Es ADCs (Antibody-Drug Conjugates) correspondent à une nouvelle stratégie thérapeutique anti-tumorale particulièrement prometteuse. Néanmoins, les ADCs actuellement utilisés en clinique sont obtenus par conjugaisons chimiques, resultant en des mixtures hétérogènes impactant négativement leurs pharmacocinétiques et leurs performances in vivo.Récemment, différentes strategies de couplage site-spécifique ont été développées afin de réduire cette hétérogénéité. Dans cette thèse, nous rapportons le développement d’une nouvelle technologie CovIsoLink™ (Covalently Isopeptide Crosslinking) permettant la génération d’ADCs par utilisation de nouveaux peptides glutamine Q-Tag présentant des affinités optimisées par rapport à des peptides disponibles (ZQG, LLQG) pour une enzyme bactérienne la transglutaminase (mTG).La preuve de concept de cette technologie a été réalisée par insertion de ces peptides Q-Tag en C-ter de la région codant pour la chaine lourde des anticorps anti-HER2 (Trastuzumab). Nous avons ainsi pu démontrer la conjugaison homogène et reproductible de différentes drogues sans contamination par des chaines d’anticorps non conjuguées. Nous avons pu montrer que l’immunoréactivité et la capacité d’internalisation de ces ADCs n’étaient pas altérées par la conjugaison et qu’ils présentaient in vitro et in vivo, des propriétés de lyse de cellules tumorales similaires au Trastuzumab emtansine (Kadcyla®), actuellement en clinique. Par ailleurs, afin de généraliser notre technologie à différents formats d’anticorps nous avons générés des fragments Fab et scFv et évalué leur fonctionnalité. Ainsi, nous avons pu prouver que l’utilisation de nouveaux peptides optimisés Q-Tag substrat de la transglutaminase permettait une stratégie de couplage alternative plus homogène par couplage de différentes molécules sans espèce contaminante non couplée / Antibody-drug conjugates (ADCs) are a powerful class of therapeutic agents, demonstrating success in the treatment of several malignancies. The currently approved ADCs are produced by chemical conjugations and exist as heterogeneous mixtures that negatively influence the pharmacokinetics and in vivo performance. Recently many of site-specific conjugation technologies have been developed to reduce heterogeneity and batch-to batch variability. Microbial transglutaminase (mTG) has been demonstrated as efficient tool for site-specific conjugation. In this thesis we report the development CovIsoLink™ (Covalently Isopeptide Crosslinking) technology for the generation of homogenous immunoconjugates using a novel glutamine donor peptides (Q-tag) with improved affinity compared to the known peptides (ZQG, LLQG). As a proof of concept, the peptides sequences were engineered into the heavy chain C-terminal of Trastuzumab antibody. We demonstrated the reproducible and homogeneous conjugation of Q-tagged Trastuzumab with different payloads, without any unconjugated species. The ADCs were evaluated in series of in vitro and in vivo assays. We confirmed that the immunoreactivity and internalisation are not altered by the conjugation. Furthermore similar in vitro and in vivo tumor cell killing potency was demonstrated than Trastuzumab emtansine (Kadcyla®), which is already used in the clinic. Morover we extend our site-specific conjugation technology to antibody fragments (Fab and scFv), evaluating their functionality by conjugation with AlexaFluor488-cadaverine and in antigen binding assays. Thus, using novel glutamine donor peptides, our technology provides an alternative enzymatic conjugation strategy for the engrafment of different payloads resulting in homogeneous batches, without unconjugated species

No description available.

The need for an unlimited source of islets and a safer method of immunosuppression has limited the widespread application of islet transplantation. To remedy the shortage of donor tissue, xenotransplantation of neonatal porcine islets (NPI) has been proposed. In this study we sought to determine if combining co-transplantation of NPI with Sertoli cells (SC) with a short-term monoclonal antibody (mAb) therapy would prevent NPI xenograft rejection. We hypothesize that this combination of treatments will lead to long-term NPI xenograft survival. Our result show a significant increase in the proportion of mice achieving long-term graft survival compared to untreated mice transplanted with NPI alone, as 7/7 mice treated with anti-LFA-1 mAb (p=0.001), 7/8 mice treated with anti-CD154 mAb (p=0.003), and 4/9 mice treated with anti-CD45RB mAb (p=0.020) achieved and maintained normoglycemia long-term. Therefore, we conclude that the combination of mAb therapy with SC is highly efficacious in preventing NPI xenograft rejection. / Experimental Surgery

No description available.

Despite the clinical importance of anaerobic bacteria in otitis media and the uncertainty regarding the proper treatment of the anaerobic focal infection, few experimental studies focused upon the role of these microorganisms in otitis media have been publis­hed. In the present investigation a guinea-pig model for the induc­tion of anaerobic monoinfections in the middle ear was described. Bacteroides fragilis and Propionibacterium acnes (4.0-10x10 colony forming units) injected via the tympanic membrane were capable of inducing clinical and histological otitis media with persistent seguele in the middle ear cavity. Bacteroides asacc-harolyticus, Peptostreptococcus micros and Peptost repto- coccus anaerobius failed to induce otitis media. B. fragilis otitis was accompanied by increased serum IgG and IgM antibody titres against the challenge organism, whereas P. acnes and P. anaerobius did not induce a humoral immune response. The results suggested true virulence of B. fragilis in guinea-pig middle ear monoinfections. Metronidazole was found to accelerate the elimination of B.fragilis from the middle ear. However, even high doses of metro­nidazole were nojt fully effective perhaps reflecting an incomplete anaerobiosis at the site of infection in some instances. At pre­sent, nitroimidazoles in chronic otitis media must be regarded as a possible alternative reguiring further study, particularly with regard to the dosage. In order to gain further knowledge of the interaction between poly­morphonuclear leukocytes and bacteria under anaerobic conditions an in vitro model was established. It was shown that P. acnes was readily phagocytosed with the aid of C3 activated either via the classical or alternative pathway and that killing of P. acnes was inefficient during anaerobiosis. The results suggest that P.acnes is maintained in the pus in chronic otitis media because it sur­vives phagocytosis. Finally, the interaction between the most common pathogen in acute purulent otitis media, Streptococcus pneumoniae, and human poly­morphonuclear leukocytes under anaerobic conditions was studied. Since purulent maxillary sinus effusion (and probably also purulent middle ear effusion), invariably has a pO^ approaching zero, such studies are highly relevant with regard to the host defence in sinuitis and perhaps also in otitis media. S. pneumoniae was killed by the phagocytes under anaerobic condi­tions although at a slower rate than in air. Degradation of pneumo­coccal teichoic acid, DNA and RNA took place after phagocytosis under aerobic as well as anaerobic conditions, whereas degradation of unsaturated cell membrane lipids took place only under aerobic conditions. Furthermore, the pneumococcal autolytic system did not participate in the killing or the degradation of the bacteria. / <p>Diss. Umeå, Umeå universitet, 1984, härtill 6 uppsatser</p> / digitalisering@umu

In the past 15-20 years, advances in antibody engineering have facilitated the generation and isolation of monoclonal antibodies (mAbs) to a wide array of antigens. Consequently, mAbs have become essential therapeutic tools and currently dominate the global protein therapeutics market. The engineering of anti-infective antibodies, however, has proven quite a challenge, despite the fact that antibodies were naturally evolved to fight infections. The identification of suitable antigens, the mode of administration and the high cost associated with the production of antibody therapeutics are some of the major hurdles for the progress of anti-infective antibodies. This dissertation addresses issues concerning the development of anti-infective antibodies against two different pathogens: SARS coronavirus (CoV) and two pathogenic species of Burkholderia bacteria. To investigate the role of affinity in viral neutralization and evolution of escape mutants, we first sought to isolate an antibody with high affinity towards the receptor binding domain (RBD) of SARS-CoV. Following high-throughput screening of a library of random mutants via the APEx display system, we isolated antibodies with affinities in the range of 0.8 nM - 0.1 nM. The affinity was further improved by additional mutagenesis and DNA shuffling, and a high affinity variant (45pM) with ~300-fold improvement over the parental antibody was isolated. Evaluation of these antibodies in an in vitro assay demonstrated that neutralization of wild-type Urbani strain of SARS-CoV correlates well with the affinity of the antibody, with higher affinity leading to greater neutralization. Moreover, the antibody exhibiting the highest affinity could neutralize SARS-CoV escape mutants that evaded neutralization by both parental and lower affinity antibodies. Another important aspect for the development of anti-infective antibodies concerns the identification of suitable antigen targets to be used in the isolation of antibodies. In an effort to develop a high-throughput screening method for the isolation of antibodies to a wide array of antigens, we used a synthetic antibody (Fab) library constructed by a minimalist approach and displayed on the surface of filamentous bacteriophage. The library was screened against antigens from Burkholderia pseudomallei and Burkholderia mallei. After only three rounds of selection and enrichment against five different antigens, we obtained Fabs specific to four of the antigens as confirmed by ELISA. These results not only demonstrate the use of a synthetic antibody library for the isolation of antibodies against infectious pathogens, but also its feasibility, and potential applicability as a high-throughput screen for a variety of antigens. / text

The ability to formulate monoclonal antibodies at high concentration in a low-viscosity form is of broad interest in drug delivery, as monoclonal antibody-based drugs are now prescribed for cancer, autoimmune disorders, and many other diseases. Herein, we create highly concentrated antibody dispersions (up to 260 mg/mL) via three different methods, utilizing proline as an interacting depletant or trehalose as a non-interacting depletant. These dispersions are able to achieve viscosities an order of magnitude lower than similarly concentrated antibody solutions over a range of formulation pHs. When diluted, these antibody dispersions return to monomer. The proline acts to minimize protein zeta potential, thus reducing the electrostatic repulsion on the protein, even when formulated 3 pH units away from the antibody pI. In addition, it acts as a depletant, forcing the monomers into cluster via osmotic effects / text

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No description available.

Taura syndrome (TS) is one of the most devastating diseases affecting the shrimp farming industry worldwide. The causative virus, Taura syndrome virus (TSV), has been identified. My work is centred on the development of monoclonal antibodies against TSV. I have also characterized a novel variant of the virus from Venezuela and evaluated the effect of hyperthermia on TSV infection. This work has resulted in 3 manuscripts, which constitute the core of this dissertation. The taxonomy throughout this dissertation is done according to Holthuis (1980).The first manuscript describes the production of a monoclonal antibody reacting with the Belize strain of TSV. The antibody, MAb 2C4, exhibits good sensitivity and specificity for TSV in immunohistochemistry (IHC) and dot blot immunoassay. MAb 2C4 reacted with the TSV-HI94, TSV-SI98 and TSV-BZ02 variants, but not with the TSV-VE05 and TSV-TH05 variants. This antibody adds and improves tools to those available for TSV diagnosis.Chapter three describes a relatively novel variant of TSV from Venezuela, which was characterized by our laboratory. By genetic sequencing, this new isolate exhibits a 94% similarity with TSV-HI94. IHC, dot blot immunoassay and bioassays were also performed. While processed samples reacted only weakly with the TSV monoclonal antibody MAb 1A1, the virus in its native state reacted strongly with the antibody. In bioassays, TSV-VE05 presented mortality comparable to TSV-HI94 in Penaeus vannamei. These data confirm the presence of TSV in Venezuela and that a new variant of the virus was responsible for the outbreak of TS.In chapter four, the behavior of TSV infection under hyperthermic conditions was examined. I compared the susceptibility of Kona stock P. vannamei to the infection by two variants of TSV under hyperthermic conditions (32oC). Shrimp, infected with TSV-HI94, were resistant to infection at high temperature. However, under the same hyperthermic conditions, the challenged shrimp were fully susceptible to the infection by TSV-BZ02. This susceptibility to TSV-BZ02 at higher temperatures was independent both of the route of infection and of the salinity of water. I conjecture that TSV-BZ02 might be a temperature permissible mutant of TSV.