Stabilization and humanization of a single-chain Fv antibody fragment specific for human lymphocyte antigen CD19 by designed point mutations and CDR-grafting onto a human framework

A single-chain Fv (scFv) fragment derived from the murine antibody 4G7, specific for human lymphocyte CD19, was engineered for stability and expression in Escherichia coli in view of future use as a therapeutic protein. We compared two orthogonal knowledge-based procedures. In one approach, we designed a mutant with 14 single amino-acid substitutions predicted to correct destabilizing residues in the 4G7-wt sequence to create 4G7-mut. In the second variant, the murine CDRs were grafted to the human acceptor framework huVκ3-huVH3, with 11 additional point mutations introduced to obtain a better match between CDR graft and acceptor framework, to arrive at 4G7-graft. Compared to 4G7-wt, 4G7-mut showed greater thermodynamic stability in guanidinium chloride-induced equilibrium denaturation experiments and somewhat greater stability in human serum. The loop graft maintained the comparatively high stability of the murine loop donor, but did not improve it further. Our analysis indicates that this is due to subtle strain introduced between CDRs and framework, mitigating the otherwise highly favorable properties of the human acceptor framework. This slight strain in the loop graft is also reflected in the binding affinities for CD19 on leukemic cells of 8.4 nM for 4G7-wt, 16.4 nM for 4G7-mut and 30.0 nM for 4G7-graft. This comparison of knowledge-based mutation and loop-grafting-based approaches will be important, when moving molecules forward to therapeutic application

Sumoylation inhibits α-synuclein aggregation and toxicity

Sumoylation of α-synuclein decreases its rate of aggregation and its deleterious effects in vitro and in vivo

Aggregation of αSynuclein promotes progressive in vivo neurotoxicity in adult rat dopaminergic neurons

Fibrillar αSynuclein is the major constituent of Lewy bodies and Lewy neurites, the protein deposits characteristic for Parkinson’s disease (PD). Multiplications of the αSynuclein gene, as well as point mutations cause familial PD. However, the exact role of αSynuclein in neurodegeneration remains uncertain. Recent research in invertebrates has suggested that oligomeric rather than fibrillizing αSynuclein mediates neurotoxicity. To investigate the impact of αSynuclein aggregation on the progression of neurodegeneration, we expressed variants with different fibrillation propensities in the rat substantia nigra (SN) by means of recombinant adeno-associated viral (AAV) vectors. The formation of proteinase K-resistant αSynuclein aggregates was correlated to the loss of nigral dopaminergic (DA) neurons and striatal fibers. Expression of two prefibrillar, structure-based design mutants of αSynuclein (i.e., A56P and A30P/A56P/A76P) resulted in less aggregate formation in nigral DA neurons as compared to human wild-type (WT) or the inherited A30P mutation. However, only the αSynuclein variants capable of forming fibrils (WT/A30P), but not the oligomeric αSynuclein species induced a sustained progressive loss of adult nigral DA neurons. These results demonstrate that divergent modes of αSynuclein neurotoxicity exist in invertebrate and mammalian DA neurons in vivo and suggest that fibrillation of αSynuclein promotes the progressive degeneration of nigral DA neurons as found in PD patients

Extracellular vesicle sorting of α-Synuclein is regulated by sumoylation

Extracellular α-Synuclein has been implicated in interneuronal propagation of disease pathology in Parkinson’s Disease. How α-Synuclein is released into the extracellular space is still unclear. Here, we show that α-Synuclein is present in extracellular vesicles in the central nervous system. We find that sorting of α-Synuclein in extracellular vesicles is regulated by sumoylation and that sumoylation acts as a sorting factor for targeting of both, cytosolic and transmembrane proteins, to extracellular vesicles. We provide evidence that the SUMO-dependent sorting utilizes the endosomal sorting complex required for transport (ESCRT) by interaction with phosphoinositols. Ubiquitination of cargo proteins is so far the only known determinant for ESCRT-dependent sorting into the extracellular vesicle pathway. Our study reveals a function of SUMO protein modification as a Ubiquitin-independent ESCRT sorting signal, regulating the extracellular vesicle release of α-Synuclein. We deciphered in detail the molecular mechanism which directs α-Synuclein into extracellular vesicles which is of highest relevance for the understanding of Parkinson’s disease pathogenesis and progression at the molecular level. We furthermore propose that sumo-dependent sorting constitutes a mechanism with more general implications for cell biology.Instituto de Investigaciones Bioquímicas de La Plat

Development of antibody-derived therapeutics for the treatment of hematopoietic neoplasias

Trotz deutlicher Fortschritte bei der Behandlung hämatopoietischer Neoplasien in den letzten Jahren, verlaufen viele dieser Erkrankungen immer noch tödlich und gängige Therapieoptionen sind häufig mit starken Nebenwirkungen assoziiert. Daher besteht ein dringender Bedarf an innovativen, tumorgerichteten Therapieformen, die gesundes Gewebe möglichst nicht schädigen. Antikörper-abgeleitete Moleküle sind aufgrund ihrer hohen Bindespezifität hierfür besonders geeignet. Obwohl das B-lymphoide Oberflächenantigen CD19 eine attraktive Zielstruktur für eine Antikörper-abgeleitete Therapie darstellt, ist trotz zahlreicher Bemühungen bislang kein gegen CD19-gerichtetes Molekül klinisch zugelassen. Zur Verbesserung eines bestehenden CD19 gerichteten scFv Antikörperfragments (4G7-wt) wurden mit Hilfe zweier unterschiedlicher Methoden des antibody engineerings, Mutanten hergestellt und funktionell charakterisiert. Die erste Variante trug 14 Aminosäuresubstitutionen an potentiell destabilisierenden Positionen, welche durch die Konsensus-Methode identifiziert wurden (4G7-mut). Bei der zweiten Variante wurden die murinen CDRs mit Hilfe der loop graft Methode auf eine stabile humane Gerüstregion transferiert. Die vergleichende Charakterisierung ergab, dass das humanisierte Antikörper-Fragment 4G7-graft ähnliche Eigenschaften wie der 4G7-wt aufwies. Die zweite Variante zeigte hingegen deutliche Verbesserungen in Expressionsausbeute und Stabilität. Beide Moleküle weisen spezielle vorteilhafte Eigenschaften gegenüber der Ausgangsvariante auf und empfehlen sich somit für weiterführende Entwicklungen CD19-spezifischer Therapeutika. Bei der aktuen myeloischen Leukämie handelt es sich um eine Stammzell-getriebene Erkrankung, die trotz moderner Behandlungsmethoden in vielen Fällen mit der Ausbildung von Rezidiven verbunden ist. Ursache hierfür sind Zellen der minimalen Resterkrankung (minimal residual disease, MRD), welche über besondere Resistenzmechanismen gegenüber aktuellen Therapieprotokollen verfügen. AML Leukämie Stammzellen (AML-LSZ) stellen zumindest einen Teil der MRD Population dar und verfügen über ein charakteristisches Expressionsprofil von Oberflächenantigenen, mit dessen Hilfe es durch Antikörper-abgeleiteter Therapeutika möglich sein sollte ein präferentielles Anvisieren dieser Zellpopulation zu ermöglichen. Zwei dieser, auf AML-LSZ vorkommenden Antigene sind CD33 und CD123. Zwei single-chain triplebodies,sctbs [123 x ds16 x 33] und [123 x ds16 x 123], gerichtet gegen CD33 und CD123 wurden konstruiert und charakterisiert. Beide Moleküle vermittelten potente Dosis- und Antigen-abhängige Lyse von AML-abgeleiteten Zelllinien in Antikörperabhängige zelluläre Zytotoxizität (ADCC)-Experimenten. Das Molekül gegen die beiden unterschiedlichen Tumorantigene, sctb [123 x ds16 x 33], vermittelte signifikant höhere Lysen bei niedrigeren EC50-Werten als das Konstrukt gegen nur ein Tumorantigen,sctb [123 x ds16 x 123]. Auch gegen primäre AML Zellen aus dem peripheren Blut zeigte sctb [123 x ds16 x 33] tendenziell, wenn auch nicht signifikant, höhere Lysen. Beide Moleküle sind attraktive Kandidaten für die Weiterentwicklung zum klinischen Einsatz. Vor allem der sctb [123 x ds16 x 33] sollte das Potential besitzen die klinisch relevanten AML-LSZ präferentiell an zu visieren.In recent years remarkable advances in the treatment of hematopoietic malignancies were achieved. However, a lot of cases still lead to death and current therapeutic options are often accompanied by severe side effects. Therefore, innovative tumor specific therapeutics sparing healthy tissues are urgently needed. Due to their binding-specificity antibody-derived molecules are highly suitable for this purpose. The B-lymphoid surface antigen CD19 is a promising target structure for an antibody derived therapy. Although various CD19-specific therapeutics were investigated,none have been approved for clinical use so far. In order to improve an existing CD19-specific antibody fragment (4G7-wt), two independent antibody engineering techniques were used to design more favorable mutant molecules. The first mutant carried 14 amino acid substitutions at potentially destabilizing positions identified by the modified consensus method (4G7-mut), whereas the second mutant was generated by loop grafting of the murine CDRs onto stable human framework regions (4G7-graft). Their comparative characterization revealed that the humanized antibody fragment 4G7-graft possessed comparable properties to the 4G7-wt. However, the 4G7-mut showed increased expression yields and stability. Both new molecules offer unique properties and thereby qualify for the design of promising therapeutic molecules towards a potential clinical application. Acute myeloid leukemia (AML) is a stem cell-driven disease showing a high frequency of relapses. Minimal residual disease cells (MRD), which possess an increased resistance against current therapeutic protocols, are responsible for these relapses. AML leukemic stem cells (AML-LSCs) are discussed to be a part of these MRD cells and can be characterized by the expression profile of their surface antigens. Therefore,antibody-derived molecules should be capable to preferentially target and eliminate this type of malignant cells. Among others, the surface antigens CD33 and CD123 characterize these cells and two single-chain triplebodies, sctbs [123 x ds16 x 33] and [123 x ds16 x 123], were designed and characterized. Both molecules induced potent dose- and antigen-dependent lysis of AML cells in antibody-dependent cellular cytotoxicity (ADCC) experiments. The dual targeting molecule sctb [123 x ds16 x 33] induced significantly higher lysis at lower EC50-values as the mono targeting construct sctb [123 x ds16 x 123]. The dual targeting molecule also showed a tendency, though not significant, to induce higher lysis of primary AML cells from the peripheral blood of AML patients. Both molecules represent promising candidates for further pre-clinical investigation. Especially the sctb [123 x ds16 x 33] has the potential for a preferential targeting of AML-LSCs

Public Key Infrastructure and Crypto Agility Concept for Intelligent Transportation Systems

Secure vehicular communication has been discussed over a long period of time. Now,- this technology is implemented in different Intelligent Transportation System (ITS) projects in europe. In most of these projects a suitable Public Key Infrastructure (PKI) for a secure communication between involved entities in a Vehicular Ad hoc Network (VANET) is needed. A first proposal for a PKI architecture for Intelligent Vehicular Systems (IVS PKI) is given by the car2car communication consortium. This architecture however mainly deals with inter vehicular communication and is less focused on the needs of Road Side Units. Here, we propose a multi-domain PKI architecture for Intelligent Transportation Systems, which considers the necessities of road infrastructure authorities and vehicle manufacturers, today. The PKI domains are cryptographically linked based on local trust lists. In addition, a crypto agility concept is suggested, which takes adaptation of key length and cryptographic algorithms during PKI operation into account

Pathophysiological Consequences of Neuronal α-Synuclein Overexpression: Impacts on Ion Homeostasis, Stress Signaling, Mitochondrial Integrity, and Electrical Activity

α-Synuclein (α-Syn) is intimately linked to the etiology of Parkinson's Disease, as mutations and even subtle increases in gene dosage result in early onset of the disease. However, how this protein causes neuronal dysfunction and neurodegeneration is incompletely understood. We thus examined a comprehensive range of physiological parameters in cultured rat primary neurons overexpressing α-Syn at levels causing a slowly progressive neurodegeneration. In contradiction to earlier reports from non-neuronal assay systems we demonstrate that α-Syn does not interfere with essential ion handling capacities, mitochondrial capability of ATP production or basic electro-physiological properties like resting membrane potential or the general ability to generate action potentials. α-Syn also does not activate canonical stress kinase Signaling converging on SAPK/Jun, p38 MAPK or Erk kinases. Causative for α-Syn-induced neurodegeneration are mitochondrial thiol oxidation and activation of caspases downstream of mitochondrial outer membrane permeabilization, leading to apoptosis-like cell death execution with some unusual aspects. We also aimed to elucidate neuroprotective strategies counteracting the pathophysiological processes caused by α-Syn. Neurotrophic factors, calpain inhibition and increased lysosomal protease capacity showed no protective effects against α-Syn overexpression. In contrast, the major watchdog of outer mitochondrial membrane integrity, Bcl-Xl, was capable of almost completely preventing neuron death, but did not prevent mitochondrial thiol oxidation. Importantly, independent from the quite mono-causal induction of neurotoxicity, α-Syn causes diminished excitability of neurons by external stimuli and robust impairments in endogenous neuronal network activity by decreasing the frequency of action potentials generated without external stimulation. This latter finding suggests that α-Syn can induce neuronal dysfunction independent from its induction of neurotoxicity and might serve as an explanation for functional deficits that precede neuronal cell loss in synucleopathies like Parkinson's disease or dementia with Lewy bodies