Propagation of transmissible spongiform encephalopathies is associated with the conversion of normal prion protein, PrP C , into a misfolded, oligomeric form, PrP Sc . Although the high-resolution structure of the PrP C is well characterized, the structural properties of PrP Sc remain elusive. Here we used MS analysis of H/D backbone amide exchange to examine the structure of amyloid fibrils formed by the recombinant human PrP corresponding to residues 90 -231 (PrP90 -231), a misfolded form recently reported to be infectious in transgenic mice overexpressing PrP C . Analysis of H/D exchange data allowed us to map the systematically H-bonded ␤-sheet core of PrP amyloid to the C-terminal region (staring at residue Ϸ169) that in the native structure of PrP monomer corresponds to ␣-helix 2, a major part of ␣-helix 3, and the loop between these two helices. No extensive hydrogen bonding (as indicated by the lack of significant protection of amide hydrogens) was detected in the N-terminal part of PrP90 -231 fibrils, arguing against the involvement of residues within this region in stable ␤-structure. These data provide long-sought experimentally derived constraints for high-resolution structural models of PrP amyloid fibrils.prion diseases ͉ transmissible spongiform encephalopathy ͉ amyloid structure ͉ mass spectrometry

Contemporary in vivo and in vitro discovery platform technologies greatly increase the odds of identifying high-affinity monoclonal antibodies (mAbs) towards essentially any desired biologically relevant epitope. Lagging discovery throughput is the ability to select for highly developable mAbs with drug-like properties early in the process. Upstream consideration of developability metrics should reduce the frequency of failures in later development stages. As the field moves towards incorporating biophysical screening assays in parallel to discovery processes, similar approaches should also be used to ensure robust chemical stability. Optimization of chemical stability in the early stages of discovery has the potential to reduce complications in formulation development and improve the potential for successful liquid formulations. However, at present, our knowledge of the chemical stability characteristics of clinical-stage therapeutic mAbs is fragmented and lacks comprehensive comparative assessment. To address this knowledge gap, we produced 131 mAbs with amino acid sequences corresponding to the variable regions of clinical-stage mAbs, subjected these to low and high pH stresses and identified the resulting modifications at amino acid-level resolution via tryptic peptide mapping. Among this large set of mAbs, relatively high frequencies of asparagine deamidation events were observed in CDRs H2 and L1, while CDRs H3, H2 and L1 contained relatively high frequencies of instances of aspartate isomerization.

The detailed structures of prion disease-associated, partially protease-resistant forms of prion protein (e.g. PrP Sc ) are largely unknown. PrP Sc appears to propagate itself by autocatalyzing the conformational conversion and oligomerization of normal prion protein (PrP C ). One manifestation of PrP Sc templating activity is its ability, in protein misfolding cyclic amplification reactions, to seed the conversion of recombinant prion protein (rPrP) into aggregates that more closely resemble PrP Sc than spontaneously nucleated rPrP amyloids in terms of proteolytic fragmentation and infrared spectra. The absence of posttranslational modifications makes these rPrP aggregates more amenable to detailed structural analyses than bona fide PrP Sc . Here, we compare the structures of PrP Sc -seeded and spontaneously nucleated aggregates of hamster rPrP by using H/D exchange coupled with mass spectrometry. In spontaneously formed fibrils, very slow H/D exchange in region ϳ163-223 represents a systematically H-bonded cross-␤ amyloid core structure. PrP Scseeded aggregates have a subpopulation of molecules in which this core region extends N-terminally as far as to residue ϳ145, and there is a significant degree of order within residues ϳ117-133. The formation of tightly H-bonded structures by these more N-terminal residues may account partially for the generation of longer protease-resistant regions in the PrP Sc -seeded rPrP aggregates; however, part of the added protease resistance is dependent on the presence of SDS during proteolysis, emphasizing the multifactorial influences on proteolytic fragmentation patterns. These results demonstrate that PrP Sc has a distinct templating activity that induces ordered, systematically H-bonded structure in regions that are dynamic and poorly defined in spontaneously formed aggregates of rPrP.

M. Damschroder (2016) Inhibition of CD73 AMP hydrolysis by a therapeutic antibody with a dual, non-competitive mechanism of action, mAbs, 8:3, 454-467, DOI: 10.1080/19420862.2016 ABSTRACT CD73 (ecto-5 0 -nucleotidase) has recently been established as a promising immuno-oncology target. Given its role in activating purinergic signaling pathways to elicit immune suppression, antagonizing CD73 (i.e., releasing the brake) offers a complimentary pathway to inducing anti-tumor immune responses. Here, we describe the mechanistic activity of a new clinical therapeutic, MEDI9447, a human monoclonal antibody that non-competitively inhibits CD73 activity. Epitope mapping, structural, and mechanistic studies revealed that MEDI9447 antagonizes CD73 through dual mechanisms of inter-CD73 dimer crosslinking and/or steric blocking that prevent CD73 from adopting a catalytically active conformation. To our knowledge, this is the first report of an antibody that inhibits an enzyme's function through 2 distinct modes of action. These results provide a finely mapped epitope that can be targeted for selective, potent, and non-competitive inhibition of CD73, as well as establish a strategy for inhibiting enzymes that function in both membrane-bound and soluble states.

Susceptibility of methionine to oxidation is an important concern for chemical stability during the development of a monoclonal antibody (mAb) therapeutic. To minimize downstream risks, leading candidates are usually screened under forced oxidation conditions to identify oxidation-labile molecules. Here we report results of forced oxidation on a large set of in-house expressed and purified mAbs with variable region sequences corresponding to 121 clinical stage mAbs. These mAb samples were treated with 0.1% HO for 24 hours before enzymatic cleavage below the hinge, followed by reduction of inter-chain disulfide bonds for the detection of the light chain, Fab portion of heavy chain (Fd) and Fc by liquid chromatography-mass spectrometry. This high-throughput, middle-down approach allows detection of oxidation site(s) at the resolution of 3 distinct segments. The experimental oxidation data correlates well with theoretical predictions based on the solvent-accessible surface area of the methionine side-chains within these segments. These results validate the use of upstream computational modeling to predict mAb oxidation susceptibility at the sequence level.