Gábor Tóth

Hungary's Constitutional Transformation

Hungary – Democratic state structure – Two-thirds parliamentary majority – First flurry of constitutional amendments of 2010 – Checks and balances – Media – Ex post facto legislation – Hungarian Constitutional Court – Judicial review – Wholesale constitutional review and Basic Law of 2011

The combination of trastuzumab and pertuzumab administered at approved doses may delay development of trastuzumab resistance by additively enhancing antibody-dependent cell-mediated cytotoxicity

ABSTRACT Although the recently concluded CLEOPATRA trial showed clinical benefits of combining trastuzumab and pertuzumab for treating HER2-positive metastatic breast cancer, trastuzumab monotherapy is still the mainstay in adjuvant settings. Since trastuzumab resistance occurs in over half of these cancers, we examined the mechanisms by which treatment of intrinsically trastuzumab-resistant and -sensitive tumors can benefit from the combination of these antibodies. F(ab′)2 of both trastuzumab and pertuzumab were generated and validated in order to separately analyze antibody-dependent cell-mediated cytotoxicity (ADCC)-based and direct biological effects of the antibodies. Compared to monotherapy, combination of the two antibodies at clinically permitted doses enhanced the recruitment of natural killer cells responsible for ADCC, and significantly delayed the outgrowth of xenografts from intrinsically trastuzumab-resistant JIMT-1 cells. Antibody dose-response curves of in vitro ADCC showed that antibody-mediated killing can be saturated, and the two antibodies exert an additive effect at sub-saturation doses. Thus, the additive effect in vivo indicates that therapeutic tissue levels likely do not saturate ADCC. Additionally, isobole studies with the in vitro trastuzumab-sensitive BT-474 cells showed that the direct biological effect of combined treatment is additive, and surpasses the maximum effect of either monotherapy. Our results suggest the combined therapy is expected to give results that are superior to monotherapy, whatever the type of HER2-positive tumor may be. The combination of both antibodies at maximum clinically approved doses should thus be administered to patients to recruit maximum ADCC and cause maximum direct biological growth inhibition.

Cell confluence induces switching from proliferation to migratory signaling by site-selective phosphorylation of PDGF receptors on lipid raft platforms

Platelet derived growth factor receptors (PDGFR) play an important role in tumor pathogenesis and are frequently overexpressed in glioblastoma. Earlier we have shown that only confluent glioblastoma cell cultures exhibit a biphasic calcium transient upon PDGF stimulation. Here, we examined how the change in cell density leads to differential cellular responses to the same PDGF stimulus. PDGF beta receptors and their specific phosphotyrosine residues were fluorescently co-labeled on A172 and T98G glioblastoma cells. The distribution in cell membrane microdomains (lipid rafts) and the phosphorylation state of PDGFR was measured by confocal microscopy and quantitated by digital image processing. Corresponding bulk data were obtained by Western blotting. Activation of relevant downstream signaling pathways was assessed by immunofluorescence in confocal microscopy and by Western blot analysis. Functional outcomes were confirmed with bulk and single cell proliferation assays and motility measurements. In non-confluent (sparse) cultures PDGF-BB stimulation significantly increased phosphorylation of Tyr716 specific for the Ras/MAPK pathway and Tyr751 specific for the phosphoinositide 3-kinase/Akt pathway. As cell monolayers reached confluence, Tyr771 and Tyr1021 were the prominently phosphorylated residues. Tyr771 serves as adaptor for Ras-GAP, which inactivates the MAPK pathway, and Tyr1021 feeds into the phospholipase C-gamma/PKC pathway. Coherent with this, MAPK phosphorylation, Ki-67 positivity and proliferation dominated in dispersed cells, and could be abolished with inhibitors of the MAPK pathway. At the same time, RhoA activation, redistribution of cortactin to leading edges, and increased motility were the prominent output features in confluent cultures. Importantly, the stimulus-evoked confluence-specific changes in the phosphorylation of tyrosine residues occurred mainly in GM1-rich lipid microdomains (rafts). These observations suggest that the same stimulus is able to promote distinctly relevant signaling outputs through a confluence dependent, lipid raft-based regulatory mechanism. In particular, cell division and survival in sparse cultures and inhibition of proliferation and promotion of migration in confluent monolayers. In our model, the ability to switch the final output of the same stimulus as a function of cell density could be a key to the balance of proliferation and invasion in malignant glioblastoma.

Validating Pharmacological Disruption of Protein–Protein Interactions by Acceptor Photobleaching FRET Imaging

Proteins are the major targets of drug discovery and many of the new drugs are designed to exert their effect by disrupting protein-protein interactions. Validation of the inhibition of molecular interactions is generally done by biochemical methods, however, these are often not feasible when the interaction is not stable enough. Fluorescence resonance energy transfer (FRET) is an excellent tool for determining direct molecular interactions between two molecules in the cell membrane or inside cells in their natural state. Although originally established as a flow cytometric approach, FRET has been adapted for microscopy, allowing for analysis of sub-cellular co-localization at the single cell level. In this chapter, we provide theoretical introduction to the phenomenon of FRET, and a protocol - including labeling techniques, measurement, and evaluation of microscopy images - of the simplest microscopic FRET approach, acceptor photobleaching FRET. This technique is generally usable for studying protein interactions and requires only a standard confocal laser scanning microscope. To demonstrate the value of image based FRET for testing pharmacological disruption of protein-protein interactions, we show how inhibition of the hetero-dimerization of ErbB2 and ErbB1 by the humanized monoclonal antibody pertuzumab can be validated using this technique.

How to use major parts of a paper previously published by others to write a new one. An allegation of plagiarism by indian authors

ABSTRACT In our present communication we prove the allegation that Indian authors plagarized the major parts and conclusions of our paper published in 1997 and used them in their paper published in this journal in 2000.

Quantitating ADCC against adherent cells: Impedance-based detection is superior to release, membrane permeability, or caspase activation assays in resolving antibody dose response

Monoclonal antibody‐based immunotherapeutics will dominate Pharmas next generation of blockbuster drugs, and Fc‐associated functions, including antibody dependent cellular cytotoxicity (ADCC) are among the highly desired activities mediated by these antibodies. Therefore, quantitative evaluation of ADCC is required during drug development. Our objective was to find the most suitable and reliable nonradioactive method for quantitative analysis of in vitro ADCC against adherent cells, which often serve as models for solid tumors. The test system was comprised the HER2 positive JIMT‐1 cells targeted by the specific therapeutic antibodies trastuzumab (Herceptin®) and pertuzumab (Perjeta®). These cells are resistant to the direct biological effects of these antibodies, and, therefore, allow the isolated assessment of ADCC. We compared fluorescein diacetate (FDA) and carboxyfluorescein diacetate succinimidyl ester (CFSE) release as a fluorescent alternative to 51Cr release; propidium iodide (PI) uptake revealing increased membrane permeability; the PanToxiLux assay measuring ADCC induced pro‐apoptotic protease activity in flow cytometry; and an impedance‐based real time cell adhesion test. We found that release assays are compromised by high spontaneous release of the label. PI uptake could not differentiate well between spontaneous NK activity and specific ADCC. The PanToxiLux assay, besides allowing for shorter assay times, offers improvement over the previous approaches in distinguishing spontaneous and antibody mediated NK action, but, probably owed to the prolonged detached state of adherent target cells, only at highly saturating antibody concentrations. In the case of adherent target cells, impedance‐based cell analysis attains functional information exclusively on the target cells without having to label them for distinguishing from effectors or assay readout. It also allows continuous monitoring for days, and specifically detects target cell detachment, as the final functional consequence of ADCC. The sensitivity of this method even allows for quantitating the additivity and saturability of ADCC as a function of antibody concentration. We conclude that impedance‐based assays are the most sensitive for quantitatively assessing in vitro ADCC on adherent target cells.

Abstract B069: The best is not enough—when it is alone: Current clinical doses of humanized anti-Her2 antibodies should be applied in combination to delay development of trastuzumab resistance by additively enhancing ADCC

Although the recently concluded CLEOPATRA trial showed clinical benefits of combining trastuzumab and pertuzumab for treating HER2-positive metastatic breast cancer, trastuzumab monotherapy is still the mainstay in adjuvant settings. Since trastuzumab resistance occurs in over half of these cancers, we examined the mechanisms by which treatment of intrinsically trastuzumab-resistant and -sensitive tumors can benefit from the combination of these antibodies. F(ab9)2 of both trastuzumab and pertuzumab were generated and validated in order to separately analyze antibody-dependent cell-mediated cytotoxicity (ADCC)-based and direct biological effects of the antibodies.Compared to monotherapy, combination of the two antibodies at clinically permitted doses enhanced the recruitment of natural killer cells responsible for ADCC, and significantly delayed the outgrowth of xenografts from intrinsically trastuzumab-resistant JIMT-1 cells. Antibody dose-response curves of in vitro ADCC showed that antibody-mediated killing can be saturated, and the two antibodies exert an additive effect at sub-saturation doses. Thus, the additive effect in vivo indicates that therapeutic tissue levels likely do not saturate ADCC. Additionally, isobole studies with the in vitro trastuzumab-sensitive BT-474 cells showed that the direct biological effect of combined treatment is additive, and surpasses the maximum effect of either monotherapy. Our results suggest the combined therapy is expected to give results that are superior to monotherapy, whatever the type of HER2-positive tumor may be.The combination of both antibodies at maximum clinically approved doses should thus be administered to patients to recruit maximum ADCC and cause maximum direct biological growth inhibition. Citation Format: Gabor Toth, Arpad Szoőr, Laszlo Simon, Yosef Yarden, Janos Szollősi, Gyorgy Vereb. The best is not enough—when it is alone: Current clinical doses of humanized anti-Her2 antibodies should be applied in combination to delay development of trastuzumab resistance by additively enhancing ADCC [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B069.

In vitro, in vivo and microscopic analysis of tumor immunotherapy using combination of two antibodies against the same ErbB2 target

Trastuzumab (Herceptin®), a humanized anti-ErbB2 antibody is a specific targeted therapy against ErbB2 positive tumors, with a history of both success and a high rate of therapy resistance. Another humanized antibody, pertuzumab (Perjeta®) inhibits ErbB2 heterodimerization. While these antibodies have been developed based on the in vitro direct cellular effect of their mouse parent antibodies, there is the possibility that their in vivo mechanism of action roots rather in antibody dependent cellular cytotoxicity (ADCC) exerted by natural killer (NK) cells. Our goal was to ascertain the extent of contribution of the direct cellular effect of the antibodies and that of the in vivo evoked ADCC to tumor growth inhibition. We generated the F(ab)2 fragments of the antibodies lacking the Fc part, that have the ability to generate direct cellular effects, but lack the ADCC component. Affinity and lack of Fc fragment on F(ab)2-s were tested with immunofluorescence in flow cytometry. In vitro EC50 was assessed with an MTT based assay. The effect on proliferation of in vitro sensitive BT-474 and resistant JIMT-1 cell lines, both ErbB2 positive, was not affected by the removal of the Fc region. Based on the EC50 values determined as single agents on BT-474 cells, isoboles for a range of combination were also measured for both the whole antibodies and the F(ab)2 fragments and no co-operativity was found. Non-radioactive in vitro ADCC assay on JIMT-1cell line was optimized with CD16.176V.NK-92 high affinity natural killer cell line. Intact antibodies mediated in vitro ADCC-based killing, while F(ab)2 fragments did not. Formation of immunological synapses was verified by confocal microscopy. NK-92 cells were able to form synapses upon recognition of whole antibodies by their high affinity FcγRIII, while owed to the lack of Fc region, synapse formation did not occur with F(ab)2-s. For in vivo ADCC study, JIMT-1 cells were inoculated s.c. in severe combined immunodeficiency mice. The whole antibodies were able to inhibit tumor growth, but their F(ab)2 fragments were ineffective. Combination of the whole antibodies showed a great degree of synergism in vivo, which possibly indicates synergism of two antibodies on the same target protein recruiting FcγRIII receptors and a consequentially better binding and activation of NK cells.