συγκεκριμένα, παρατηρήθηκε ότι το mAb 4C5 αντιδρούσε έντονα με την α ισομορφή της HSP90 και σε μικρότερο βαθμό με τη β ισομορφή

ch-4C5 inhibis cancer cell invasion in <i>vitro</i>.

<p><b>A</b>. Wound healing assay. Photographs represent phase-contrast images obtained at zero time (left panel) and at 24 h (right panel) after scratch formation, showing MDA-MB-453 cell migration either in control cultures or cultures including anti-HSP90α, mAb 4C5, rec-4C5 or ch-4C5. Scale bar: 200 µm. <b>B</b>. Quantitative effect of antibodies on the closure of the wound. Addition of 200 µg/ml of anti-HSP90α and mAb 4C5 in the culture medium resulted in a 48% and 55% reduction of wound closure, respectively when compared to control cultures that were considered as resulting in 100% wound closure. Addition of 200 µg/ml rec-4C5 and ch-4C5 in the culture medium resulted in a 46% and 46% inhibition of wound closure, respectively. Statistical significance of differences was assessed by Student's t test. The presence of anti-HSP90α, mAb 4C5, rec-4C5 or ch-4C5 had a statistically significant effect on the wound closure (p<0.01 in each case). <b>C</b>. Phase-contrast images obtained at zero time (left panel) and at 24 h after scratch formation (right panel), showing B16 F10 melanoma cell invasion in a wound healing assay in the presence of 200 µg/ml of ch-4C5. Scale bar: 200 µm. <b>D</b>. Quantitative effect of increasing concentrations of ch-4C5 on the invasion level of B16 F10 melanoma cells. Presence of 50 µg/ml ch-4C5 resulted in 15% inhibition of invasion, while addition of 100 µg/ml and 200 µg/ml ch 4C5 resulted in 21% and 43% inhibition of migration, respectively when compared to control cultures that were considered as resulting in 100% wound closure. <b>E</b>. Visualization of dead cells using trypan blue dye. In ch-4C5 treated cultures, the cell death incidence is similar to that observed in the control cultures. In contrast, in the cultures treated with the anti-HSP90α antibody, a much greater number of cells are stained with Trypan blue, indicating an increased incidence of cell death Scale bar, 30 µm. <b>F</b>. Control and ch-4C5 treated cells were fixed permeabilized and stained with fluorescently labelled phalloidin. Scale bar 40 µm <b>G</b>. Higher magnification showing phalloidin staining (F-actin). Ch-4C5 effectively blocks spreading of lamellipodia. Scale bar: 16 µm.</p

Nucleotide and aminoacid sequences of mAb 4C5.

<p>Nucleotide and amino acid sequences of the L-chain variable region of mAb 4C5.</p

mAb 4C5 is a kappa L- chain dimer.

<p><b>A</b>. Electrophoretic analysis of mAb 4C5, followed by immunoblotting with an anti-mouse kappa chain, an anti-mouse Fab and an anti-Fcγ antibody. Intact IgG1 immunoglobulin produced by the 2D10 hybridoma cells serves as positive control. Under reducing electrophoresis followed by western blot with the anti-Fab antibody, a single 25 kDa-immunoreactive band is observed, instead of the 25- and 50-kDa bands corresponding to the L- and H-chain, respectively, of an intact IgG1. This 25 kDa band is identical to the band corresponding to the kappa L-chain as shown by western blot with the anti-mouse kappa chain antibody. Under-non reducing electrophoresis followed by western blot with both the anti-kappa and the anti-Fab antibodies, mAb 4C5 is shown to migrate at approximately 50 kDa, and not at 150 kDa as expected for an intact IgG1 molecule. No mAb 4C5 immunoreactivity is detected after electrophoresis under both reducing and non-reducing conditions followed by western blot with an anti-Fcγ antibody. In the case of the IgG1 immunoglobulin under the same conditions a 50 kDa and a 150 kDa band is observed, respectively. <b>B</b>. No radioactivity is detected after northern blot analysis of 4C5 hybridoma-derived RNA with a H-chain radiolabelled probe. RNA derived from the IgG1-producing 2D10 hybridoma cells and the NSO myeloma cells served as positive and negative control, respectively.</p

Electrophoretic mobility and specificity of antibodies.

<p><b>A</b>. <i>Left panel:</i> SDS-PAGE of purified antibodies under reducing conditions followed by Coomasie Brilliant Blue-R staining revealed in all cases an approximately 25 kDa band corresponding to the L-chain. <i>Right panel:</i> Under non-reducing conditions the antibodies are shown to migrate as a L-chain dimer. <b>B</b>. Western blot of MDA-MB-453 cell lysates using mAb 4C5, rec-4C5, ch-4C5 and a commercial anti-HSP90α antibody, serving as positive control. In all cases a single 90 kDa immunoreactive band corresponding to HSP90 is observed. <b>C</b>. Immunoprecipitation in MDA-MB-453 cell lysates with anti-HSP90α, followed by immunoblotting with either the murine or the recombinant antibodies. In all cases a single immunoreactive band is observed. <b>D</b>. Reverse immunoprecipitation experiments in MDA-MB-453 cell lysates using mAb 4C5, rec-4C5 and ch-4C5, followed by western blot with anti-HSP90α. In all cases a single immunoreactive band is observed indicating that both recombinant antibodies recognize HSP90.</p

ch-4C5 inhibits the metastatic deposition of MDA-MB-453 cancer cells into the lungs of SCID mice.

<p>Control or ch-4C5 treated MDA-MB-453 cells were labeled with the fluorescent dye DiO and injected into SCID mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023906#s4" target="_blank">Materials and Methods</a>. Evaluation of metastatic deposits was performed several hours later. A. Representative cryosections of the lungs of control and ch-4C5 treated mice. The arrows show MDA-MB-453 cells stained with DiO present in the lung tissue. A significant decrease in the deposition of cancer cells was observed in the lungs of ch-4C5 treated mice. Scale bar: 100 µm <b>B</b>. Quantitative effect of ch-4C5 on the metastatic deposition of MDA-MB-453 cells into the lungs, showed a 38% inhibition when compared to control animals. The bars represent the average of two independent experiments ±SEM. Statistical significance of differences was tested by Student's t test (p<0.01).</p

ch-4C5 binds to the surface pool of HSP90 and is not internalized by MDA-MB-453 cells.

<p><b>A</b>. Immunofluoresence labelling of MDA-MB-453 cells using both the rec-4C5 and ch-4C5. The punctuate immunolabeling indicates the surface pool of HSP90. Negative controls were performed using an antibody against the intracellular protein β tubulin (data non shown). Scale bar: 20 µm <b>B</b>. Immunofluoresence detection of intracellular HSP90 in fixed MDA-MB-453 cells, permeabilized with 0.1% Triton X-100. Similarly to the murine antibody, rec-4C5 and ch-4C5 recognize the intracellular pool of HSP90. Scale bar: 20 µm <b>C</b>. For detection of antibody internalization, live cells were incubated with the antibodies for various time intervals, then fixed, permeabilized and fluorescently labelled. No internalization of mAb4C5, rec-4C5 and ch-4C5 is observed even after 24 h of incubation. In contrast the anti-HSP90α antibody could be detected intracellularly after 24 h of incubation. Scale bar: 20 µm <b>D</b>. MDA-MB-453 cell lysates, treated with mAb 4C5, rec-4C5, ch-4C5 and anti-HSP90α were analyzed by western blot using antibodies against ErbB2, Akt, cRaf and HSP90α. Actin served as a loading control. Presence of mAb 4C5, rec-4C5 and ch-4C5 did not affect the levels of the kinases compared to controls. In contrast the cell permeable anti-HSP90α antibody significantly reduced the levels of ErbB2, Akt and cRaf.</p

Hepatitis B virus X protein upregulates HSP90alpha expression via activation of c-Myc in human hepatocarcinoma cell line, HepG2

<p>Abstract</p> <p>Background</p> <p>The Hepatitis B Virus X protein (HBx) plays a major role in hepatocellular carcinoma (HCC) development, however, its contribution to tumor invasion and metastasis has not been established so far. Heat shock protein 90 alpha (HSP90alpha) isoform is an ATP-dependent molecular chaperone that maintains the active conformation of client oncoproteins in cancer cells, which is abundantly expressed in HCC, especially in hepatitis B virus (HBV)-related tumors, might be involved in tumor progression.</p> <p>Methods</p> <p>The levels of HSP90alpha, extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2) and c-Myc in HBx-transfected HepG2 cells were determined by western blots analysis. The endogenous ERKs activity was demonstrated by ELISA assay. The regulation of c-Myc-mediated HSP90 alpha promoter transactivation by HBx was evaluated through electrophoretic mobility shift analysis (EMSA). The c-Myc-mediated HSP90alpha transcription was analysed by promoter assay. The HBx-expressing cells were transfected with specific small interference RNA (siRNA) against c-Myc. The <it>in vitro </it>invasion potentials of cells were evaluated by Transwell cell invasion assay.</p> <p>Results</p> <p>HBx induces HSP90alpha expression at the transcription level. The induction effect of HBx was inhibited after treatment with c-Myc inhibitor, 10058-F4. In addition, the luciferase activity of the HSP90alpha promoter analysis revealed that the HBx is directly involved in the c-Myc-mediated transcriptional activation of HSP90alpha. Furthermore, HBx induces c-Myc expression by activation of Ras/Raf/ERK1/2 cascades, which in turn results in activation of the c-Myc-mediated HSP90alpha promoter and subsequently up-regulation of the HSP90alpha expression. Overexpression of HSP90alpha in HBx-transfected cells enhances tumor cells invasion. siRNA-mediated c-Myc knockdown in HBx-transfected cells significantly suppressed HSP90alpha expression and cells invasion <it>in vitro</it>.</p> <p>Conclusion</p> <p>These results demonstrate the ability of HBx to promote tumor cells invasion by a mechanism involving the up-regulation of HSP90alpha and provide new insights into the mechanism of action of HBx and its involvement in tumor metastasis and recurrence of HCC.</p