Final Report Summary - SERPINOPATHIES (Determination of the structure of the pathological neuroserpin polymer and development of an intrabody strategy to prevent disease-associated inclusions in cell and animal models of disease) ad Aim 1 - Structure analysis of pathological serpins by epitope mappingThe most common pathological variant associated with α1-antitrypsin (AAT) deficiency is the Z allele (E342K) which leads to accumulation of AAT as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z AAT lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Monocolonal antibody (mAb) technology can identify interactors with Z AAT that comply with both requirements. Prof. Lomas’ group has developed an array of conformational specific serpin antibodies, including the novel 4B12 antibody  which binds with high affinity to AAT in its monomeric form and interestingly, blocks its polymerization in vitro as well as in a cell model of disease.The first part of this project aimed at mapping the epitope of the monoclonal 4B12 antibody on monomeric AAT. The objectives were to (i) reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of AAT and (ii) to further understand the mechanism of pathological AAT polymerisation which will provide new angles for drug development.Epitope mapping was achieved by mutating twenty amino acid residues of AAT to cysteine and conjugating the resulting variants to PEG5000 at the modified positions. In subsequent 4B12 binding studies by non-denaturing gel electrophoresis and ELISA, we identified three positions (residues 32, 43 and 306) that showed less efficient binding to 4B12 when PEGylated. A histidine residue at position 43 was found to be of particular importance for antibody recognition: no binding was detected upon mutation to cysteine, even in the absence of a PEG conjugate. The conformational integrity of all mutants was confirmed in order to ensure that the observed effects after PEGylation are due to steric hindrance and not due to perturbance of secondary structure. This was done by enzyme inhibitory tests and gel based protection assays in which 4B12 is bound to AAT prior to pegylation. Residues involved in antibody recognition do not pegylate after 4B12 binding, confirming the mapped epitope.Experiments were designed to test the hypothesis that the 4B12 antibody prevents polymerization by stabilizing a structural element known as β-sheet A. During polymerization this β-sheet accepts an additional β-strand which causes a pronounced increase in the stability of the protein. Despite the unexpected location of the epitope of the antibody, this mechanism was the most likely based on prevailing evidence from our and other groups, and served as a useful point of reference.The first approach was to use a peptide mimetic of the additional β-strand. This process can be used to monitor a change in the ability of β-sheet A to incorporate an extra strand, which in turn reflects changes in its stability and dynamics. Unexpectedly, the binding of the 4B12 antibody increased the rate at which β-sheet A accepted the peptide, despite its ability to prevent the process of polymerization. This is contrary to the behavior predicted by any of the dominant models proposed for the polymerization mechanism.The site of the epitope includes the ‘I helix’, which is a structural element at the edge of β-sheet A. There is evidence in the literature that mutations in the ‘I helix’ affect the function of antitrypsin as a protease inhibitor . Experiments were therefore designed to test the ability of antitrypsin to block the activity of a model protease, trypsin. While the ratio of antitrypsin needed to inhibit trypsin was unaffected by the presence of the antibody, in a more sensitive test of inhibitor behavior it was found that the normally irreversible interaction between antitrypsin and trypsin was destabilized. Because this destabilization could be induced by adding the antibody after the inhibitory complex was formed, this indicated there was a local conformational change that was being transmitted to the binding site of the protease.Based on these observations, I have been able to conclude (1) opening of β-sheet A to accept a β-strand is necessary but not sufficient for polymerization to occur; (2) remodeling of helix I or a nearby structural element such as β-strand 6A is a necessary step in the polymerization mechanism; (3) despite the inhibitory complex being hyperstable, local conformational change can still occur that is capable of destabilizing the interaction with the protease; (4) the vicinity of helix I is a potential target for the binding of therapeutic molecules for the purpose of preventing polymerization.The results of this epitope mapping effort are currently being summarized in a first author manuscript for publication, describing (i) a method for epitope mapping and (ii) the epitope of the polymerization blocking antibody 4B12 (iii) new insight into the polymerization mechanism of AAT. These new findings will subsequently be used for drug development screens.ad Aim 2 - Intrabody targeting of mutant serpins to prevent the formation of intracellular polymers based on a monoclonal serpin antibodyIntrabodies are gene-engineered antibodies that can alter the fate of their target molecule inside cells while offering conformational specificity. They can be generated from a monoclonal antibody by cloning its FAB portion into a suitable expression vector  and their efficacy can be tested in cell or in vivo models of disease.Invertebrate genetic model organisms such as Caenorhabditis elegans offer fast, economical in vivo models in which to triage therapeutic tools prior to validation in vertebrate models [4, 5]. Their well-studied genetics, fast regeneration time, reproductive capacity and ease of large-scale culture are complemented by access to automated behavioural analysis [6, 7]. Several C. elegans models of neuromuscular and neurodegenerative diseases have been validated by the effectiveness of drugs currently used in the clinic [8, 9]. Recently, Z AAT has been expressed in the intestine of C. elegans and this animal used as a platform for high-throughput drug screening . Using this model, the drug fluphenazine was shown to reduce proteotoxicity and reverse the phenotypic effects of Z AAT accumulation.The second part of this project aimed at (i) generating an intrabody scFv-4B12 suitable for expression in C.elegans (ii) generating a C.elegans model of AAT deficiency, and (iii) validating in vivo our 4B12 intrabody as well as small molecule compounds that can block polymerisation.The single chain variable fragment of the 4B12 mAB was amplified by PCR from RNA extracted from hybridoma cells by using species- and isotype specific, generic primers and then cloned into a series of expression vectors, including those suitable for C.elegans. The efficacy of the cloned region was confirmed in a CHO cell model of disease where it reduced the intracellular polymerisation of Z α1-antitrypsin by 60% when assessed by ELISA, electrophoresis, and pulse-chase assays .It is known that the expression of Z AAT in the intestine of C. elegans results in viable strains that accumulate AAT polymers [10, 11]. To extend this previous model, here we targeted protein expression to muscle tissue and to specialized cells that are found adjacent to somatic musculature in the pseudocoel body cavity, called the coelomocytes. Coelomocytes accumulate different macromolecules from the body cavity fluid and are thought to exert a similar function as macrophages in vertebrates as they can phagocytose and digest cellular debris and pathogens.Exploiting this feature, we have generated an improved C. elegans model of AAT deficiency that allows for easy screening of accumulation or clearance of polymers and evaluation of protein secretion. We have created several new lines of C. elegans that express M and Z AAT fused to RFP. Transgenic animals were identified utilizing the fluorescent protein tag and cultured at a permissive (15°C) and elevated (25°C) temperature. The animals are viable and express fluorescently labeled protein under both conditions. Wild type (M) AAT is efficiently secreted from the muscle into the pseudocoel and then endocytosed by the coelomocytes. Z AAT is retained in the muscle where it forms punctae, apparently within the endoplasmic reticulum. The conformational identity of the expressed polymers was confirmed by protein extraction from animals and analysis by ELISA using our polymer specific antibody, 2C1 . Furthermore, Z lines grown at elevated temperature were found to have an impaired phenotype with reduced mobility and decelerated development. A detailed characterization of the phenotype assessing survival, longevity, brood size and locomotor function is currently ongoing and the polymer load, subcellular accumulation and secretion efficiency will be assessed by fluorescent microscopy and immunocytochemistry using the 2C1 monoclonal antibody in the future.In future steps, small molecules that were shown to block polymerisation in vitro and in cell models of AAT deficiency  will be tested in our model. If they are able to exert their blocking function then the deleterious effects of AAT accumulation on survival, longevity, brood size and locomotor function will be reversed. The clearance of polymers will be observed at a cellular level by fluorescent microscopy and immunocytochemistry using the 2C1 monoclonal antibody. In a different strategy, we will use the newly generated C.elegans lines to test the in vivo efficacy of the scFv fragment of the 4B12 monoclonal antibody by generating expression vectors to co-express the fluorescently tagged scFv fragment of the 4B12 monoclonal antibody with M or Z AAT. The 4B12 scFv is expected to co-localize with AAT and to block polymerisation. We will assess the phenotypic effects of a reduced polymer load on survival, longevity, brood size and locomotor function as well as the clearance pattern at a cellular level using the same methods as described above.Socio-economic impact: AAT deficiency is the most common hereditary disease, with the Z allele being the most common severe deficiency allele in Europe (4% of individuals are heterozygous (PI*MZ) and around 1 in 1700 homozygous (PI*Z) ) . Accumulation of mutant AAT starts in utero  with 1 in 10 infants developing cholestatic jaundice in the first few months of life, approximately 15% of whom will progress to juvenile cirrhosis [16, 17]. The mortality rate from liver disease in PI*Z children during childhood is 2–3% [18, 19], whereas nearly half the AAT-deficient adults over 50 have pathological features of liver cirrhosis and occasionally hepatocellular carcinoma [19, 20]. The concomitant lack of circulating M AAT also predisposes these adults to early onset emphysema. Deficiency of α1-antitrypsin is the most important genetic factor in the development of COPD (chronic obstructive pulmonary disease) and is found in 1–2% of affected individuals .Severe cases of emphysema are currently addressed by AAT replacement therapy where purified AAT is given as a weekly intravenous infusion. The cost of replacement is estimated to be above $100,000 per annum, per patient, and is a lifelong treatment . To date, no alternative therapies that prevent lung or liver damage are available.To address this unmet medical need, the Lomas group has been woking in close collaboration with the pharmaceutical industry (GlaxoSmithKline) on a drug discovery program. Aiding the development of new drugs through this work will (i) help patients and decrease costs for the public health care system and (ii) generate economic growth by development of a new pharmaceutical product.References:1. Ordonez, A., et al., A single-chain variable fragment intrabody prevents intracellular polymerization of Z alpha1-antitrypsin while allowing its antiproteinase activity. FASEB J, 2015.2. Knaupp, A.S. et al., The roles of helix I and strand 5A in the folding, function and misfolding of alpha1-antitrypsin. PLoS One, 2013. 8(1): p. e54766.3. Zhou, C. and S. Przedborski, Intrabody and Parkinson's disease. Biochim Biophys Acta, 2009. 1792(7): p. 634-42.4. Sleigh, J.N. and D.B. 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