14 research outputs found
A novel method for in silico assessment of Methionine oxidation risk in monoclonal antibodies: Improvement over the 2-shell model
Over the past decade, therapeutic monoclonal antibodies (mAbs) have established their role as valuable agents in the treatment of various diseases ranging from cancers to infectious, cardiovascular and autoimmune diseases. Reactive groups of the amino acids within these proteins make them susceptible to many kinds of chemical modifications during manufacturing, storage and in vivo circulation. Among these reactions, the oxidation of methionine residues to their sulfoxide form is a commonly observed chemical modification in mAbs. When the oxidized methionine is in the complementarity-determining region (CDR), this modification can affect antigen binding and thus abrogate biological activity. For these reasons, it is essential to identify oxidation liabilities during the antibody discovery and development phases. Here, we present an in silico method, based on protein modeling and molecular dynamics simulations, to predict the oxidation-liable residues in the variable region of therapeutic antibodies. Previous studies have used the 2-shell water coordination number descriptor (WCN) to identify methionine residues susceptible to oxidation. Although the WCN descriptor successfully predicted oxidation liabilities when the residue was solvent exposed, the method was much less accurate for partially buried methionine residues. Consequently, we introduce a new descriptor, WCN-OH, that improves the accuracy of prediction of methionine oxidation susceptibility by extending the theoretical framework of the water coordination number to incorporate the effects of polar amino acids side chains in close proximity to the methionine of interest
Representation of the two-shell water coordination number (WCN) and the WCN-OH variant.
(A) For a methionine with the side chain exposed to the solvent, the water molecules within 6 Ã… from the sulfur atom are shown. (B) For a methionine with the side chain only partially exposed to the solvent, water molecules and side chains containing a hydroxyl group are shown. Carbon atoms are colored in cyan, oxygen in red, hydrogen in gray, nitrogen in blue and sulfur in yellow. The sphere represents the 2-solvation shell (radius 6 Ã…).</p
Confusion matrices for the in silico prediction of the oxidation events of the 7 mAbs and 2 ADCs.
Confusion matrices reports TP, FN on the first row and FP, TN on the second row. Sensitivity calculated as TP/(TP+FN); Specificity calculated as TN/(TN+FP). TP = True Positive, TN = True Negative, FP = False Positive, FN = False Negative. Error for the sensitivity and specificity were estimated from 50 bootstrap replicates.</p
Immunodominance in T cell responses elicited against different domains of detoxified pneumolysin PlyD1
Detoxified pneumolysin, PlyD1, is a protein vaccine candidate that induces protection against infections with Streptococcus pneumoniae in mouse models. Despite extensive knowledge on antibody responses against PlyD1, limited information is available about PlyD1 induced T cell recognition. Here we interrogated epitope breadth and functional characteristics of the T cell response to PlyD1 in two mouse strains. BALB/c (H-2d) and C57BL/6 (H-2b) mice were vaccinated with Al(OH)3-adjuvanted or non-adjuvanted PlyD1, or placebo, on day 0, 21 and 42 and were sacrificed at day 56 for collection of sera and spleens. Vaccination with adjuvanted and non-adjuvanted PlyD1 induced anti-pneumolysin IgG antibodies with neutralizing capacity in both mouse strains. Adjuvantation of PlyD1 enhanced the serological responses in both strains. In vitro restimulation of splenocytes with PlyD1 and 18-mer synthetic peptides derived from pneumolysin revealed specific proliferative and cytokine responses. For both mouse strains, one immunodominant and three subdominant natural epitopes were identified. Overlap between H-2d and H-2b restricted T cell epitopes was limited, yet similarities were found between epitopes processed in mice and predicted to be immunogenic in humans. H-2d restricted T cell epitopes were localized in pneumolysin domains 2 and 3, whereas H-2b epitopes were scattered over the protein. Cytokine responses show mostly a Th2 profile, with low levels of Th1 cytokines, in both mouse strains. In conclusion, PlyD1 evokes T cell responses in mice directed against multiple epitope regions, that is dependent on Major Histocompatibility Complex (MHC) background. These results are important to understand human PlyD1 T cell immunogenicity, to guide cell mediated immunity studies in the context of vaccine development
3D modelling of T cell epitopes in Ply for BALB/c and C57BL/6 mice.
<p>Yellow spheres represent the C-ß atoms of the ID epitope and orange spheres represent the C-ß atoms of the subD epitopes. The dotted circle represents the overlap region between both mouse strains. Colors of the protein structure display the different domains of Ply: cyan indicates domain 1 (amino acids 1–21, 58–147,198–243,319–342), green indicates domain 2 (amino acids 22–57,343–359), red indicates domain 3 (amino acids 148–197,244–318), and purple indicates domain 4 (amino acids 360–469).</p
Ply sequence with localization of BALB/c and C57BL/6 T cell-immunogenic regions and predicted HLA-DR binding regions.
<p>Immunodominant epitopes of both BALB/c and C57BL/6 mice are indicated with a #, and subdominant epitopes with a *. ¶ shows the Epi-bars that are <i>in silico</i> predicted for HLA-DR-binding epitopes in humans. Mutated aa residues of PlyD1 are squared and positions with allelic variation as described by Jeffries <i>et al</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193650#pone.0193650.ref008" target="_blank">8</a>] are underlined. Colors, cyan, green, red and purple, indicate domains 1, 2, 3, and 4 of Ply, respectively.</p
Identified immunodominant and subdominant T cell regions in PlyD1.
<p>Identified immunodominant and subdominant T cell regions in PlyD1.</p
Correlations between Cytotoxicity and Topography of Some 2-Arylidenebenzocycloalkanones Determined by X-ray Crystallography
Determination of ID and subD PlyD1 T cell epitopes.
<p>Splenocytes from mice immunized with PlyD1, PlyD1+Al(OH)<sub>3</sub> or PBS placebo were tested, freshly in BALB/c mice and after thawing in C57BL/6 mice, against PlyD1 and individual peptides from positively tested smart pools. ID regions are peptides with highest proliferation, subD regions are epitopes with subsequent proliferative levels. Proliferation is expressed as mean SI with standard deviation per group. * p value < 0.05 compared to the placebo group. # p value < 0.05 comparing PlyD1 with and without Al(OH)<sub>3</sub>.</p
Serological responses after 3 immunizations with PlyD1.
<p>BALB/c and C57BL/6 mice were immunized 3 times with PlyD1, with or without aluminum hydroxide, or with PBS placebo. Ply-specific antibody titers are shown for total IgG (A), IgG1 (B), IgG2a for BALB/c mice and IgG2c for C57BL/6 mice, respectively (C). Neutralizing capacity of antibodies against hemolytic activity of Ply was determined using HIMA (D). Geometric means of (reciprocal) titers at 50% ODmax with standard deviations are displayed per group.</p