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PDF file - 470K, Calculation of tumor growth inhibition. Tumor growth inhibition was calculated compared with respective controls using a standard formula. Surface plasmon resonance. Calculation of kinetic parameters ka (1/Ms), kd (1/s) and KD (M) of the antibody/receptor domain. Supplementary Table 1. Surface plasmon resonance - Kinetic data (ka, kd, and KD) for the binding of RG7116 and cetuximab to their respective antigen across the entire temperature gradient investigated. Supplementary Table 2. Surface plasmon resonance - Detailed analysis of the Van't Hoff and Eyring association/dissociation thermodynamic parameters for the binding of RG7116/HER3 and cetuximab/HER1. Supplementary Figure S1. Van't Hoff and Eyring association/dissociation plots showing the linear transformation of affinity (KD) and kinetic (ka and kd) parameters varying with reaction temperature for RG7116/HER3 and cetuximab/HER1. Structure of the HER3-ECD::Fab(RG7116) complex - Preparation and crystallization. Detailed methodology for the preparation and crystallization of the Fab fraction of RG7116 bound to HER3-ECD Structure of the HER3-ECD::Fab(RG7116) complex - Data collection, model building and refinement. Detailed methodology for the processing of X-ray diffraction data and solving of the Supplementary Table 3. Data collection and refinement statistics for the HER3-ECD::Fab(RG7116) complex. Supplementary Figure S2. Comparison of apo- and Fab(RG7116) bound HER3-ECD, 2Fo-Fc electron density composite omit map and electrostatic surface calculation of anti-HER3 binding epitope. Sensitivity of models investigated in vivo to therapeutic agents. Details of the common chemotherapeutic agents that the cell lines/tissue fragments used in this study were resistant to.

The LST1 gene is located in the MHC class III cluster between the MHC class I and II regions. While most genes in this cluster have been sufficiently characterised, a definitive function and expression pattern for LST1 still remains elusive. In the present review we describe its promotor, gene organisation, splice variants and expression in human tissues, cell lines and cancer. We focus on LST1 expression in inflammation and discuss known correlations with autoimmune diseases and cancer. Current data on LST1 polymorphisms and their known associations with pathologies are also discussed in detail. We summarize the potential functions that have been described for the full-length LST1 protein including its function as a transmembrane adaptor protein with inhibitory signal transduction and its role as a membrane scaffold facilitating the formation of tunnelling nanotubes. We also discuss further potential functions by compiling all known LST1-interacting proteins. Furthermore, we address knowledge gaps and conflictive issues regarding disease association, non-hematopoietic expression and the discrepancy between RNA and protein expression data.

A number of agents designed for immunotherapy of Acute Myeloid Leukemia (AML) are in preclinical and early clinical development. Most of them target a single antigen on the surface of AML cells. Here we describe the development and key biological properties of a tri-specific agent, the dual-targeting triplebody SPM-2, with binding sites for target antigens CD33 and CD123, and for CD16 to engage NK cells as cytolytic effectors. Primary blasts of nearly all AML patients carry at least one of these target antigens and the pair is particularly promising for the elimination of blasts and leukemia stem cells (LSCs) from a majority of AML patients by dual-targeting agents. The cytolytic activity of NK cells mediated by SPM-2 was analyzed in vitro for primary leukemic cells from 29 patients with a broad range of AML-subtypes. Blasts from all 29 patients, including patients with genomic alterations associated with an unfavorable genetic subtype, were lysed at nanomolar concentrations of SPM-2. Maximum susceptibility was observed for cells with a combined density of CD33 and CD123 above 10,000 copies/cell. Cell populations enriched for AML-LSCs (CD34pos and CD34pos CD38neg cells) from 2 AML patients carried an increased combined antigen density and were lysed at correspondingly lower concentrations of SPM-2 than unsorted blasts. These initial findings raise the expectation that SPM-2 may also be capable of eliminating AML-LSCs and thus of prolonging survival. In the future, patients with a broad range of AML subtypes may benefit from treatment with SPM-2.

Triplebody 19-3-19, an antibody-derived protein, carries three single chain fragment variable domains in tandem in a single polypeptide chain. 19-3-19 binds CD19-bearing lymphoid cells via its two distal domains and primary T cells via its CD3-targeting central domain in an antigen-specific manner. Here, malignant B-lymphoid cell lines and primary cells from patients with B cell malignancies were used as targets in cytotoxicity tests with pre-stimulated allogeneic T cells as effectors. 19-3-19 mediated up to 95 % specific lysis of CD19-positive tumor cells and, at picomolar EC₅₀ doses, had similar cytolytic potency as the clinically successful agent Blinatumomab. 19-3-19 activated resting T cells from healthy unrelated donors and mediated specific lysis of both autologous and allogeneic CD19-positive cells. 19-3-19 led to the elimination of 70 % of CD19-positive target cells even with resting T cells as effectors at an effector-to-target cell ratio of 1 : 10. The molecule is therefore capable of mediating serial lysis of target cells by a single T cell. These results highlight that central domains capable of engaging different immune effectors can be incorporated into the triplebody format to provide more individualized therapy tailored to a patient's specific immune status.

// Christian B. Schiller 1, * , Todd A. Braciak 2, * , Nadja C. Fenn 1, * , Ursula J. E. Seidel 3, * , Claudia C. Roskopf 2, * , Sarah Wildenhain 1, * , Annemarie Honegger * , Ingo A. Schubert 5 , Alexandra Schele 1 , Kerstin Lammermann 2 , Georg H. Fey 6 , Uwe Jacob 6 , Peter Lang 3 , Karl-Peter Hopfner 1, # , Fuat S. Oduncu 2, # 1 Department of Biochemistry and Gene Center, Ludwig-Maximilians-University, Munich, Germany 2 Division of Hematology and Oncology, Medizinische Klinik und Poliklinik IV, Klinikum der Universitat Munchen, Munich, Germany 3 Department of General Paediatrics, Oncology/Haematology, University Children’s Hospital Tubingen, Tubingen, Germany 4 Department of Biochemistry, University of Zurich, Zurich, Switzerland 5 Department of Biology, University of Erlangen-Nuremberg, Erlangen, Germany 6 Westend-Innovation, Munich, Germany * CBS, TAB, NCF, UJES, CCR and SW are co-first authors in this study # KPH and FSO are co-senior authors in this study Correspondence to: Claudia C. Roskopf, email: Claudia.Roskopf@med.uni-muenchen.de Keywords: single chain triplebody, antibody-dependent cellular cytotoxicity, gamma delta T cell, leukemia, immunotherapy Received: June 30, 2016 Accepted: October 03, 2016 Published: November 04, 2016 ABSTRACT Triplebodies are antibody-derived recombinant proteins carrying 3 antigen-binding domains in a single polypeptide chain. Triplebody SPM-1 was designed for lysis of CD19-bearing malignant B-lymphoid cells through the engagement of CD16-expressing cytolytic effectors, including NK and γδ T cells. SPM-1 is an optimized version of triplebody ds(19-16-19) and includes humanization, disulfide stabilization and the removal of potentially immunogenic sequences. A three-step chromatographic procedure yielded 1.7 - 5.5 mg of purified, monomeric protein per liter of culture medium. In cytolysis assays with NK cell effectors, SPM-1 mediated potent lysis of cancer-derived B cell lines and primary cells from patients with various B-lymphoid malignancies, which surpassed the ADCC activity of the therapeutic antibody Rituximab. EC 50 -values ranged from 3 to 86 pM. Finally, in an impedance-based assay, SPM-1 mediated a particularly rapid lysis of CD19-bearing target cells by engaging and activating both primary and expanded human γδ T cells from healthy donors as effectors. These data establish SPM-1 as a useful tool for a kinetic analysis of the cytolytic reactions mediated by γδ T and NK cells and as an agent deserving further development towards clinical use for the treatment of B-lymphoid malignancies.

DNA double-strand breaks are repaired by two major pathways, homologous recombination or nonhomologous end joining. The commitment to one or the other pathway proceeds via different steps of resection of the DNA ends, which is controlled and executed by a set of DNA double-strand break sensors, endo- and exonucleases, helicases, and DNA damage response factors. The molecular choreography of the underlying protein machinery is beginning to emerge. In this review, we discuss the early steps of genetic recombination and double-strand break sensing with an emphasis on structural and molecular studies.

The regulation of gene expression depends on the interplay of multiple factors at the transcriptional and translational level. Upstream open reading frames (uORFs) play an important role as translational repressors of main ORFs and their presence or usage in transcripts can be regulated by different mechanisms. The main objective of the present study was to assess whether uORFs regulate the expression of the MHC class III gene LST1. We report that expression of LST1 is tightly regulated by alternative transcription initiation and the presence of an uORF in the 5'-UTR of transcripts. Specifically, using EGFP reporter constructs in human HeLa and HEK-293T cells and flow cytometry as well as western blot analysis we found the uORF to reduce the expression of the main ORF by roughly two-thirds. Furthermore, we were able to correlate a previously detected increase in LST1 protein expression during monocyte differentiation with an increase of transcription initiation at an alternative exon that does not contain an uORF.

Meeting abstracts The design of antibody derivatives with higher tumour cell selectivity is hoped to improve cancer immunotherapy and to decrease side effects. One promising molecular format that we have developed is the triple body. A triple body allows for selective lysis based on its 'dual

Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the "CrossMab" format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the "knobs-and-holes" approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding.

Carefully orchestrated intercellular communication is an essential prerequisite for the development of multicellular organisms. In recent years, tunneling nanotubes (TNT) have emerged as a novel and widespread mechanism of cell-cell communication. However, the molecular basis of their formation is still poorly understood. In the present study we report that the transmembrane MHC class III protein leukocyte specific transcript 1 (LST1) induces the formation of functional nanotubes and is required for endogenous nanotube generation. Mechanistically, we found that LST1 induces nanotube formation by recruiting the small GTPase RalA to the plasma membrane and promoting its interaction with the exocyst complex. Furthermore, we determined that LST1 recruits the actin-crosslinking protein filamin to the plasma membrane and interacts with M-Sec, myosin and myoferlin. These results allow us to suggest a molecular model for nanotube generation. In this proposal LST1 functions as a membrane scaffold mediating the assembly of a multimolecular complex, which controls the formation of functional nanotubes.

The LST1 gene is located in the human MHC class III region and encodes transmembrane and soluble isoforms that have been suggested to play a role in the regulation of the immune response and are associated with inflammatory diseases such as rheumatoid arthritis. Here we describe the generation and characterization of the first monoclonal antibodies against LST1. Two hybridoma lines secreting monoclonal antibodies designated 7E2 and 8D12 were established. The 7E2 antibody detects recombinant and endogenous LST1 by Western blot analysis while 8D12 reacts with recombinant and endogenous LST1 in immunoprecipitation and flow cytometry procedures. The newly established antibodies were used to survey LST1 protein expression in human cell lines, which was found to be tightly regulated, allowing the expression of transmembrane isoforms but suppressing soluble isoforms.

Summary The MR ( M re11 nuclease and R ad50 ABC ATPase) complex is an evolutionarily conserved sensor for DNA double-strand breaks, highly genotoxic lesions linked to cancer development. MR can recognize and process DNA ends even if they are blocked and misfolded. To reveal its mechanism, we determined the crystal structure of the catalytic head of Thermotoga maritima MR and analyzed ATP-dependent conformational changes. MR adopts an open form with a central Mre11 nuclease dimer and two peripheral Rad50 molecules, a form suited for sensing obstructed breaks. The Mre11 C-terminal helix-loop-helix domain binds Rad50 and attaches flexibly to the nuclease domain, enabling large conformational changes. ATP binding to the two Rad50 subunits induces a rotation of the Mre11 helix-loop-helix and Rad50 coiled-coil domains, creating a clamp conformation with increased DNA-binding activity. The results suggest that MR is an ATP-controlled transient molecular clamp at DNA double-strand breaks. PaperFlick