left to the speaker's name in the program to display the corresponding abstract of main talks and short communications. For poster abstracts you have to browse through this page.ABSTRACTS FOR TALKS AND POSTERS
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CATEGORIES:
Generation and design of targeted toxins
Targeted human proteins as effectors
Innovative tools to improve targeted tumor therapies
Targeted tumor therapies in clinical applications
Category: Generation and Design of Targeted Toxins
Luzie Fabisch Memorial Lecture
TALK: Wednesday 21 March 2012, 11:00
Genetic Engineering New targeting Agents to Cancer Cells and Cancer Stem Cells
Daniel A. Vallera1, Seunguk Oh2, John Ohlfest*2, Nathan Waldron2, David Zelmer2, Suresh Swaminathan2, Jayanth Panyam*2
1Radiation Oncology, University of Minnesota Cancer Center, Minneapolis, MN 55455, USA; 2University of Minnesota Masonic Cancer Center, Minneapolis, MN 55455, USA
* These authors contributed equally to this work
Cancer stem cells (CSC) are one of the most interesting and controversial topics in cancer biology. Evidence is mounting that cancers contain a small subset of stem-cells that can self-renew and facilitate tumor initiation. These cells are believed to be the source of chemotherapy drug resistance. Of the known markers associated with CSC, CD133 is perhaps the most controversial. Identified as a marker of CSC in many tumors, CD133 has been used to enrich for CSC in breast and colorectal cancer. CD133 is a 5-transmembrane glycoprotein that localizes to epithelial cell membrane protrusions and mAb recognizing CD133 enrich for human hematopoietic and endothelial stem cells. Arguments against CD133 as a valid stem cell target have been clouded by studies showing that cancer cell differentiation is not likely unidirectional and commercial anti-CD133 binding is influenced by glycosylation related to changes in the cell cycle. To address these concerns, we used a new mAb developed to recognize only the extracellular peptide backbone of CD133. We then created a single chain drug consisting of this scFV spliced to deimmunized PE. In testing, we observed that the anti-CD133 scFV was capable of enriching tumor initiating cells. The drug was selectively toxic in vitro likely due to its rapid internalization. Surprisingly, the drug was effective in vivo in two independent xenograft carcinoma models. Also, it was effective in combination with other anti-carcinoma agents. Together, these findings indicate that selectively attacking cancer stem cells may be a valid therapeutic approach.
TALK: Wednesday 21 March 2012, 11:50
In Vitro and In Vivo Efficacy of an anti-PSMA Immunotoxin against Prostate Cancer
Philipp Wolf
Urologie, Universitätsklinikum Freiburg, 79106 Freiburg, Germany
We generated monoclonal antibodies (mAbs) against the Prostate Specific Membrane Antigen (PSMA) as basic molecules for new targeted diagnostic and therapeutic approaches against prostate cancer.
An anti-PSMA single chain antibody fragment (scFv), called D7, was generated from the mAb 3/F11 by phage display. The recombinant immunotoxin D7-PE40 was designed by C-terminal ligation of the gene sequence of the toxic domain of Pseudomonas aeruginosa (PE40) to the gene of D7. D7-PE40 was bacterially expressed and showed high serum stability. It specifically bound to PSMA expressing cells and was able to ADP-ribosylate the eukaryotic elongation factor 2 (eEF-2). Moreover, the immunotoxin exhibited a high cytotoxicity (IC50 = 140 pM) against C4-2 target cells. In vivo, D7-PE40 was well tolerated in SCID mice up to a single dose of 20 µg, whereas higher doses induced severe hepatotoxicity. Immunotoxin treatment of mice bearing C4-2 xenografts caused a significant inhibition of tumor growth, whereas PSMA negative tumors remained unaffected. Current strategies comprise the structural optimization of the immunotoxin and its application in combination therapies.
TALK: Thursday 22 March 2012, 08:30
PDZ Domains and Membrane Nucleolin in the Engineering and Production of Chimeric Toxins Against Cancer Cells
Francesco Giansanti, Luana Di Leandro, Giuseppina Pitari, Elisabetta Benedetti, Annamaria Cimini, Rodolfo Ippoliti
Department of Basic and Applied Biology, University of L'Aquila, I-67100 L'Aquila, Italy
In the research of new engineered toxins to target cancer cells, we have studied PDZ protein domain as possible tools for the production, activation and cellular targeting of the ribosome inactivating protein saporin. PDZ domains are found in proteins involved in cellular processes, from the signal transduction to the transport and particularly in synapse formation in mammals. They recognize and bind short peptide sequences located at the C-terminus of a protein. Recognition of the C-termini represents a ‘non-invasive’ interaction, very well suited to mediate organization of transport, localization, sorting and spatial arrangement of proteins exposing their individual C-terminal tails that can be recognized and handled by various PDZ domains. We have recently used the second PDZ domain from murine PTP-BL protein for the co-expression of this protein within bacteria together with a saporin derivative (VSAV). We have now focussed our attention to the PDZ domain from hCASK (Human calcium/calmodulin-dependent serine protein kinase) that has been demonstrated to bind extracellular CD98 in epithelial cells, recognized as a marker for several human tumors. We produced fusion of hCASK-PDZ with saporin and tested it on human glioblastoma cells. Furthermore we are exploring nucleolin expressed on the cell membrane of cancer cells as a specific target of human glioblastoma, since its membrane expression in gliomas is stricltly correlated with their malignancy and can be exploited to deliver toxic substances.Possible applications of these targeting strategies will be discussed
G-01
POSTER
Anti-mesothelin Immunoconjugates for Pancreatic and Ovarian Cancer Imaging and Therapy
Cristina Anselmi1, Giulio Fracasso1, Sara Cingarlini2, Elisabetta Grego2, Stefano Eleuteri1, Silvia Zanini1, Marcella Pinto1, Marco Colombatti1
1Pathology and Diagnostic- Immunology, University of Verona, 37134 Verona, Italy; 2Azienda Ospedaliera Universitaria Integrata Verona, Medical Oncology Unit, 37134 Verona, Italy
Introduction: Mesothelin is a GPI linked membrane overexpressed in same tumors (pancreatic, ovarian and mesothelioma). These diseases are often detected at an advanced stage, which generally results in a poor prognosis. New therapeutic approaches could help us in the diagnosis of the early stage diseases as well as provide targets to improve the treatments. The aim of this study is to develop antibodies (Abs) recognizing Mesothelin, which can be used naked or armed with trackers/cytotoxic substances for immunodiagnosis and immunotherapy.
Results: The Mesothelin cDNA obtained from an ovarian cancer explant was cloned into the vector pET30b for expression in bacteria; the recombinant protein was purified by affinity chromatography and the purity verified by SDS-PAGE. Also HEK293-Mesothelin positive cells were produced by transfection with a pcDNA3.1-plasmid. The aforementioned recombinant protein and transfected cells were used to immunize Balb/c mice. One hybridoma supernatant stains in flow cytometry Ag+ cells. This hybridoma has been cloned and some mAbs have been obtained. One of these Abs shows high specificity in flow cytometry and its binding ability is superior to the binding property of the commercial Ab K1. A chemical immunoconjugated was synthesized by linking mAb with nRTA; this IT was in vitro assessed on Mesothelin +/- cell lines. We observed that nRTA cytoxicity was increased of about 1,000 times after conjugation to the mAbs. The killing specificity was confirmed by coincubation of IT with an excess of free Ab.
Funded by Fondazione Cariverona, AIRC (M.C.)
TALK: Wednesday 21 March 2012, 12:25
DeBouganin: A de-immunized toxin payload and its applications in oncology
Jeannick P.A. Cizeau, Shilpa Chooniedass, Arjune Premsukh, Joycelyn Entwistle, Glen MacDonald
Research, Viventia Biotechnologies Inc., Winnipeg, Manitoba R3T 3Z1, Canada
Immunotoxin fusion proteins represent a highly potent alternative to conventional anti-cancer agents. However, the clinical development of immunotoxins in the treatment of solid tumors has been impeded, in part, by the induction of an immune response directed primarily against the toxin moiety. Bouganin is a plant-derived type I ribosome-inactivating protein (RIP). To prevent an anti-bouganin response, T-cell epitopes were identified and mutated to create an equally potent yet non-immunogenic variant, deBouganin. To demonstrate the successful de-immunization, deBouganin was genetically-linked to an anti-EpCAM Fab fragment creating VB6-845. After in vitro characterization and pre-clinical studies demonstrating the potency, specificity and safety profile of the molecule, a Phase I clinical trial was initiated. VB6-845 was administered weekly at 1 and 2 mg/kg dose to patients with EpCAM-positive solid tumor and the sera analyzed for the presence of anti-deBouganin antibodies at 4 and 8 weeks post-treatment. Of the 12 patients treated with VB6-845, 10 showed no immune response to deBouganin whereas two patients had a titre just above the threshold of the assay. The presentation will also include the engineering and biologic characterization of deBouganin linked with other types of antibody formats.
G-03
POSTER
NPY as targeting component of immunotoxins
Franziska Gehle1, Annette Beck-Sickinger2, Hauke Lilie1
1Institut für Biochemie und Biotechnologie, MLU Halle, 06120 Halle, Germany; 2Institut für Biochemie, Universität Leipzig, 04103 Leipzig, Germany
The Y1 receptor belonging to the class of G protein-coupled receptors has been revealed as tumour marker. In this context, a Y1 receptor-specific neuropeptide Y analogue (NPY) has successfully been used in the diagnosis of breast cancer. A further approach is the treatment of Y1 receptor-overexpressing cells by using this analogue as targeting component of immunotoxins.
Here, we describe the design and functional characterization of such a conjugate (NPY-PE38) containing the NPY analogue and a Pseudomonas exotoxin A variant. Coupling was achieved via polyionic linker peptides and the formation of a disulfide bond. On two Y1 receptor-positive cell lines, a cytotoxic activity of NPY-PE38 with EC50 concentrations in the low nanomolar range has been measured. The toxic effect could be impeded by a Y1 receptor-specific antagonist proofing the specific activity of this conjugate.
G-04
POSTER
Recombinant Immunotoxin Targeting the CD38 Antigen Inhibits Proliferation and Promotes Apoptosis of Neoplastic B-lymphocytes
Monica Castagna1, Matteo Pasetto1, Pietro Della Cristina1, David J. Flavell2, Sopsamorn U. Flavell2, M. Serena Fabbrini3, Aldo Ceriotti3, Luana Di Leandro4, Francesco Giansanti4, Rodolfo Ippoliti4, Wijnand Helfrich5, Marco Colombatti1
1Pathology and Diagnostic-Immunology, University of Verona, 37134 Verona, Italy; 2The Simon Flavell Leukaemia Research Unit, Leukaemia Busters, Southampton General Hospital, Southampton SO16 6YD, United Kingdom; 3Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, 22100 Milan, Italy; 4Department of Basic and Applied Biology, University of L'Aquila, 67010 L'Aquila, Italy; 5Surgical Research Laboratory, Department of Surgery, University Medical Center Groningen, 9713 Groningen, The Netherlands
The success of conventional anti-tumor chemotherapy and radiotherapy has been limited due to factors such as chemoresistance and peripheral toxicity as a result of lack of specificity. For this reason the interest in targeted therapies using immunotoxins (ITs), especially for the treatment of haematological malignancies is increasing. Here we report on the construction of a novel therapeutic fusion protein, AT13.5-PE40, designed to induce target antigen-restricted apoptosis in human B-cell neoplasms. The target of our immunotoxin is CD38, a surface molecule highly expressed by B lymphocytes belonging to a particularly aggressive sub-group of Chronic Lymphocytic Leukemia (CLL) leading to the prognostically unfavorable Richter’s Syndrome and by the neoplastic immature plasma cells in Multiple Myeloma (MM). AT13.5-PE40 consists of a CD38-specific single-chain antibody fragment (scFv) fused to the 40 kDa truncated form of the exotoxin A of Pseudomonas aeruginosa (PE40), a potent toxin which inhibits protein synthesis and causes cell death. AT13.5-PE40 blocks proliferation of CD38-positive cell lines at nanomolar concentrations but does not have any cytotoxic activity against CD38- cell lines. AT13.5-PE40 can also induce apoptosis of CD38+ RPMI myeloma cells in a dose and time-dependent manner and displays synergistic toxicity when used in combination with all-trans retinoic acid (ATRA), a potent inducer of CD38 expression. In conclusion AT13.5-PE40 is a novel recombinant protein with a potential therapeutic value for the treatment of some B-cell haematological neoplasms.
SHORT COMMUNICATION: Wednesday 21 March 2012, 14:15
Targeting Rhabdomyosarcomas with a Novel Immunotoxin Directed Against the Fetal Form of the Acetylcholine Receptor
Stefan Gattenlöhner1, Mehmet K. Tur1, Alexander Marx2, Stefan Barth3
1Experimental Pathology and Immunotherapy, University Hospital Gießen and Marburg (UKGM) GmbH, 35392 Gießen, Germany; 2Institute of Pathology, University of Mannheim, 68167 Mannheim, Germany; 3Institute of Applied Medical Engineering, Helmholtz-Institute, RWTH Aachen, 52074 Aachen, Germany
Rhabdomyosarcomas (RMS) are the most common soft tissue sarcomas of childhood. Despite intensified multimodality treatment approaches, the overall survival rates of RMS patients have remained at 5% to 20% over the last decades.
In work aimed at identifying tumor specific antigens for the diagnosis and treatment of RMS, we established that, in contrast to healthy muscle tissue or any other type of malignancy, the fetal form of the acetylcholine receptor (fAChR) is strongly expressed on the surface of RMS cells. Moreover, we showed that the rate of fAChR-expression is increased in residual RMS cells after chemotherapy and that the detection of fAChR-expression can be utilized to sensitively identify minimal residual RMS.
Based on our previous findings, we constructed an immunotoxin capable of targeting RMS cells in vitro and in vivo by binding to fAChR and Pseudomonas-A exotoxin-mediated lysis. Moreover, we generated T-cells with chimeric T-cell receptor complexes capable of directly binding and lysing RMS cells in vitro and in vivo.
Currently, we are in the process of developing bi-specific antibodies designed to stimulate NK-cells to redirected lysis of RMS cells.
Although targeting fAChR has yet to prove a successful approach in the therapy of RMS in the clinical setting, our results indicate that there is reason to hope that this goal may be achieved in future.
G-06
POSTER
Improved Efficacy of HER2-targeted Immunotoxins Using Photochemical Internalization
Maria Berstad1, Kristian Berg1, Michael Rosenblum2, Yu Cao2, James Marks3, Anette Weyergang1
1Radiation Biology, Institute for Cancer Research, Oslo University Hospital, 0310 Oslo, Norway; 2Immunopharmacology and Targeted Therapy Laboratory, Department of Experimental Therapeutics, MD Anderson Cancer Center, Houston, TX 77054, USA; 3Department of Anaesthesia, University of California, San Fransisco, CA 94143, USA
HER2 is overexpressed in several cancers and is an attractive target for delivery of cancer therapeutics. However, both primary and acquired resistance to current anti-HER2 therapies is of major clinical concern and calls for more effective HER2-targeted treatment strategies. We here present photochemical internalization (PCI) of HER2-targeted ITs as a novel treatment strategy for HER2-positive cancers. PCI mediates release of drugs entrapped in endocytic vesicles. The method is based on photosensitizers (PSs) which localize to the membranes of endocytic vesicles, and subsequent membrane rupture upon light exposure so that the drugs can escape into the cytosol. The purpose of this study was to evaluate PCI of HER2-targeted ITs with various binding sites and affinities. ITs were constructed by coupling the HER2 antibody, trastuzumab, to a type I ribosome inactivating protein (RIP), saporin, through a biotin-streptavidin linkage, or by genetically fusing human single-chain antibodies (scFv) to recombinant gelonin, another type I RIP. The cytotoxicity of PCI of the ITs was assessed against HER2 positive Zr-75-1 and BT-20 cells and HER2 negative MDA-MB231 cells. The present data confirm synergistic cytotoxicity of PCI of all ITs compared to treatment with ITs and photodynamic therapy (PDT) alone (PS+light). At LD50-PDT, a subtoxic dose of 100 pM of the different ITs was sufficient to kill 75-85% of Zr-75-1 cells. LD95 of PCI of the recombinant fusions was detected between 1 and 10 nM of all 4 ITs (subtoxic dosage) at a PDT dose reducing the viability of the BT-20 cells to 40%.
G-07
POSTER
Shiga Toxin-sensitive Pancreatic Cancer Cells: Glycosphingolipid Receptors as Potential Targets for Tumor Therapy?
Wiebke Storck1, Iris Meisen1, Kathrin Gianmoena2, Michael Mormann3, Hans-Ulrich Humpf2, Jörg Haier4, Helge Karch1, Johannes Müthing1
1Institute for Hygiene, University of Münster, 48149 Münster, Germany; 2Institute of Food Chemistry, University of Münster, 48149 Münster, Germany; 3Institute of Medical Physics and Biophysics, University of Münster, 48149 Münster, Germany; 4Department of General Surgery, University Hospital Münster, 48149 Münster, Germany
Targeted therapies based on bacterial or plant toxins which recognize specific glycosphingolipids (GSLs) on the cell surface of malignant cells might prove to be promising clinical applications. Shiga toxins (Stxs) are composed of an enzymatically active A subunit (StxA) and the pentameric B subunit (StxB) which specifically binds to the GSL globotriaosylceramide (Gb3Cer/CD77). Gb3Cer has been previously identified as a tumor-associated GSL in human pancreatic cancer. Thus, overexpression of Stx-receptor Gb3Cer in pancreatic cancer tissue opens for the possibility of using either the holotoxin or the Gb3Cer-binding StxB subunit coupled to therapeutic drugs in targeted therapy. This prompted us to investigate the expression of Gb3Cer in human pancreatic cancer cell lines originating from primary tumors as well as liver, ascites and lymph node metastases. Using immunochemical solid phase binding assays and mass spectrometry, all of the investigated cell lines were found to express Gb3Cer showing substantial lipoform heterogeneity owing to variability in the fatty acyl chain of the ceramide moieties. Stx-challenged tumor cell lines exhibited distinct susceptibility towards the cytotoxic action of the holotoxin that may rest upon varying receptor lipid composition suggesting different mechanisms of Stx-membrane-interaction, endocytosis of the Stx-Gb3Cer-complex or trafficking of the toxin to intracellular targets. Our preliminary data will stimulate us to unravel the molecular mechanisms underlying the cytotoxic activity of Stx in human pancreatic cancer cells.
G-08
POSTER
Generation of AML-internalizing Antibody Fragments and in Vitro Characterization of a New Recombinant Immunotoxin C3(scFv)-ETA`
Jenny Fitting1, Stefan Barth*2, 1, Mehmet K. Tur*3
1Experimental Medicine and Immunotherapy, Institute of Applied Medical Engineering, Helmholtz-Institute for Biomedical Engineering; University Hospital RWTH Aachen, 52074 Aachen, Germany; 2Department of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, 52074 Aachen, Germany; 3Experimental Pathology and Immunotherapy, University Hospital Gießen and Marburg (UKGM) GmbH, 35392 Gießen, Germany
* These authors contributed equally to this work
Acute myelogenous leukemia (AML) is a cancer of the myeloid line of blood cells and is characterized by an uncontrolled overproduction of non-functional blast cells in the peripheral blood. The first line treatment consists of a high dosis polychemotherapy resulting in 50-80 % complete remission rate in adult patients. However, the 5-year survival rate is only 24 %. This is due to treatment resistant residual tumor cells that trigger relapse, the main cause for high mortality. Therefore, an adjuvant immunotherapeutic strategy targeting remaining leukemic blast cells would be favorable to prolong the relapse free survival and decrease mortality. We used the Tomlinson library and generated a panel of highly specific cell surface-internalizing antibody fragments to AML M2-derived Kasumi-1 cells by a subtractive whole-cell-panning strategy. Specific binding to Kasumi 1 cells were demonstrated by ELISA and flow cytometry. No cross-reactive binding was detectable to healthy peripheral blood cells. We picked the best binder for recombinant immunotoxin construction by fusion of clone C3(scFv) to a truncated version of Pseudomonas exotoxin A (ETA`), designated as C3(scFv)-ETA`. This molecule was shown to inhibit cell proliferation and induce apoptosis in AML-derived Kasumi-1 cells, demonstrating a putative novel immunotherapeutic approach for adjuvant AML therapy.
POSTER and SHORT COMMUNICATION: Friday 23 March 2012, 11:00
Human Kappa Light Chain Targeted Pseudomonas Exotoxin A – Identifying Human Antibodies with Favorable Characteristics for Antibody-drug Conjugate Development
Christian Kellner1, Bart E.C.G. de Goeij2, Wim K. Bleeker2, Katja Klausz1, Stefanie Derer1, Jeroen J. Lammerts van Bueren2, Jan G.J. van de Winkel2, Thomas Valerius1, Martin Gramatzki1, Paul W.H.I. Parren2, Matthias Peipp1
1Division of Stem Cell Transplantation and Immunotherapy, Christian-Albrechts-University Kiel, 24105 Kiel, Germany; 2Genmab, 3584 CM Utrecht , The Netherlands
Antibody-drug conjugates (ADC) represent promising agents for targeted cancer therapy. To allow rational selection of human antibodies with favorable characteristics for ADC development a screening tool was designed obviating the need of preparing individual covalently linked conjugates. Therefore, α-kappa-ETA was designed as a fusion protein consisting of a human kappa light chain binding antibody fragment and a truncated version of Pseudomonas exotoxin A. Antibody-redirected α-kappa-ETA specifically inhibited proliferation of antigen-expressing cell lines at low toxin and antibody concentrations. Selected antibodies that efficiently delivered α-kappa-ETA were used to generate covalently linked immunotoxins and efficiently triggered apoptosis of target cells, indicating that antibodies identified in our assay system can be converted to functional immunoconjugates. The assay system was further used for screening panels of EGFR- and Her2-specific antibodies - demonstrating favorable characteristics of selected antibodies. In the Her2 model our data suggest that the formation of HER2/ErbB-heterodimers is important for HER2 antibody-mediated delivery of the kappa-directed model toxin ETA’ in tumor cells. In conclusion, antibodies with potential for ADC development can be identified using the novel α-kappa-ETA conjugate.
G-10
POSTER
Systematic Comparison of Anti-CD22 Single-chain Fusion Toxin Contructs Containing Saporin or PE40 Produced in Different Microbial Expression Systems.
David J. Flavell1, Sopsamorn U. Flavell1, Suzanne E. Holmes1, Rodolfo Ippoliti2, Alessio Lombardi3, Luana Leandro2, Francesco Giansanti2, Pietro Della Cristina4, Monica Castagna4, Aldo Ceriotti3, Giovanni Tagliabue3, M. Serena Fabbrini3, Marco Colombatti4
1The Simon Flavell Leukaemia Research Unit, Leukaemia Busters, Southampton General Hospital, Southampton SO16 6YD, United Kingdom; 2Department of Basic & Applied Biology, University of L'Aquila, 67010 L'Aquila, Italy; 3Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, 20133 Milan, Italy; 4Department of Pathology & Diagnostics, University of Verona, 37134 Verona, Italy
The aim of this study was to identify novel recombinant single chain antibodies showing good affinity for the CD22 antigen, a known marker for B-cell lymphomas and some leukaemias, that could be used for the construction of recombinant immunotoxins (IT) based on the ribosome inactivating protein saporin or bacterial pseudomonas exotoxin (PE40) and expressed in Pichia pastoris or E. coli, respectively.Our first attempts to screen naive phage display libraries for anti-CD22 scFvs failed and for this reason, we resorted to the standard strategy of isolating the variable domains of the antibody light and heavy chains from 4KB128, an established anti-CD22 secreting hybridoma, We obtained potential candidate recombinant scFvs that showed affinities estimated to be in the nanomolar range. We progressed to assaying different formats of recombinant constructs after producing a series of model recombinant immunotoxins (IT) based on saporin or pseudomonas exotoxin 40 (PE40) by fusing the anti-CD22 scFv to each toxin. Comparison of the saporin and PE-based scFv ITs in a protein synthesis inhibition assay revealed similar performances with observed IC50 values of 3.6 x 10-10M and 1 x 10-9M , for the saporin and PE40 constructs, respectively. Saponinum albans saponins (SA) augmented the PE40 based IT by only 170-fold whilst the saporin-based IT was augmented more than one million-fold by SA. These results reveal the very major differences in augmentative effect of SA on PE40 and saporin based ITs, an observation which likely reflects differences in the intracellular routing of each.
Category: Targeted human proteins as effectors
TALK: Wednesday 21 March 2012, 14:50
Cell Surface Delivery of TRAIL Strongly Augments the Tumoricidal Activity of T Cells
Wijnand Helfrich
Surgery, University Medical Center Groningen, 9713 GZ Groningen, The Netherlands
In general, current adoptive T-cell therapies fail to induce meaningful anticancer responses in patients with solid tumors. Here, we present a novel approach designed to selectively enhance the tumoricidal activity of T cells by the targeted delivery of TNF-related apoptosis-inducing ligand (TRAIL) to the T-cell surface. To this end, we constructed two recombinant fusion proteins, designated anti-CD3:TRAIL and K12:TRAIL, directed at the T-cell surface markers CD3 and CD7, respectively. Tumoricidal activity of T cells in the presence of these fusion proteins was assessed using solid tumor cell lines, primary patient-derived malignant cells, and in a murine xenograft model.
When added to T cells, fusion proteins K12:TRAIL and anti-CD3:TRAIL selectively bind to the T-cell surface antigens CD3 and CD7, respectively. T cell surface accretion of anti-CD3:TRAIL and K12:TRAIL increased the tumoricidal activity of T cells toward cancer cell lines and primary patient-derived malignant cells by more than 500-fold. Furthermore, T-cell surface delivery of TRAIL strongly inhibited tumor growth and increased survival time of xenografted mice more than 6-fold.
In conclusion, targeted delivery of TRAIL to cell surface antigens of T cells potently enhances the tumoricidal activity of T cells. This approach may be generally applicable to enhance the efficacy of adoptive T-cell therapy.
TALK: Wednesday 21 March 2012, 15:25
Targeted Induction of Tumor Cell Death by Gene-Modified Killer Cells
Winfried S. Wels1, Kurt Schönfeld1, Christiane Sahm1, Torsten Tonn2, Manuel Grez1, Robert Jabulowsky1, Pranav Oberoi1, Sabrina Genßler1, Congcong Zhang1
1Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, 60596 Frankfurt/Main, Germany; 2German Red Cross Blood Donation Service East, 01307 Dresden, Germany
Natural killer (NK) cells are the body´s first line of defense against viral infections and malignant cells, and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also continuously growing killer cells such as the clinically applicable human NK cell line NK-92 hold promise for cancer immunotherapy. To further enhance their therapeutic utility and provide NK-92 cells with pre-determined tumor cell specificity, we generated genetically modified variants that express chimeric antigen receptors (CAR). These CAR are composed of a single-chain antibody fragment (scFv) for recognition of antigens on the tumor cell surface, fused to the CD3 zeta chain or a composite CD28-CD3 zeta sequence as an intracellular signaling moiety. We could demonstrate that NK cells carrying CAR with specificity for tumor-associated surface antigens such as ErbB2 (HER2), EpCAM and GD2 selectively recognize and kill otherwise NK-resistant cancer cells which express the respective target molecules. Unlike unmodified NK cells, the retargeted killer cells specifically localized to tumors in vivo. For development of ErbB2-specific NK-92 cells towards clinical applications, we generated an optimized lentiviral CAR vector, and established GMP-compliant transduction and expansion protocols. Ongoing work also aims at the generation of optimized CAR for alternative target structures, and tumor-specific NK-cell variants that function independently of exogenous cytokines.
TALK: Wednesday 21 March 2012, 16:00
First and Second Generation Human anti-ErbB2 Immunoagents
Gennaro Riccio, Carmine Fedele, Marianna Borriello, Paolo Laccetti, Giuseppe D'Alessio, Claudia De Lorenzo
Department of Structural and Functional Biology, University of Naples Federico II, 80126 Napoli, Italy
Overexpression of ErbB2 receptor is a sign of malignancy and poor prognosis of breast cancer. Herceptin, a humanized anti-ErbB2 antibody, is effective in the therapy of breast carcinoma, but it engenders cardiotoxicity and many breast cancer patients are resistant to Herceptin-treatment.
Two human antitumor immunoconjugates were engineered in our laboratory by fusion of a human anti-ErbB2 scFv, termed Erbicin, with either human pancreatic RNase (HP-RNase) or the Fc region of a human IgG1 (Erb-hcAb). Both Erbicin-Derived Immunoagents (EDIA) are selectively cytotoxic for ErbB2-positive cancer cells in vitro and in vivo.
EDIA recognize an epitope different from that of Herceptin, are active on some Herceptin-resistant cancer cells both in vitro and in vivo, and are not cardiotoxic. Indeed, EDIA, differently from Herceptin, do not show toxic effects in vitro on rat and human cardiomyocytes, and do not impair cardiac function in vivo in a mouse model.
Second generation EDIA were obtained either by the fusion of Erb-hcAb with HP-RNase (Erb-hcAb-RNase) or by the conjugation of Erbicin with a dimeric variant of HP-RNase (Erb-HHP-RNase).
Both the novel immunoRNases retain the RNase enzymatic activity and specifically bind to ErbB2-positive cells. Moreover, the novel EDIA are endowed with an effective and selective cytotoxic action for ErbB2-positive tumor cells more potent than that of the first generation ImmunoRNases.
Thus, EDIA could fulfil the therapeutic need of cancer patients ineligible to Herceptin treatment due to cardiac dysfunction and to Herceptin-resistance.
TALK: Thursday 22 March 2012, 15:25
Recombinant Cytolytic Fusion Proteins for the Treatment of CD64-positive Diseases
Theo Thepen1, Michael Huhn2, Mehmet K. Tur3, Grit Hehmann4, Georg Melmer4, Stefan Barth1, 2
1Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, 52074 Aachen, Germany; 2Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, 52074 Aachen, Germany; 3Experimental Pathology and Immunotherapy, University Hospital Gießen and Marburg (UKGM) GmbH, 35392 Gießen, Germany; 4Pharmedartis, 52074 Aachen, Germany
Under pathological conditions, activated and dysregulated macrophages play a decisive role in the development of chronic inflammation. Because of their versatile functions, e.g. powerful control over other cells and directly inflicting severe damage, they provide a good target for therapeutic intervention. One characteristic feature for activated macrophages is specific and high expression of CD64, the high affinity receptor for IgG. The primary function of CD64 is internalization, which qualifies this receptor as a prime target for therapeutic intervention. To specifically target cell-receptors like CD64, monoclonal antibodies, or derivatives thereof are highly suitable. Using these ligands for selective elimination by combining them with e.g. a cytotoxic compound might result in potent therapeutics to treat CD64-expressing diseases. Proof of concept was provided by experiments targeting activated macrophages in chronic disease using chemically linked immunotoxins based on anti-CD64 antibody. There are however potential side effects, like immunogenicity and non-specific toxicity, associated with these classically constructed IT. Therefore, we designed a number of recombinant cytolytic fusion proteins, consisting of either the bacterial toxin ETA or human enzymes including angiogenin or granzyme B fused to single chain antibody fragments against CD64. This presentation will summarize the latest results on our next generation fully human recombinant cytolytic fusion proteins.
TALK: Thursday 22 March 2012, 16:00
Targeting Cancer Amino Acid Auxotrophy with Pegylated Recombinant Human Amino Acid Hydrolases
Arthur E. Frankel1, Jeremy Mauldin1, Vaidehi Agrawal1, Jung Hee Woo1, Everett Stone2, George Georgiou2
1Department of Medicine, Cancer Research Institute of Scott & White, Temple, TX 76502, USA; 2Chemical Engineering, University of Texas, Austin, TX 78712, USA
Human tumors are frequently auxotrophic for individual amino acids due to deficiency of synthetic enzymes or increased utilization of amino acids. ALLs are deficient in asparagine synthetase and sensitive to L-asparaginase therapy. A fraction of melanomas, mesotheliomas and HCCs lack argininosuccinate synthetase (ASS) and are L-arginine auxotrophs. We prepared a recombinant cobalt-substituted, pegylated human arginase I (Co-ArgI-PEG). The kcat/Km was 3,070 mM-1s-1 and Mr was 100 kDa consistent with homotrimer. Lysyl conjugated PEG yielded 8-10 PEGs/monomer. Co-ArgI-PEG at 1 µM or 6 mg/kg reduced serum L-arginine levels to less than 5 µM for >60 h in vitro and in vivo. The agent was selectively cytotoxic to ASS deficient tumor cells in the presence of L-citrulline. In mice, the MTD was 8 mg/kg IP/wk and the DLT was weight loss and marrow necrosis. In xenograft models of human melanoma, pancreas cancer and HCC, Co-ArgI-PEG at 6-8 mg/kg IP/wk. A clinical batch of Co-ArgI-PEG was prepared. Neuroblastomas, SCLCs, and pancreatic carcinomas are methionine auxotrophs secondary to rapid metabolism and/or deficiency of methionine synthetase or methylthioadenosine phosphorylase. Because of the lack of a human methioninase enzyme, we engineered cystathione γ-lyase to change its substrate specificity. CGL-NLV-PEG and a second generation molecule display improved methionine degradation. Studies in tissue culture show moderate tumor cell selectivity across a range of tumor types and tumor growth inhibition in vivo combined with a Met(-) diet with human neuroblastoma xenografts.
SHORT COMMUNICATION: Friday 23 March 2012, 14:20
Antibodies and Antibody RNase Fusion Proteins Targeting CD30+ Lymphoma
Thomas Schirrmann
Institute of Biochemistry / Biotechnology, Technische Universität Braunschweig, 38108 Braunschweig, Germany
Antibody fusion proteins offer the perspective to improve the efficacy of antibody tumor therapies by adding novel effector functions. However, this approach has been hampered for decades by the lack of appropriate human fusion partners as effectors to overcome immunogenicity and/or unspecific toxicity as well as difficult recombinant production. A now well established class of effectors are ribonucleases of the RNase A protein superfamily which are mostly non-toxic while in circulation but become highly effective in cell killing after internalization and cytoplasmic release. We engineered an entirely human immunoRNase directed to CD30+ tumors by fusing a single chain (sc)Fv-Fc antibody with human pancreatic RNase. The scFv-Fc-RNase was produced in suspension HEK293-EBNA cells with yields of more than 400 mg/L. The scFv-Fc-RNase protein showed the same antigen binding like the parental scFv-Fc antibody, and ribonucleolytic activity was comparable to human pancreatic RNase. RNase activity was reduced in the presence of cytosolic RNase inhibitor (RI) but did not decrease below 20% residual activity even in 150 times molar access of RI. The scFv-Fc-RNase specifically inhibited the growth of CD30+ lymphoma cells at low nanomolar concentrations.
POSTER and SHORT COMMUNICATION: Friday 23 March 2012, 14:00
Human Recombinant Arginase I (Co)-PEG5000 is Selectively Cytotoxic to Human Acute Myeloid Leukemia (AML) Cell lines
Ralph J. Abi-Habib1, Amira Bekdash*1, Rita Tanios*1, Elie Kassab1, Everett Stone2, George Georgiou2, Arthur E. Frankel3
1Department of Biology, Lebanese American University, 1102 2801 Beirut, Lebanon; 2Department of Chemical Engineering, University of Texas, Austin, TX 78712, USA; 3Cancer Research Institute of Scott & White, Temple, TX 76502, USA
* These authors contributed equally to this work
Though combination induction and consolidation chemotherapy induce complete remission in a high proportion of AML patients, most eventually relapse. Alternative approaches with more selective mechanisms for targeting AML are needed. We attempt to target potential Arginine auxotrophy of AML cells using pegylated recombinant human Arginase I cobalt [HuArgI (Co)-PEG5000].
Potency and selectivity of HuArgI (Co)-PEG5000 were tested on 8 human AML cell lines and on human monocytes using a proliferation inhibition assay. Cell cycle analysis was carried out by propidium iodide (PI) staining and type of cell death was determined by AnnexinV/PI staining using flow cytometry.
7 AML cell lines were sensitive to HuArgI (Co)-PEG5000 at 48-hour incubation (IC50 = 14-550 pM). Human monocytes were not sensitive to HuArgI (Co)-PEG5000 (IC50 > 10,000 pM). Incubation times of 48 and 72 hours had similar potency but were 7-fold more potent than 24-hour incubation. There was no impact of treatment on cell cycle with both the G0/G1 and G2/M peaks decreasing significantly with increased concentrations of HuArgI (Co)-PEG5000, along with a significant increase in the pre-G0/G1 peak (90% of total events). AnnexinV/PI staining showed a dose-dependent increase in cells stained with both annexinV and PI (4.5% and 55% in non-treated vs treated cells) with no increase in cells staining with annexinV alone, indicating that HuArgI (Co)-PEG5000 induces necrotic cell death in AML cells.
These findings demonstrate potency and selectivity of HuArgI (Co)-PEG5000 as a potential targeted therapy for AML.
H-03
POSTER
Surface Charge-Modification Prevents Sequestration and Enhances Tumor-Cell Specificity of a Recombinant Granzyme B - TGFα Fusion Protein
Robert Jabulowsky, Pranav Oberoi, Hayat Bähr-Mahmud, Benjamin Dälken, Winfried S. Wels
Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, 60596 Frankfurt/Main, Germany
The pro-apoptotic serine protease granzyme B (GrB) plays an important role in the immune defense mediated by cytotoxic lymphocytes. Recombinant GrB derivatives fused to tumor-targeting ligands hold promise for cancer therapy, but their applicability may be limited by promiscuous binding to non-target tissues via electrostatic interactions.
Here we investigated cell binding and specific cytotoxicity of chimeric molecules consisting of wildtype or surface charge-modified human GrB and the natural EGFR ligand TGFα for tumor targeting. We mutated two cationic heparin-binding motifs responsible for electrostatic interactions of GrB with cell surface structures, and fused the resulting GrBcs derivative to TGFα for expression in the yeast Pichia pastoris.
Purified GrBcs-TGFα (GrBcs-T) and a corresponding fusion protein employing wildtype GrB (GrB-T) displayed similar enzymatic activity and targeted cytotoxicity against EGFR-expressing breast carcinoma cells. However, unspecific binding of the modified GrBcs-T variant to EGFR-negative cells was dramatically reduced, preventing sequestration by non-target cells in mixed cell cultures and increasing tumor-cell specificity. Likewise, modification of the GrB domain alleviated unspecific extracellular effects such as cell detachment indicative of extracellular matrix degradation. Our data demonstrate improved selectivity and functionality of targeted GrB employing a surface charge-modified effector domain, suggesting this strategy as a general approach for the development of optimized GrB fusion proteins for therapeutic applications.
H-04
POSTER
Genetically Modified Natural Killer Cells that Express Tumor-Specific Granzyme B Fusion Proteins
Pranav Oberoi, Robert Jabulowsky, Hayat Bähr-Mahmud, Winfried S. Wels
Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, 60596 Frankfurt/Main, Germany
Immunotoxin-like molecules that combine selective recognition of tumor cells with a cell death-inducing activity of human origin hold promise as targeted therapeutics for cancer treatment. One such approach is based on derivatives of the pro-apoptotic serine protease granzyme B (GrB) that harbor peptide ligands or antibody domains for tumor-specific cell recognition. GrB is naturally produced by cytotoxic lymphocytes, where it resides together with other granzymes and the pore-forming protein perforin in cytotoxic granules. Upon degranulation GrB is released, enters target cells along with perforin, and rapidly induces apoptosis via caspase-dependent and caspase-independent mechanisms. Here we investigated natural killer (NK) cells as a system for the expression of tumor-specific GrB fusion proteins. Since NK cells naturally express wildtype GrB, they may represent an ideal environment for processing, packaging and activation of retargeted GrB variants, and allow combining such tumor-specific molecules with the NK cells natural cytotoxicity.
To establish proof of concept, GrB fusion proteins that carry either an ErbB2-specific scFv antibody fragment or the EGFR-specific peptide ligand TGFalpha for cell targeting were successfully expressed in NK cells. Induction of degranulation resulted in the release of functionally active chimeric GrB proteins together with endogenous granular proteins. Potential cooperation of the chimeric molecules with endogenous granular constituents from NK cells in tumor cell killing is currently being investigated.
H-05
POSTER
Antibody-fusion proteins targeting the intracellular domain of epidermal growth factor receptor
Nina Müller1, Cord Hartmann1, Joachim Koch1, Yonathan Deribe2, Ivan Dikic2, Winfried S. Wels1
1Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, 60596 Frankfurt/Main, Germany; 2Institute of Biochemistry II, Goethe University , 60590 Frankfurt/Main, Germany
Aberrant activation of the epidermal growth factor receptor (EGFR) has been found in cancers of various origins, and has been implicated in cancer pathogenesis. We previously generated scFv antibody fragments specific for the intracellular domain of EGFR, which retained specific binding upon expression as cytoplasmic intrabodies. Here we identified the binding epitope of the scFv antibodies, which recognized an identical linear peptide close to the C-terminus of EGFR in peptide array analysis. To investigate the potential utility of the scFv molecules for intracellular targeting of heterologous protein domains to EGFR, we generated chimeric scFv-Cbl fusion proteins. The E3 ubiquitin-ligase c-Cbl is an important regulator of intracellular signaling pathways, and is critically involved in EGFR downregulation. We fused the RING-finger domain (RING) or the complete C-terminal domain (ALL) of c-Cbl, both lacking the intrinsic phosphotyrosine binding domain, to a selected EGFR-specific scFv (scFv30). The resulting scFv30-Cbl fusion proteins displayed specific binding to EGFR, and recombinant scFv30-RING mediated ubiquitination of a GST-EGFR fusion protein in a cell-free assay. In addition, expression of scFv30-RING or scFv30 ALL fusion proteins in EGFR-dependent MDA-MB468 breast carcinoma cells resulted in transient growth retardation, and in the case of scFv30 ALL decreased EGFR surface levels after receptor stimulation. Ongoing work now aims at a detailed characterization of the effects of scFv30-Cbl fusion proteins in living cells utilizing an inducible expression system.
H-06
POSTER
Anthrax Lethal Toxin (LeTx) Induces Both Cytotoxicity and Cell Cycle Arrest in Acute Myeloid Leukemia (AML) Cell Lines
Ralph J. Abi-Habib1, Manal Darwish*1, Zahra Timsah*1, Elie Kassab1, Shihui Liu2, Stephen H. Leppla2, Arthur E. Frankel3
1Department of Biology, Lebanese American University, 1102 2801 Beirut, Lebanon; 2National Institute of Allergies and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD20892, USA; 3Cancer Research Institute, Scott&White Memorial Hospital, Temple, TX76502, USA
* These authors contributed equally to this work
In this study, we attempt to target the MAPK pathway in AML cells using recombinant anthrax lethal toxin. LeTx consists of protective antigen (PrAg), and lethal factor (LF). PrAg binds cells, is cleaved by furin, heptamerizes, binds 3 molecules of LF and undergoes endocytosis, releasing LF into the cytosol. LF is a protease that cleaves all mitogen-activated protein kinase (MAPK) kinases.
Potency of LeTx was tested on 11 human AML cell lines using a proliferation inhibition assay. Cell cycle analysis was carried out by propidium iodide (PI)-staining and type of cell death was determined by Annexin V/PI staining.
7 AML cell lines were sensitive to LeTx (IC50 = 13-94 pM and percent cell death > 70%). LeTx-resistant cell lines were sensitive to the PI3K inhibitor LY294002. Co-treatment with LeTx and LY294002 led to marginal increase in sensitivity. Co-treatment of LeTx-sensitive cells with both LeTx and LY294002 also led to marginal increase in sensitivity, indicating that AML cells are sensitive to the inhibition of either the MAPK or the PI3K/Akt pathway, but not to both. LeTx caused cell cycle arrest in 3/4 LeTx-resistant cell lines. AnnexinV/PI and caspase staining showed an increase in the percentage of cells stained with annexinV alone and an increase in cells positive for active caspases indicating that LeTx treatment leads to apoptotic cell death in AML cells.
Results indicate that a majority of AML cell lines can be targeted through LeTx-mediated inhibition of the MAPK pathway with a subset being sensitive to the inhibition of the PI3K/Akt pathway.
H-07
POSTER
The Human Epithelial Cell Adhesion Molecule (EpCAM) as Target for Molecular Imaging
Markus Heine
Institute of Anatomy and Experimental Morphology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
Molecular imaging is a technique to determine in a non invasive way the amount of molecules in vivo. So far, little is known about the relation of the amount of target sites and their accessibility for molecular imaging tracers.
In this study, we systematically investigated the presence of EpCAM as target molecule ranging from human cell culture grown cells to xenograft tumours of several human malignant entities by different techniques. These results were correlated to the binding of i.v. applicated anti-EpCAM antibody MOC31 and the nonspecific diffusion of Evans Blue into HT29 xenograft tumour. We could show that EpCAM is both expressed in vivo and in vitro. However, far less binding sites are accessible for the antibody if applied in vivo as compared to tissue sections. Investigation of the fine structure of the HT29 tumour blood vessels showed that they are immature and thus prone for higher fluid flux into the interstitial space. Consistent with this hypothesis, a higher interstitial fluid pressure of about 12 mbar was measured in the HT29 primary tumour via a “wick in needle” technique which explains the limited diffusion of the antibody into the tumour observed in immunohistochemistry. To the best of our PubMed searches, this is the first work demonstrating both antibody binding site distribution and measurement of the interstitial fluid pressure explaining the results of the antibody distribution experiments.
SHORT COMMUNICATION: Wednesday 21 March 2012, 14:35
Efficacy of Bifunctional TRAIL-KV10.1 Potassium Channel Antibody in Xenograft Models
Luis A. Pardo, Franziska Hartung
Max-Planck-Institute of Experimental Medicine, 37075 Göttingen, Germany
The potassium channel KV10.1 (Ether-á-go-go) is attractive as target since this surface protein is virtually not detected in normal tissue outside the central nervous system, but is expressed in approximately 70% of tumors from different origins. We designed a single-chain antibody against an extracellular region of KV10.1 (scFv62) and fused it to the human soluble TRAIL. Prostate cancer cells, either positive or negative for KV10.1 were treated with the purified construct. After sensitization with cytotoxic drugs, scFv62-TRAIL induced apoptosis only in KV10.1-positive cancer cells, but not in non-tumor cells, nor in tumor cells lacking KV10.1 expression. In co-cultures with KV10.1-positive cancer cells the fusion protein also induced apoptosis in bystander KV10.1-negative cancer cells, while normal prostate epithelial cells were not affected when present as bystander. In mouse xenograft models, administration of scFv62-TRAIL in combination with doxorubicin induced a reduction in tumor mass after the first injection; ex vivo tumor weight was significantly smaller in mice treated with scFv62-TRAIL plus doxorubicin than in those receiving either agent alone.
Category: Innovative Tools to Improve Targeted Tumor Therapies
TALK: Wednesday 21 March 2012, 17:35
Facile functionalization of DARPins for tumor targeting
Uwe Zangemeister-Wittke, Manuel Simon, Nikolas Stefan, Andreas Plückthun
Institute of Pharmacology, University of Bern, 3010 Bern, Switzerland and Institute of Biochemistry, University of Zurich, 8057 Zürich, Switzerland
For tumor targeting antibodies and antibody fragments have practical limitations due to poor expression yield, low stability, aggregation tendency and limited freedom for engineering. A solution might come with the use of non-IgG scaffolds like Designed Ankyrin Repeat Proteins (DARPins). DARPins consist of repeat sequence motifs forming a contiguous protein domain for target binding flanked by N- and C-terminal capping repeats. Due to their modular design, high stability and favorable biophysical properties DARPins tolerate engineering procedures not applicable to antibodies. Moreover, they are devoid of cysteines, which can be introduced for site-specific modifications, and contain only 1-2 methionines, which can be replaced by clickable surrogates.
We have generated affinity matured DARPins recognizing EpCAM as a representative tumor-associated antigen overexpressed in carcinomas. To expand our arsenal of proteins for tumor targeting, DARPins and fusion toxins derived thereof were engineered for facile functionalization with molecules designed to improve blood half-lives, tumor accumulation and in vivo imaging, using Cu-free click and thiol chemistry. Since functionalization with hydrophilic polymers like poly(ethyleneglycol) often comes at the expense of efficient ligand binding and toxin activity, we also devised a strategy for reversibly veiling bioactive domains in the cell binding and toxin moiety using clickable linker constructs sensitive to cleavage by proteases expressed in or co-targeted to tumors.
TALK: Wednesday 21 March 2012, 18:10
Single Chain Triplebodies for the Elimination of Leukemic Cells
Georg H. Fey1, Ingo Schubert1, Markus Kuegler1, Christoph Stein1, Christian Kellner2, Nadja Fenn3, Karl-Peter Hopfner3, Fuat Oduncu4, Bernhard Stockmeyer5, Andreas Mackensen5
1Biology, University of Erlangen-Nuremberg, 91058 Erlangen, Germany; 2Division of Stem Cell Transplantation and Immunotherapy, University Medical Center, University of Schleswig-Holstein, 24105 Kiel, Germany; 3Biochemistry, Genecenter, Ludwig Maximilians University, 81377 Munich Martinsried, Germany; 4Hematology and Oncology, University Medical Center, Ludwig Maximilians University, 80336 Munich, Germany; 5Internal Medicine V, Hematology/Oncology, University Medical Center Erlangen, 91054 Erlangen, Germany
To enhance selectivity of targeting, we have developed a new format of antibody-derivatives, single-chain triplebodies, consisting of 3 single-chain Fv binding sites, joined into a single polypeptide chain. The distal sites are specific for tumor cell surface antigens, the central site binds a trigger on an effector cell, for example CD16 on NK-cells and macrophages or CD3 on T-cells. A single triplebody molecule can simultaneously bind 2 antigens on the cancer cell and trigger the effector function.
The prototype 19-16-19 with specificity for CD19 eliminated malignant B-lymphoid cells in redirected lysis (ADCC) experiments with NK cell effectors and an EC50 in the low picomolar range. It has a useful plasma retention time in mice, low molecular mass, good avidity for cancer cells and good stability. Preclinical development is under way.
The second prototype, 33-16-123, targets CD33 and CD123 on the surface of malignant myeloid cells. It was designed for a preferential elimination of acute myeloid leukemia stem cells (AML-LSCs), which carry a greater cell surface density of CD123 than bulk AML cells and healthy myeloid cells. This agent efficiently eliminated AML-derived cell lines and primary cells from AML patients.
In a model situation we have shown that dual targeting created preferential binding to antigen double-positive over antigen single-positive cells, even if the latter were present in numerical excess. Triplebody 33-16-123 is also under preclinical development, and the format can be used to build other agents for dual-targeting of a variety of cancers.
TALK: Thursday 22 March 2012, 09:10
Recombinant Human Antibodies for Target Identification
Stefan Dübel
Institute of Biochemistry / Biotechnology, Technische Universität Braunschweig, 38108 Braunschweig, Germany
Recombinant antibodies are now widely accepted as targeting domains for the treatment of cancer, infectious diseases or other conditions. Today, we master the generation of human antibodies, and can improve many of their properties. For example, our system for the generation of human antibodies by antibody phage display has yielded binders to more than 170 antigens, including more than 110 human proteins. The control of the biochemical conditions during the selection process allowed to make antibodies e.g. specific for a single phosphorylation or to antigens in complex membrane preparations. Binding properties and stability of the antigen binding domains can be improved by in vitro evolution, as demonstrated for a MUC1 antibody. With the technology for the generation of human targeting domains being matured now, it is evident that identification of disease related targets becomes the limiting factor. Here, progress in miniaturization and automation of antibody phage display allows to tackle the generation of binders to the entire human proteome, facilitating a systematic search for new targets. Our integrated pipeline for the in vitro generation of monoclonal antibodies by phage display not only allows to increase the throughput but also delivers the antibody gene, which now can be used for functional analysis, like functional knock down of the target antigen, as demonstrated for receptors on immune and nerve cells. Once an interesting target has been identified, the human antibody lead for further development into a drug is available right away.
Fusion Constructs Targeting Metastatic Spread, Skeletal Tumors and Tumor Vasculature – A Multifaceted Approach with One Molecule
Michael Rosenblum
Experimental Therapeutics, MD Anderson Cancer Center, Houston, TX 77030, USA
The development of tumor neovasculature defines successful establishment of tumor metastases. The same process occurs in normal events such as wound healing and embryogenesis and is central to pathological states such as diabetic retinopathy and rheumatoid arthritis. The growth factor VEGF and its R1 and R2 receptors are mediators of normal and pathological neovascularization processes. We designed a construct containing VEGF121 and rGel toxin. The VEGF/rGel construct was highly cytotoxic to endothelial cells expressing the R2 receptor. Intoxication of cells occurred quickly through a necrotic mechanism. VEGF/rGel localized in the vasculature of tumor xenografts. Against 9 different orthotopic, metastatic and subcutaneous tumor models VEGF/rGel showed impressive antitumor effects. IHC analysis showed impressive damage to tumor vascular bed integrity. Skeletal metastases represent a major cause of morbidity and mortality in a number of tumor types. Against an osteolytic and osteoblastic prostate tumor model, VEGF/rGel showed impressive in vivo efficacy through targeting osteoclast and osteoblast precursor cells. Clinical Trial design will be presented and discussed. In addition a novel theragnostic application of this agent for PET imaging using 64Cu-DOTA VEGF/rGel will be presented in animal studies and a Phase 0 clinical trial design will be presented and discussed. Finally, data will be presented on the impact of VEGF/rGel in the formation of metastatic lesions in the lung and the use of targeted therapeutics on disrupting the early events in tumor metastatic spread.
TALK: Thursday 22 March 2012, 11:15
Half-life Extension Strategies to Improve Targeted Delivery of Antibodies
Roland Kontermann
Institute of Cell Biology and Immunology, University of Stuttgart, 70569 Stuttgart, Germany
With a growing number of small protein therapeutics being developed, including recombinant antibodies and the emerging class of novel scaffold proteins, half-live extension strategies have become increasingly important to improve their pharmacokinetic and pharmacodynamic properties. In order to improve the pharmacokinetic properties of a small recombinant antibody, we have investigated several half-life extension strategies including PEGylation, N-glycosylation, fusion to albumin or an albumin-binding domain (ABD). This comparative study revealed that fusion to albumin or an ABD to a 55 kDa bispecific single-chain diabody (scDb) results in a similar increase in circulation time as PEGylation, while N-glycosylation only moderately increased half-lives. The prolonged half-life especially of the ABD fusion protein also translated into an increased tumor accumulation of the scDb-CEACD3 directed against carcinoembryonic antigen (CEA) and CD3. Recently, we have extended the half-life extension strategies to immunoglobulin-binding domains (IgBD). Fusion of various IgBDs derived from protein A, protein G and protein L to bispecific single-chain diabodies and scFv fragments prolonged the plasma half-life in mice to varying extent. A remarkably long half-life was mediated by domain C3 of streptococcal protien G (SpGC3), which makes this domain a promising module for further studies and optimization. In summary, our data provide a rational for the modulation of pharmacokinetic properties and therapeutic efficacy of small-sized protein therapeutics.
TALK: Thursday 22 March 2012, 14:15
Superior Anti-tumoral Activity of Dimerized Targeted Single-chain TRAIL Fusion Proteins under Retention of Tumor Selectivity
Martin Siegemund1, Nadine Pollak1, Oliver Seifert1, Kristin Wahl2, Kristin Hanak2, Arndt Vogel2, Andreas Nussler3, Doris Göttsch1, Sabine Münkel1, Heike Bantel2, Roland Kontermann4, Klaus Pfizenmaier1
1Cell Biology and Immunology, 70569 Stuttgart, Germany; 2Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 30625 Hannover, Germany; 3Department of Trauma Surgery, Eberhard Karls University Tübingen, 72076 Tübingen, Germany; 4Institute of Cell Biology and Immunology, University of Stuttgart, 70569 Stuttgart, Germany
Due to legal reasons this abstract is not available here.
TALK: Thursday 22 March 2012, 14:50
Manipulating Apoptosis and Autophagy to Improve Targeted Toxin Therapy
Andrew Thorburn
Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
Like most anti-cancer drugs, tumor cell-targeted toxins work by killing cancer cells and in most cases these drugs kill cells by inducing apoptosis or some combination of apoptosis and non-apoptotic forms of cell death. Using an Epidermal Growth Factor Receptor-targeted diphtheria toxin molecule (DT-EGF) we have been studying cell death mechanisms and the results of manipulating these mechanisms on tumor cells. DT-EGF can kill cells by both apoptotic and non-apoptotic mechanisms and it is also able to regulate autophagy in the dying cells. Autophagy is now widely viewed as a mechanism whereby cell death can be regulated although both pro-and anti-death effects can be seen. In this presentation I will argue that autophagy does something else – it controls the characteristics of dying cells. Additionally I will present data showing that by manipulating the way that tumor cells are killed (by allowing or inhibiting autophagy) it is possible to alter the growth of neighboring tumor cells that are resistant to the treatment. Thus m,anipulation of death mechanisms can be used to maximize the effects of an anti-tumor treatment not because one alters the number of tumor cells being killed but instead because one alters the response of resistant cells to the effects of the dying cells.
TALK: Friday 23 March 2012, 08:30
Enhancers of Endosomal Escape for Improved Targeted Drug Delivery
Diana Bachran1, Christopher Bachran2, Alexander Weng1, Mayank Thakur1, 3, Matthias F. Melzig3, Hendrik Fuchs1
1Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité – Universitätsmedizin Berlin, 12200 Berlin, Germany; 2Laboratory of Parasitic Diseases, Microbial Pathogenesis Section, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA; 3Institut für Pharmazie-Pharmazeutische Biologie, Freie Universität Berlin, 14195 Berlin, Germany
Targeted tumor therapies, consisting of a cytotoxic moiety fused to an antibody or ligand, are a promising approach for the treatment of cancer to overcome some chemotherapy-based problems. A lot of new strategies have been developed in the last years and were successfully tested in clinical trials. However, targeted therapies are not free of side effects, are often not suitable for the treatment of solid tumors and show a low cytosolic uptake. Here, we describe the synergistically increase in cytotoxicity of a targeted toxin due to the combined application with glycosylated triterpenoids (saponin) of plant origin. The targeted toxin (SA2E) consisted of saporin as toxic agent, an adapter for reduction of side effects and the epidermal growth factor (EGF) as targeting moiety. In vitro studies revealed a cell-depended clearly synergistic increase in toxin-mediated cytotoxicity up to 20,000-fold. Interestingly, the usage of inhibitory agents of different processes involved in the uptake of EGF receptor had no effect on the targeted toxin SE (saporin and EGF without the adapter) alone, while they diminished the cytotoxicity of SE when combined with saponin. In in vivo studies the combinational treatment of TSA-EGFR tumors grown in BALB/c mice resulted in 94% tumor volume reduction. Furthermore, SA2E was used in a 50-fold lower concentration compared to a treatment with SA2E alone. Side effects were, due to the low concentration of SA2E, only moderate and usually reversible demonstrating the impressive potential of a combinational treatment with saponins.
TALK: Friday 03 April 2009, 10:50
Improving Immunotoxin Potency with Triterpenoid Saponins
David J. Flavell
The Simon Flavell Leukaemia Research Laboratory, Southampton General Hospital, SO16 6YD Southampton, United Kingdom
A major limiting step that determines the cytotoxic potency of immunotoxins (IT) and cytotoxins (CT) is the efficiency with which the toxin component is internalised and appropriately routed to target ribosomes in the target cell cytosol. There are several small molecules described in the literature that modify IT trafficking, degradation and/or translocation across the endosomal membrane into the cytosol of the target cell. However, the majority of molecules described only exert relatively modest augmentative effects on IT activity and most possess unacceptable toxicities. Development of alternative non-toxic strategies to improve IT or CT potency would represent a major step forward for the useful clinical utility of this class of drug.
Triterpenoid saponins derived from a variety of plant sources have been shown to potently augment IT and CT cytotoxicity by a factor that can be up to several million-fold for relevant ligand bearing target cells. The degree of augmentation varies depending on the cellular target molecule and/or cell type, an observation that possibly reflects differences in lipid/cholesterol composition of membrane microdomains associated with a particular protein target molecule. Experimental data will be presented that describes, amongst other things, the importance of membrane cholesterol content and the cell membrane permeabilising properties of saponins for their IT augmentative characteristics. These and other findings will be discussed in the context of the potential value of saponins as agents used to augment IT activity in a clinical setting.
TALK: Friday 23 March 2012, 15:10
Treating Solid Tumors with Protease-activated Anthrax Toxin
Shihui Liu1, Diane Peters2, Stephen H. Leppla1, Thomas Bugge2
1Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA; 2Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD 20892, USA
A majority of human melanomas contain a BRAF V600E activating mutation. These tumors depend on this BRAF oncogene and thus are sensitive to MEK inhibitors as well as anthrax lethal factor (LF), due to its proteolytic activities toward MEKs. However, anthrax lethal toxin, consisting of cellular binding moiety protective antigen (PA) and LF, is a major virulence factor of Bacillus anthracis. In order to decrease its in vivo toxicity, we generated mutated PA proteins that can only be activated by matrix metalloproteinases (MMPs) or urokinase plasminogen activator (uPA) that are overproduced by tumor tissues. We found, surprisingly, that both the MMP-activated and uPA-activated lethal toxins have potent anti-tumor activity not only to human melanomas with the BRAF mutation, but also to a wide range of other tumor types regardless of the BRAF status. This is largely due to the indirect targeting of tumor angiogenesis.
PA forms a heptamer in which the binding site for LF spans two adjacent monomers. PA mutants were produced having mutations in different LF binding subsites and containing either uPA or MMP cleavage sites. Individually, these PA mutants had low toxicity due to impaired LF binding, but when administered together to uPA- and MMP-expressing tumor cells, they assembled into functional LF-binding heteroheptamers. The mixture of two complementing PA variants and LF had further reduced toxicity to mice and was highly effective in treatment of human head and neck squamous cell carcinomas in mouse xenograft models, thereby achieving high therapeutic indices.
SHORT COMMUNICATION: Friday 23 March 2012, 14:45
Ubiquitin-Anthrax Toxin Fusion Proteins for the Modulation of Toxin Release and Cytotoxicity of Tumor-Targeted Anthrax Toxins.
Christopher Bachran, Suzanne Abdelazim, Shihui Liu, Stephen H. Leppla
Laboratory of Parasitic Diseases, Microbial Pathogenesis Section, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA
Anthrax toxin constitutes a highly efficient protein delivery system to the cytosol of tumor cells. However, the ultimate anti-tumor efficacy of these agents depends on their resistance to inactivation and degradation within the cells. It is evident from the low lysine to arginine ratios in many bacterial toxins that avoidance of ubiquitinylation is a key factor controlling their potency. We created fusion proteins containing modified ubiquitins having C-terminal fusions to the catalytic domain of Pseudomonas Exotoxin A (PEIII). The potency of these proteins was highly dependent on the number of lysines retained with the ubiquitin domain, and on whether they retained the C-terminal ubiquitin sequence cleaved by cytosolic deubiquitinating proteases (DUBs). Thus, fusions in which all seven native lysines of ubiquitin were retained whereas the DUB site was removed resulted in lower cytotoxicity, apparently due to rapid ubiquitination and proteasomal degradation. In contrast, fusions in which all lysines of ubiquitin were substituted by arginine and the DUB site was retained had high potency, exceeding that of a simple fusion lacking ubiquitin. It appears that rapid cytosolic release of a cytotoxic enzyme (e.g., PEIII) that is itself resistant to ubiquitination, is an effective strategy for enhancing the potency of tumor-targeting toxins.
SHORT COMMUNICATION: Thursday 22 March 2012, 10:20
Photochemical Internalization – an Innovative Tool to Improve Targeted Tumor Therapies
Pål K. Selbo1,2, Anette Weyergang1, Monica Bostad1, Marius Eng1, Maria Berstad1, Anders Høgset2, Kristian Berg1
1Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, 0310 Oslo, Norway; 2PCI Biotech AS, 1366 Lysaker, Norway
Photochemical internalization (PCI) was developed at the Norwegian Radium Hospital as a method for light-enhanced cytosolic release of membrane-impermeable molecular therapeutics entrapped in endocytic vesicles. Briefly, the drug of interest colocalizes with a photosensitizer in endocytic organelles. Light-activation of the photosensitizer results in reactive oxygen species-mediated rupture of the membranes of these vesicles with the subsequent release of the drug to cytosol. PCI has been shown to be a specific and efficient technology for induced endolysosomal escape for several cancer targeted therapeutics and to increase the efficacy of some conventional anti-cancer agents. As drug models we have used type I ribosome-inactivating proteins (RIPs), including saporin and gelonin, and immunotoxins based on type I RIPs targeting EpCAM, EGFR and CSPG4/HMW-MAA. Site specific laser-activation of a tumor-accumulated photosensitizer that co-localizes with an immunotoxin makes this therapeutic strategy highly selective and efficient. PCI can also improve the delivery of nanoparticles and improve gene therapy. PCI has been demonstrated to be a feasible drug delivery technology in >80 different cancer cell lines and >10 different animal models. A successful Phase I clinical study (http://clinicaltrials.gov/ct2/show/NCT00993512) was completed at the University Collage Hospital in London in May 2011. Recently, we initiated PCI-based targeting of cancer stem cell markers which will be a main focus of this presentation.
T-03
POSTER
Targeting of Epidermal Growth Factor Receptor with m-THPC Loaded Nanoparticles for Photodynamic Therapy
Marcella Pinto1, Giulio Fracasso1, Silvia Zanini1, Cristina Anselmi1, Daniela Segat2, Regina Tavano2, Sara Cingarlini3, Fabrizio Mancin4, Francesco Selvestrel4, Emanuele Papini2, Marco Colombatti1
1Pathology and Diagnostic-Immunology, University of Verona, 37134 Verona, Italy; 2Biomedical Sciences, University of Padova, 35100 Padova, Italy; 3Azienda Ospedaliera Universitaria Integrata Verona, Medical Oncology Unit, 37134 Verona, Italy; 4Chemical Sciences, University of Padua, 35100 Padova, Italy
Photodynamic therapy (PDT) is a new therapeutic approach for cancer treatment. One of the more effective photosensitizers employed in PDT is meta-tetra(hydroxyphenyl)chlorin (m-THPC, Foscan®), which induces high phototoxicity at very low concentrations. The use of nanoparticles as carriers for photosensitizers delivery is useful both to protect hydrophobic m-THPC from aqueous environment of biological fluids and to selectively accumulate high photosensitizer concentration in cancer tissues. The uptake at the tumor sites can be improved by nanoparticle surface modifications linking on the outer nanoparticle shell targeting molecules as antibodies recognizing tumor associated antigens or growth factors whose receptors are overexpressed in the tumor tissues.
In this work organically-modified silica (ORMOSIL) nanoparticles loaded with m-THPC were conjugated with a specific targeting molecule, i.e. anti-EGFR monoclonal antibody Cetuximab (Erbitux®), and assessed for in vitro photodynamic applications. Selective internalization of these nanoparticles in tumor cells expressing epidermal growth factor receptor (EGFR) was demonstrated. Preliminary experiments showed a good potential for m-THPC loaded nanoparticles in photodynamic applications, since the conjugation with the targeting antibody Cetuximab induced a more selective mortality effect on EGFR-expressing cells compared with control m-THPC loaded nanoparticles. Further studies will focus on defining the optimal conditions to increase system efficiency for in vivo future photodynamic application.
POSTER and SHORT COMMUNICATION: Friday 23 March 2012, 16:00
Mechanistic Studies of the Augmentative Effect of Gypsophila Saponins on Saporin-based Immunotoxins
Suzanne E. Holmes, Jennifer E. Adcott, Sopsamorn U. Flavell, David J. Flavell
The Simon Flavell Leukaemia Research Unit, Leukaemia Busters, Southampton General Hospital, Southampton SO16 6YD, United Kingdom
Saponinum album (SA), a mixture of triterpenoid saponins derived from Gypsophila paniculata has been shown to augment the ligand specific cytotoxicity of immunotoxins (IT) and cytotoxins based on the ribosome inactivating protein saporin. The extent of augmentation varies widely depending on the target cell and/or the target membrane ligand. Saponins bind non-covalently to cholesterol in the cell membrane and we speculate that the lipid/cholesterol composition of membrane micro-domains may account for the variations observed.
We demonstrate here that varying the level of cholesterol in the culture medium negatively affects both the permeabilising and augmentative activity of SA on IT cytotoxicity. Thus, culturing CD38+ human Daudi lymphoma cells in medium containing delipidated serum renders these cells increasingly less sensitive to permeabilisation by SA in a time dependent manner. Daudi cells cultured in delipidated medium become progressively depleted of membrane cholesterol and SA augmentation of IT activity concomitantly decreases in these cells. In addition we have conducted membrane resealing experiments with lytic and sub-lytic concentrations of SA which suggest that the membrane repair response may contribute to its augmentative properties. This is evidenced from calcium flux experiments and cell surface expression of CD107b (LAMP-1) indicating that membrane pores created by SA may be quickly sealed following exocytosis of lysosomal membranes. These preliminary results hint at the likely mechanistic complexity behind the augmentative effect of SA on IT activity.
POSTER and SHORT COMMUNICATION: Friday 23 March 2012, 10:00
Augmentative Activities of Saponin Species Derived from Various Natural Sources on Immunotoxin Cytotoxicity for Human Leukemia and Lymphoma Cell Lines
Emma Dearing, Suzanne E. Holmes, Jennifer E. Adcott, Sopsamorn U. Flavell, David J. Flavell
The Simon Flavell Leukaemia Research Unit, Leukaemia Busters, Southampton General Hospital, Southampton SO16 6YD, United Kingdom
It has previously been shown that saponins derived from Gypsophila species (Saponinum Album, SA), can, at sub-toxic dose levels, augment the activity of targeted toxins that are based on the ribosome inactivating protein saporin, when directed against specific ligand expressing human cell lines. We have investigated the ability of three other triterpenoid or steroidal saponin species, quillaja, digitonin and dioscin at augmenting the immunospecific cytotoxic activity of a saporin-based anti-CD38 immunotoxin (OKT10-Saporin) directed against CD38+ human leukaemia and lymphoma cell lines.
Comparative studies revealed that the ability to augment the cytotoxic action of the immunotoxin varied widely between different saponin species, as determined by XTT assay and Annexin V/PI staining. The augmentative activitiy of each saponin on OKT10-Saporin cytotoxicity could be ranked as quillaja > SA > digitonin > dioscin whilst their permeabilising properties as defined by PI exclusion measured flow cytometrically were ranked as dioscin > quillaja > digitonin > SA revealing an uncoupling between augmentative and permeabilising properties. Our results suggest that quillaja may prove to be more effective at augmenting immunotoxin activity than SA and may therefore have greater potential for clinical development, in particular because purified quillaja fractions are currently in clinical use as vaccine adjuvants. Further work is currently in progress to investigate saponins derived from other natural sources in order to make informed decisions about any subsequent development work.
POSTER and SHORT COMMUNICATION: Wednesday 21 March 2012, 17:15
Monitoring of Therapeutic Efficacy and Immune Responses Using Bispecific Antibodies in Mouse Leukemia/Lymphoma Models
Hossein Panjideh, Mathias Koch, Frank Wilde, Jörg Roßbacher, Felix Oden, Gerd Müller, Martin Lipp
Max Delbrück Centrum für Molekulare Medizin, 13125 Berlin, Germany
Bispecific antibodies targeting tumor cells as well as cytotoxic T cells are promising candidates in cancer therapy. Based on the observation that about 90% of NHL express high levels of CXCR5, a chemokine receptor normally expressed by B cells and a subset of CD4 T cells, we are currently developing a bispecific antibody targeting CXCR5 as well as the T cell receptor-associated molecule CD3. The simultaneous binding of cytotoxic T cells and CXCR5+ target cells should promote the elimination of the CXCR5-positive tumor cells. The bispecific antibody consists of a heavy and light chain from the parental anti-CXCR5 and anti-CD3 antibodies (rat IgG2b and mouse IgG2a, respectively). It was generated by the quadroma technology.
Particular important in the development of therapeutic antibodies is the establishment of reliable animal models and monitoring systems. To test for the therapeutic efficacy, we have developed subcutaneous and systemic leukemia/lymphoma models in NOD/SCID mice. We aim at assessing the therapeutic efficacy of bispecific antibodies in vivo by monitoring tumor growth/recession using a bioluminescence monitoring system. To this end we have constructed lentiviral vectors encoding a Luciferase and eGFP or a Luciferase and mCherry that can be used for the transduction of tumor cell lines. Following adoptive transfer, transduced lymphoma cells can be detected and monitored via bioluminescence. In addition, immune and potential inflammatory responses during antibody therapy will be monitored by the measurement of cytokine release and T cell activation.
T-07
POSTER
Gypsophila Saponins Significantly Augment the Immunospecific Cytotoxicity of Saporin-based Immunotoxins Directed Against Multiple Targets on Human Leukemia and Lymphoma Cells
Suzanne E. Holmes1, Emily L. Gibbs1, Jennifer E. Adcott1, Christopher Bachran2, Hendrik Fuchs3, Alexander Weng3, Matthias F. Melzig4, Sopsamorn U. Flavell1, David J. Flavell1
1The Simon Flavell Leukaemia Research Unit, Leukaemia Busters, Southampton General Hospital, Southampton SO16 6YD, United Kingdom; 2National Institutes for Health, National Institute for Allergy and Infectious Diseases, 33 North Drive, Bethesda, MD 20892; 3Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité – Universitätsmedizin Berlin, D-12200 Berlin, Germany; 4Institut für Pharmazie, Freie Universität Berlin, D-14195 Berlin
The therapeutic value of immunotoxins (ITs) for clinical use is diminished by their dose limiting toxicities. We investigated the use of triterpenoid saponins from Gypsophila paniculata L., (Saponinum album, SA) to augment saporin-based ITs directed against different target molecules on human leukaemia and lymphoma cells lines. The cytotoxicity of saporin-based ITs directed against human CD19, CD22, CD38 and CD71 was determined for three human leukaemia or lymphoma cell lines in the absence or presence of a sub-toxic concentration of SA.
We observed a high degree of variability in the augmentative effect of SA on IT activity, not just between different cell lines but also between different target molecules expressed by the same cell line with increases in cytotoxicity ranging from a remarkable 550-million-fold to as little as 2,500-fold. Crucially we show that SA substantially increased the rate of IT mediated protein synthesis inactivation in the target cell. We observed that SA does brings about a reduction in the immunospecificity of IT-mediated cytotoxicity but that this is vastly offset by the magnitude of the augmentative effect that SA exerts on immunospecific IT cytotoxicity exemplified by the n-fold gain values obtained following correction for non-specific effects for non-targeted saporin alone. To explain the variations that SA exerts on IT augmentation we propose a model that is based on the varied lipid/cholesterol contents of membrane micro-domains into which different target molecules partition following ligation by IT.
POSTER and SHORT COMMUNICATION: Friday 23 March, 09:45
The Effects of Gypsophila Saponins on the Internalization of an Anti-CD22 Immunotoxin
Jennifer E. Adcott, Suzanne E. Holmes, Sopsamorn U. Flavell, David J. Flavell
The Simon Flavell Leukaemia Research Unit, Leukaemia Busters, Southampton General Hospital, Southampton SO16 6YD, United Kingdom
Gypsohila paniculata saponins (SA) have been shown to specifically augment the cytotoxicity of various immunotoxins (IT) and cytotoxins, yet the mechanism underlying this effect is poorly understood. Here we follow the internalisation of an anti-CD22, 4KB128-Saporin, IT targeted against the human Burkitt’s lymphoma cell line, Daudi, in the presence of a sub-toxic yet highly augmentative concentration of SA.
Experiments were conducted in order to demonstrate that sub-toxic concentrations of chlorpromazine, an inhibitor of clathrin-mediated endocytosis, does not affect IT internalisation but does partially abrogate the augmentative effect of SA on IT cytotoxicity, indicating the possibility of multiple internalisation pathways.
Kinetic flow cytometric studies alongside confocal imaging were used to examine the internalisation pathway(s) through the cell. SA does not appear to have any major effect on IT routing within the target Daudi cell, and co-localisation analysis of IT with sub-membranous EEA1+ early endosomes failed to reveal an increased release of IT cargo from the endosomal compartment into the cytosol. Whilst these preliminary results have provided interesting data further experimental work and analysis is required for a valid interpretation.
T-09
POSTER
Theranostic Antibody-targeted Gold Nanoparticles for Tumor Cell Detection and Killing
Giulio Fracasso*1, Anita Boscaini*1, Alessia Scarsi2, Lucio Litti2, Cristina Anselmi1, Vincenzo Amendola2, Sara Cingarlini3, Moreno Meneghetti2, Marco Colombatti1
1Pathology and Diagnostic -Immunology, University of Verona, 37134 Verona, Italy; 2Chemical Sciences , University of Padova, 35100 Padova, Italy; 3Medical Oncology Unit , Azienda Ospedaliera Universitaria Integrata Verona, 37134 Verona, Italy
* These authors contributed equally to this work
Nanomaterials have emerged as promising tools to address limitations of diagnostic and therapeutic approaches of solid tumors (scarce selectivity, low efficacy, severe side effects). Association of nanostructures with targeting moieties (e.g. antibodies, mAbs) would greatly increase selective accumulation of tracking/therapeutic molecules in tumor tissues, allowing to develop theranostic (therapeutic+diagnostic) devices.
In our work we evaluated, in a preclinical setting, the potential applications of mAbs-targeted nanostructures (e.g. gold nanoparticles, Au-NPs) as diagnostic devices and as therapeutic tools, when loaded with SERS (surface enhanced Raman scattering) molecules and the drug Doxorubicin, respectively. SERS signals are superior to the fluorescence detection, which suffers of bleaching and is characterized by bands with large bandwidths. Anti-PSMA (Prostate Specific Membrane Antigen) and anti-PSCA (Prostate Stem Cell Antigen) mAbs were conjugated to Au-NPs and loaded with the SERS molecules Texas Red and Malachite green, respectively. Multiplexed Raman spectroscopy performed in in vitro cell cultures allowed us to discriminate with high specificity between double positive, single positive and double negative cells. The availability of such high sensitivity detection method will improve tumor cell identification, phenotyping and the diagnostic capabilities. Moreover when loaded whit Doxorubicin, we observed that these mAb-driven NPs killed antigen positive cells with a slightly lower efficacy with respect to the drug alone but they showed more specificity.
SHORT COMMUNICATION: Wednesday 21 March 2012, 17:00
Dual Antigen-Restricted Complementation of a Trispecific Antibody Construct for Targeted Immunotherapy of Blood Cancer
Agnes Banaszek1, Gernot Stuhler2
1Molekulare Innere Medizin, Universitätsklinikum Würzburg, 97070 Würzburg, Germany; 2Zentrum für Blutstammzelltransplantation, Universitätsklinikum Würzburg, Medizinische Klinik und Poliklinik II, 97080 Würzburg, Germany
Bispecific antibodies have been shown to improve effector-cell recruitment for antibody therapy. For cancer immunotherapy, bispecific T cell-engaging antibodies are well characterised. This study aims at treating leukaemia and other haematologic malignancies in the context of haematopoietic stem cell transplantation. An HLA-identical donor is very rare. For an HLA-mismatched situation between patient and donor, we try to exploit the HLA disparity for targeted treatment by using a trispecific HLA-A2 × CD45 × CD3 antibody construct. More precisely, we developed two single-chain antibody constructs: 1) a single-chain variable-fragment (scFv) specific for HLA-A2 in cases where the patient but not the donor is HLA-A2 positive, and 2) a scFv recognising CD45, a pan-haematopoietic marker. Each scFv is linked to a VH and a VL domain of a CD3ε-specific antibody, respectively. After the binding of each scFv to HLA-A2 and CD45 on the same cell, the unpaired variable domains assemble to a functional scFv against CD3ε. This assembly should exclusively occur on the recipient’s haematopoietic cells, leading to T cell-mediated cell destruction. Both constructs were periplasmically expressed in E. coli and purified via His tag. Stimulatory properties were assayed by measuring IL-2 release following incubation with HLA-A2- and CD45-expressing cells and PBMCs. The trispecific antibody construct mediates T-cell activation in a concentration-dependent manner at 3 - 27 nM. This effect is dual antigen-restricted, as it could be blocked by prior incubation of either HLA-A2- or CD45-specific scFv.
SHORT COMMUNICATION: Friday 23 March 2012, 10:15
Isolation Techniques of Saponins and their In Vivo Evaluation as Enhancers in Targeted Tumor Therapy
Mayank Thakur1, 2, Katharina Mergel1, Alexander Weng1, Alexandra Pieper1, Benedicta von Mallinckrodt1, Roger Gilabert-Oriol1, Matthias F. Melzig2, Hendrik Fuchs1
1Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité – Universitätsmedizin, 12200 Berlin, Germany; 2Department of Pharmaceutical Biologie, Institute of Pharmacy, Free University, 14195 Berlin, Germany
Saponins elicit a potential synergistic enhancer effect on the toxicity of TT consisting of saporin (Sap-3) and EGF (SE). Isolation of saponins in purified form is very difficult and time consuming [1]. In the present study a rapid strategy for isolation of pure saponin SpnS, its evaluation in real time cell culture studies and testing in vivo in combination with SE is reported. Isolation of SpnS was done by using a preparative HPLC, while an electrophoresis-based technique was also utilized. The impact of a combination of SpnS with either SE or Sap-3 was determined in cytotoxicity assays on murine cell lines over expressing EGF receptor (HER14 and TSA-EGFR) using MTT assay. Different concentration of 10-14 M to 10-8 M of toxins were tested and impedance based monitoring was performed. Finally the saponin was tested for acute toxicity. Thereafter, the non-toxic saponin concentration was tested for synergistic toxicity enhancement in combination with SE in-vivo in an artificially adapted syngenic mouse model [2]. A less cumbersone electrophoresis-based technique was developed for saponin m/z 1861 (SpnS) [3]. The enhancement factor for TSA-EGFR cells was more than a million fold. SpnS showed no major toxic effects at 100 µg /treatment and below. The combination of SpnS and SE could lead to a significant reduction in tumor volume ( p<0.05) wherein the tumor volume in control was >1800 mm3 compared to a tumor volume of less than 350 mm3 in the treated group. The TT-Saponin combination with SpnS (m/z 1861) has high clinical potential.
[1] Weng, A., et al., Pharmazie, 2011. 66(10): p. 744-746.
[2] Bachran, C., et al., Br J Pharmacol, 2009. 159(2): p. 345-352.
[3] Thakur, M., et al., Electrophoresis, 2011. 32(21): p. 3085-3089.
SHORT COMMUNICATION: Friday 23 March 2012, 15:45
Therapeutic Potential of a Combinatorial Anti-tumor Therapy: Role of the Toxin Moiety
Alexander Weng1, Mayank Thakur1, 2, Figen Beceren-Braun1, Roger Gilabert-Oriol 1, Diana Bachran1, Matthias F. Melzig2, Hendrik Fuchs1
1Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité – Universitätsmedizin, 12200 Berlin, Germany; 2Freie Universität Berlin, Pharmazeutische Biologie, 14195 Berlin, Germany
Tumor-targeted protein toxins are composed of a toxic enzyme coupled to a specific cell binding domain, which targets cancer associated antigens. The therapeutic future prospects of targeted toxins in humans are largely dependent on the possibility to reduce side effects.
A crude mixture of triterpenoid saponins was shown to increase the anti-tumoral efficacy of a particular targeted toxin by decreasing its dosage and concomitantly decreasing side effects.
Here we demonstrate the therapeutic potential of characterized saponins for different anti-tumor toxins varying in the toxic moiety and targeting the epidermal growth factor receptor (EGFR). As toxin moieties served truncated variants of Pseudomonas exotoxin, Diphtheria toxin and ricin from Ricinus communis L., dianthin-30 from Dianthus caryophyllus L. and saporin-3 from Saponaria officinalis L.
All EGFR-targeted toxins were characterized and combined with a purified saponin (SA1641), isolated from Gypsophila paniculata L. The SA1641-mediated efficacy increase was investigated on EGFR positive HER-14 cells. We observed a strong dependency of the SA1641-mediated efficacy increase on the nature of toxin used for the construction of the targeted toxin. Especially dianthin-30 and saporin-3 based targeted toxins benefited most from the combination with saponins. This saponin dependent effect was not based on SA1641-induced modulation of plasma membrane permeability. Surface plasmon resonance measurements point to an intracellular interaction of the toxin part of targeted toxins and SA1641.
T-13
POSTER
Saporin-HRP Endo/Lysosomal Escape Enhancement in Isolated Organelles
Roger Gilabert-Oriol1, Alexander Weng1, Mayank Thakur1, 2, Katharina Mergel1, Benedicta von Mallinckrodt1, Diana Bachran1, Matthias F. Melzig2, Hendrik Fuchs1
1Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité – Universitätsmedizin, 12200 Berlin, Germany; 2Pharmazeutische Biologie, Freie Universität Berlin, 14195 Berlin, Germany
Saporin, a plant ribosome-inactivating protein (RIP) from Saponaria officinalis L., has been used in the development of targeted tumor therapies. Saponins, which are triterpenoidal plant secondary metabolites, synergistically enhance the cytotoxicity of saporin-based targeted toxins. This effect is related to the increase of endo/lysosomal escape of fusion proteins bearing saporin. In order to investigate the endo/lysosomal escape enhancement in isolated organelles, saporin was chemically linked to the reporter horse radish peroxidase. The expected synergistic toxicity enhancement of saporin-HRP (SH) and SA1641 (saponin isolated from Gypsophila paniculata L.) was analyzed by cytotoxicity assays. A crude fraction containing endosomes and lysosomes was isolated by cell homogenization and differential centrifugation for the subsequent assays. The effect on the endo/lysosomal membrane integrity of three saponins: digitonin (lytic), α-hederin (non-lytic) and SA1641, was measured by detection of the released lysosomal enzyme β-N-acetylglucosaminidase. To study the specific endo/lysosomal escape enhancement of SH by SA1641, cells were incubated with SH for 6 h and organelles were isolated. They were incubated with saponins used before and the released amount of SH was detected by peroxidase activity. An increased amount of released SH in the presence of SA1641 indicated a specific interaction of the two compounds in the process of saporin translocation. Further experiments with SH will help to get insights into the specific interactions between saporin and SA1641 saponin.
SHORT COMMUNICATION: Wednesday 21 March 2012, 18:45
Targeting of Solid Tumors by Antibody-directed Enzyme Prodrug Therapies
Nicole Schellmann1, Hendrik Fuchs1, P. Markus Deckert2
1Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité – Universitätsmedizin, 12200 Berlin, Germany; 2Innere Medizin II - Gastroenterologie / Onkologie, Städtisches Klinikum Brandenburg GmbH Akademisches Lehrkrankenhaus der Charité, 14770 Brandenburg an der Havel, Germany
Within the last few decades many approaches have been developed in the field of targeted tumor therapies. The Antibody-directed enzyme prodrug therapy (ADEPT) is one of these. It is based on a fusion protein that contains a prodrug-converting enzyme and a targeting molecule. Following the ADEPT principle we designed the fusion protein antiA33-CDy, which is capable of binding to the human glycoprotein A33 (gpA33), a well known cell surface protein on gastrointestinal cells and 95% of colon cancers. The enzymatic acitivity of cytosine deaminase derived from yeast (CDy) converts the prodrug 5-fluorocytosine into the chemotherapeutic 5-fluorouracil used in adjuvant colon cancer therapy in general. We have previously published data about its biodistribution and therapy efficacy in nude mice.
The in vivo studies showed promising results; therefore we established a BALB/c colon carcinoma mice model with gpA33 transfected murine CT26 cells to investigate whether the immunsystem has an influence on the therapy outcome or not. The transfected cells showed no remarkable difference in cell growth and an excellent gpA33 expression on the cell surface, proven by flow cytometry and fluorescencemicroscopy. The in vivo studies displayed a superior concentration dependend response in the targeted therapy approach with antiA33-CDy and 5-fluorocytosine in BALB/c.
POSTER
Tumor-specific Accumulation of Radiosensitizers via Cationic Transporters
Christin Bednarek1, Dania Kendziora2, Ljiljana Fruk2, Daniel Fürniß3, Stefan Bräse3, Marlon Veldwijk4, Frederik Wenz4, Ute Schepers1
1Institute of Toxicology and Genetics (ITG), Karlsruhe Institute of Technology (KIT), 76344 Eggenstein-Leopoldshafen, Germany; 2Center for Nanostructures, Karlsruhe Institute of Technology (KIT), 76131 Karlsruhe, Germany; 3Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), 76131 Karlsruhe, Germany; 4Department of Radiation Oncology, University Medical Center Mannheim, University of Heidelberg, 68167 Mannheim, Germany
In recent years the efforts increased to develop drugs not only for systemic but for specific application in certain diseased tissues. This applies not only for the application in individual organs, but especially for the targeting of tumors and metastases. As not all of the drugs are soluble drug carriers are often used. Widely used systems are liposomes. They have the advantage to change or cover the physicochemical properties of drugs that are not able to penetrate cell membranes. Thus, they are made available to tissues and cells. The intention of targeted therapies is to minimize side effects on healthy tissues. Thus, numerous studies are performed to improve drug targeting and also to combine different therapeutic approaches to improve cancer therapies. The aim of this study is to develop a drug delivery system in the form of a multifunctional liposomal system. It is thought to combine two conventional types of tumor therapy with a tumor-specificity of the system at the same time. To achieve this objective a 4-Component-Concept is developed.
POSTER
ForSaTum: An animal research platform for the development and implementation of novel concepts for cancer therapy
Thomas Nachreiner*, Katharina Kolberg*, Alessa Pardo, Stefan Barth
Experimentelle Medizin und Immuntherapie, RWTH Aachen - Helmholtz Institut für Angewandte Medizintechnik, 52074 Aachen, Germany
* These authors contributed equally to this work
Many approaches for diagnosis and therapy of cancer fail due to high development costs and low clinical efficacy. Additionally, promising scientific academic approaches are not pursued by industry as these approaches are considered not validated enough or financially too risky. The main reason for this is the lack of efficient preclinical facilities for standardized longitudinal studies of new cancer drugs in relevant tumor models. Our project ForSaTum (Forschungssatellit Tumorforschung) has the objective to establish a unique structure for preclinical cancer research. Research partners of the RWTH Aachen University and the University Bochum have joined with both established and emerging companies in North-Rhine Westphalia to allow faster and better implementations of novel concepts for cancer therapy. Diagnostic imaging is an important part of medical technology innovations and is intensively used for preclinical development of therapeutics as well as in patient care. In particular the molecular imaging technology which enables non-invasive visualisation of biological and cellular processes at the molecular level will enhance the quality of preclinical studies and the success of clinical trials. Our goal is to combine molecular imaging technologies with the development of molecular diagnostic probes and therapeutics on a specialized animal research platform, supported by an IT platform network to enhance the quality of preclinical studies and subsequently the success of clinical trials.
Category: Targeted Tumor Therapies in Clinical Applications
TALK: Thursday 22 March 2012, 11:50
Serum Proteins as Carriers for Drugs, Toxins and Antibodies – from Preclinical to Clinical Application
Felix Kratz
Macromolecular Prodrugs, Tumor Biology Center, 79106 Freiburg, Germany
Among serum proteins albumin and transferrin have attracted the most interest as drug carriers. The use of transferrin has clinically been primarily restricted to the diagnosis of cancer using radiolabeling with suitable metal ions and therapeutically to immunotoxins. Albumin-based drug delivery systems ranging from albumin drug nanoparticles, albumin fusion proteins, prodrugs and peptide derivatives that bind covalently to albumin as well as physically binding antibody fragments are in advanced clinical trials or approved products. In oncology, Abraxane, an albumin paclitaxel nanoparticle as a water-soluble galenic formulation, transports paclitaxel through passive targeting as an albumin paclitaxel complex to the tumor site and is superior to conventional Taxol against metastatic breast cancer. INNO-206, an albumin-binding doxorubicin prodrug accumulates in solid tumors and releases the parent drug through acid cleavage, either intra- or extracellularly, is undergoing phase II studies against soft tissue sarcoma. Fusion antibody-based proteins that incorporate albumin instead of the Fc portion of conventional IgG immunoglobulins are also in clinical studies against breast cancer. Because albumin cannot only be used to improve the pharmacokinetic profile of therapeutically relevant peptides and the targeting moiety of antibodies but also for low-molecular weight drugs to inflamed or malignant tissue, it is anticipated that R&D efforts of academia and the pharmaceutical industry in this field of drug delivery will lead to further approved drugs.
TALK: Thursday 22 March 2012, 12:25
Immunotoxin Combination Therapy with ABT-737/263
Antonella Antignani1, Abid Mattoo1, Emily Weiss1, Ira Pastan1, Alan Wayne2, Robert Kreitman1, David Fitzgerald1
1Laboratory of Molecular Biology, CCR, National Cancer Institute, Bethesda, MD 20892, USA; 2Pediatric Oncology Branch, CCR, National Cancer Institute, Bethesda, MD 20892, USA
Immunotoxins are antibody-toxin fusion proteins targeted to kill cancer cells. Biochemically, these antibody-based agents are cytotoxic because they inhibit protein synthesis leading to apoptosis in many cell types. But some cells exhibit resistance to apoptosis despite toxin-mediated inhibition of protein synthesis. To investigate reasons for this resistance, we employed the BH3-mimetic, ABT-737, and the closely related clinical compound, ABT-263. These BH3-mimetics act in mammalian cells to neutralize the prosurvival proteins, Bcl-2, Bcl-xl and Bcl-w and are lethal for cells that depend on one of these proteins for survival. Resistance to immunotoxin- or ABT-mediated apoptosis was overcome by the use of combinations of immunotoxin and ABT-737/263. Initial observations were made in epithelial cells where synergy was clearly evident. Early experiments were expanded to include B-cell malignancy and Small Cell Lung Cancer cell lines. Here we report that for these cell types combinations of immunotoxin and ABT-737/263 were either synergistic or additive. While the cellular factors that distinguish synergy from additivity are currently under investigation, existing preclinical data support further development of these agents leading to a clinical trial of immunotoxin-ABT-263 combinations.
TALK: Friday 23 March 2012, 11:15
Immunotoxins in targeted cancer therapy, from bench to bedside
Yvonne Andersson, Merete T. Wiiger, Karianne Risberg, Kjersti Flatmark, Olav Engebraaten, Øystein Fodstad
Tumor Biology, Cancer Research, 0310 Oslo, Norway
Our PE-based ITs have shown promising preclinical effects and one IT has been in a phase I clinical trial. However, the anti-IT antibody response hinders the use of repeated IT-administration in patients, which we think is a necessity for optimal anti-metastatic effect. Recently, a phase I trial with IT and the immunosuppressor cyclosporine (CsA) has been completed. The results from these clinical trials will be discussed.
Further improvement in the IT field, however, is clearly dependent on increased knowledge of the molecular mechanisms by which the ITs affect the cancer cells. We have found that the ITs induce several parallel signalling events in targeted cancer cells. The obtained knowledge has been used with the objective to further improve IT efficacy. We have found that combinations of IT with different small anti-cancer molecules caused a remarkable synergistic effect in killing cancer cells. The mechanisms underlying the synergistic effects in vitro have been thoroughly examined and will be discussed. Furthermore, several of the combination therapies were shown to increase the symptom-free life span and the fraction of long time survivors in animal models for human cancer.
In conclusion, our research efforts might have improved the clinical potential of ITs in patients who have dismal prognosis with available treatment options, such as peritoneal carcinomatosis from colorectal and ovarian cancer.
TALK: Friday 23 March 2012, 11:50
Endoradiotherapy with peptides – clinical application and new developments
Uwe Haberkorn
Nuclear Medicine, University Hospital Heidelberg and DKFZ Heidelberg, 69120 Heidelberg, Germany
With the advances in molecular biology and biochemistry new imaging and treatment modalities based on the biological properties of tissues have been developed. In oncology, the major progress has been achieved using peptide and antibody targeting vectors. When labeled with beta-emitting radioisotopes these agents are suitable for endoradiotherapy and exploit the targeting potential for highly specific therapeutic applications which has been realized for antibodies against CD20 or peptides binding to somatostatin receptors. This novel class of pharmaceuticals offers the potential to develop patient specific therapies and might provide the means to go beyond the possibilities of current chemotherapy and radiation therapy. Furthermore, these achievements can be seen as proof of principle and encourage more research towards the identification of new specifically binding molecules.
The development of new biomolecules may be done based on a rational design or a black box approach. In contrast to rational design the central idea of combinatorial chemistry is to synthesize a vast library of possible variants of the molecule of interest and screen the population for the few variants that show the property of interest. The attracting feature of the concept arises from the huge number of candidate molecules that can be used for further evaluation. After the characterization of the structure-function relationships for the lead compounds found in this process further improvement by rational design of analogs can be performed.
TALK: Friday 23 March 2012, 12:25
Targeting Severe Herpes Simplex Virus (HSV) Infections in Immunocompromised Hosts with a Monoclonal Antibody
Adalbert Krawczyk1, 2, Anna-Maria Eis-Hübinger3, Martin Däumer3, Ulf Dittmer2, Robert Schwarzenbacher4, Karl Schneweis3, Dirk Jäger1, Michael Roggendorf2, Jürgen Krauss1, Michaela Arndt1
1Medical Oncology, National Center for Tumor Diseases, 69120 Heidelberg, Germany; 2Institute of Virology, University of Duisburg-Essen, 45147 Essen, Germany; 3Institute of Virology, University Medical Center Bonn, 53105 Bonn, Germany; 4Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria
We investigated the mode of action of a monoclonal antibody (mAb 2c) for neutralizing HSV-1 and HSV-2. MAb 2c binds to a type-common discontinuous epitope of the viral glycoprotein B (gB) which is an integral part of the multicomponent fusion system being required for virus entry and cell-cell fusion. Binding of the antibody resulted in highly efficient neutralization of HSV at the postbinding/prefusion stage and completely abrogated the viral cell-to-cell spread in vitro. Mapping of the antigenic site recognized by mAb 2c to the recently solved crystal structure of gB revealed that its discontinuous epitope is only partially accessible within the observed multidomain trimer conformation of gB, likely representing its postfusion conformation. To investigate how mAb 2c may interact with gB during membrane fusion, we characterized the properties of monovalent versus bivalent derivatives of mAb 2c. Our data show that the neutralization capacity of mAb 2c is dependent on cross-linkage of gB trimers. As a result, only bivalent derivatives of mAb 2c exhibited high neutralizing activity in vitro. Notably, bivalent mAb 2c not only was capable of preventing mucocutaneous disease in severely immunodeficient mice upon vaginal HSV-1 challenge but also protected animals even with neuronal HSV infection. We also report for the first time that an anti-gB specific mAb prevents HSV-1-induced encephalitis entirely independently from complement activation and antibody dependent cellular cytotoxicity. This indicates the potential for further development of mAb 2c as a novel anti-HSV drug.