17 research outputs found
MOESM2 of Enhancing full-length antibody production by signal peptide engineering
Additional file 2: Table S2. Strains and plasmids used in this study
MOESM3 of Enhancing full-length antibody production by signal peptide engineering
Additional file 3: Figure S1. Western blot analysis of light chain secretion
Reorienting the Fab Domains of Trastuzumab Results in Potent HER2 Activators
<div><p>The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(abā²)<sub>2</sub>-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs) of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules.</p> </div
Agonist activity of a trastuzumab isomer.
<p>(a) A brief description of the bis-Fab synthesis process is illustrated here. The thio-Fabs of interest are produced by engineering an unpaired cysteine into the Fab region of an antibody followed by recombinant expression and isolation of the thio-Fab containing the unpaired cysteine. A homobifunctional crosslinking reagent is then used in a two-step process to efficiently couple two thio-Fabs together. A more detailed explanation of the procedure can be found in the Experimental Methods and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051817#pone.0051817.s001" target="_blank">Figure S1</a></b>. (b) A matrix combination of thioFabs using the two-step synthesis process was used to produce a panel of bis-Fab molecules for use in biological and biochemical assays. Two Fabs targeting EGFR (Ī±-HER1-a targets domain III and Ī±-HER1-b targets domain III) and two Fabs targeting HER2 (Ī±-HER2-a derived from trastuzumab targets domain IV and Ī±-HER2-b derived from pertuzumab targets domain II) were used to construct the matrix. (c) Trastuzumab and bis-Fab 1321, consisting of two trastuzumab Fabs linked together at position 110 in the light chain, were examined for their effect on cell growth. Increasing concentrations of trastuzumab (blue line) or bis-Fab 1321 (orange line) were added to BT474 cells and cell proliferation was measured after five days using AlamarBlue staining. The relative fluorescence units are reported for the different treatment concentrations. Individual data points for two independent experiments are shown in the plot as well as an average of the two, which are represented by the lines and open shapes. Trastuzumab analog (bis-Fab 1321) showed agonistic activity as measured by increased cell proliferation, whereas trastuzumab inhibited cell proliferation. (d) The schematic illustrates the site of covalent attachment between the Fabs of the parent antibody trastuzumab and bis-Fab 1321. Although the global conformations of the Fab domains are unknown, this figure highlights the distinct difference in the points of connection. The native interchain disulfides (hinge region, near HC-228) in the heavy chains of trastuzumab provided the covalent attachment site for the Fab arms in the antibody. In contrast, bis-Fab 1321 was covalently linked through light chains at LC-110 using a bis-maleimido crosslinker. The resultant molecules presented Fab Fv-regions in different relative orientations.</p
Changes in HER2 phosphorylation are associated with agonist activity.
<p>(a) This panel compares phosphorylation differences at fifteen sites in HER2 after treatment with either the agonist bis-Fab 1325 or trastuzumab. The top panel shows differences between agonist treatment and no treatment (basal phosphorylation). Seven of the sites showed statistically significant increases in phosphorylation (red dots above the line), five sites showed no change, and one site decreased. The same comparison is made between trastuzumab (antagonist) and no treatment shown in the second panel. A third comparison is made between bis-Fab 1325 and trastuzumab. Here, sites that show statistically significant differences between the treatments are indicated by red dots. Those phosphorylation sites that are significantly higher than trastuzumab treatment are highlighted with green background. The results were analyzed with a Tukey-Kramer all-pairwise test and the error bars indicate the 95% confidence level. Methods used are described in detail in the Supplementary Information. <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051817#pone.0051817.s005" target="_blank">Figure S5</a></b> shows the raw data consisting of nine mass spec measurements for each treatment and phosphorylation site. Red color is used to indicate a statistically significant difference between the two samples with a P-value of <0.05. (b) This illustration of the HER2 intracellular domains shows phosphorylation sites identified by mass spectrometry. Phosphorylation sites colored orange denote sites with measureable increases, blue for decreases and uncolored for no change after agonist bis-Fab 1325 treatment. Phosphorylation sites that are significantly higher in agonist treatments compared to trastuzumab treatments are indicated by arrows.</p
Quantification of HER2 phosphorylation and the differences after treatment.
*<p>Phosphorylation sites were identified by HER2 phosphomapping.</p>ā <p>Quantitative mass spectrometry (MS) was done to determine the basal level of phosphorylation at each site. The absolute amount of phosphorylated peptide was compared to the total of phosphorylated and non-phosphorylated peptides to calculate the percent phosphorylation (% Phosph.)</p>ā”<p>Ten minutes after treatment with the indicated molecule, quantitative MS was done to determine the % phosphorylation. This was subtracted from the % phosphorylation in the basal state to provide the mean difference after treatment reported here. This time point was chosen because maximal phosphorylation of AKT was shown to occur within ten minutes after agonist treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051817#pone-0051817-g004" target="_blank"><b>Figure 4</b></a>).</p
Trastuzumab bis-Fab structural analogs show a spectrum of cell-proliferation activities.
<p>(a) Four different thio-Fabs derived from trastuzumab were used to make ten bis-Fab analogs. Each thio-Fab mutant (LC-110, LC-205, HC-118, and HC-228) was reacted with the bis-maleimido crosslinker and recombined in a matrix format described in the Experimental Methods. Fabs were derived from several sources shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051817#pone.0051817.s002" target="_blank">Figures S2A</a></b> and S<b>2B</b>. Each chain of the Fab is represented by a different color (dark blue - heavy chain and light blue - light chain) and the position of the cysteine used in coupling is denoted by the red dot. (b) The matrix-generated bis-Fab linkage analogs are shown diagrammatically in this Figure. Color coded numbers indicate the type of activity observed, where tan signifies antagonist, dark blue signifies agonist, and light blue signifies no activity. (c) BT474 cells were incubated with the indicated concentrations of bis-Fabs shown in (b). The degree of cell proliferation was assessed after 5 days using AlamarBlue staining. The results are reported as a percentage of maximum proliferation relative to untreated controls. The measured values for each test sample, as well as individual replicates, are shown as raw data in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051817#pone.0051817.s003" target="_blank">Figure S3</a></b>. Color codes are the same as in (b). (d) A model of the complex formation between two HER2 extracelluar domains (ECD) and either an agonist (1321) or antagonist (1324) bis-Fab. Here is shown two light chain connected bis-Fabs with the heavy chain colored dark blue, the light chains colored lighter blue and the HER2 ECD colored magenta. In the left panel the two complexes are shown looking up at the membrane. The point of contact between the Fv-region of the Fab and the ECD is near the membrane. The HER2 protein terminates in this structure just prior to the point at which the transmembrane domain begins. The complex models are rotated 90 degrees on both the horizontal and vertical axis to produce this viewpoint. The plane of the membrane runs perpendicular to the page. The PBD ID Code used for model building was 1N8Z.</p
Cell-surface binding and solution state properties of HER2 agonists and antagonists.
ā <p>All molecules in this table are derived from trastuzumab. LCā=ālight chain; HCā=āheavy chain. Numbers indicate the cysteine substitution position.</p>ā”<p>Activity was determined by cell proliferation assays using BT474 cells.</p><p><b><i>R<sub>hyd</sub></i></b>ā=āhydrodynamic radius determined by size exclusion chromatography coupled-light scattering.</p><p>Standard deviation is represented by +/ā values in the table.</p
Analysis of bis-Fab agonist activity in BT474 cells.
<p>(a) A time course of cell growth activity in BT474 cells in the presence of 100 nM trastuzumab, 100 nM bis-Fab 1325, or 10 nM heregulin. BT474 were cultured in media containing 10% fetal bovine serum for up to 84 hours. At 12-hour intervals total number of cells were determined; three plates from each treatment group were counted for the total number of cells and plotted as the mean cell count. The error bars indicate standard deviation from the mean. At approximately 60 hours (indicated by the arrow) the cells reached confluence in the agonist treatment groups. (b) BT474 cells were treated with 100 nM of trastuzumab, 100 nM of bis-Fab 1325, or 100 nM of bis-Fab 1329 for 10, 30 and 120 minutes. At times indicated, cell lysates were prepared and analyzed by immunoblotting using phospho-specific antibodies for HER3, AKT, and MAPK as well as antibodies recognizing total protein. Data are representative of three independent experiments. (c) Quantification of AKT phosphorylation after treatment with 100 nM bis-Fab 1325, 100 nM trastuzumab, 2 nM heregulin and a non-specific control antibody (anti-gD) by PathScan p-AKT1 (S473) ELISA. All data points were collected in triplicates and the mean of the triplicate absorbance values were used to calculate the percent change in pAKT compared to untreated control group. The error bars indicate standard deviation from the mean. Data are representative of three independent experiments. (d) A model for the HER2 dimerization patterns induced by either the agonist antibody-analogs or trastuzumab. The diagram depicts three potential dimer conformations; 1) the basal state induced by high cell surface density, 2) the activated state induced by the bis-Fab agonist, and 3) the inhibited state stabilized by trastuzumab. The cell growth activity of agonist bis-Fabs may be due to stabilization of an allosterically activated conformation between HER2āHER2 dimers. Trastuzumab's antagonistic activity may arise from dimer orientations that favor the inactive allosteric interactions between kinases. A non-stabilized dimer may represent the basal state where interactions between the juxtamembrane (black line) loop of the activator kinase and the C-terminal lobe of the receiver kinase are not fully stabilized without agonist binding.</p
Structural and Functional Characterization of a HoleāHole Homodimer Variant in a āKnob-Into-Holeā Bispecific Antibody
Bispecific
antibodies have great potential to be the next-generation
biotherapeutics due to their ability to simultaneously recognize two
different targets. Compared to conventional monoclonal antibodies,
knob-into-hole bispecific antibodies face unique challenges in production
and characterization due to the increase in variant possibilities,
such as homodimerization in covalent and noncovalent forms. In this
study, a storage- and pH-sensitive hydrophobic interaction chromatography
(HIC) profile change was observed for the holeāhole homodimer,
and the multiple HIC peaks were explored and shown to be conformational
isomers. We combined traditional analytical methods with hydrogen/deuterium
exchange mass spectrometry (HDX MS), native mass spectrometry, and
negative-staining electron microscopy to comprehensively characterize
the holeāhole homodimer. HDX MS revealed conformational changes
at the resolution of a few amino acids overlapping the C<sub>H</sub>2-C<sub>H</sub>3 domain interface. Conformational heterogeneity was
also assessed by HDX MS isotopic distribution. The holeāhole
homodimer was demonstrated to adopt a more homogeneous conformational
distribution during storage. This conformational change is likely
caused by a lack of C<sub>H</sub>3 domain dimerization (due to the
three āholeā point mutations), resulting in a unique
storage- and pH-dependent conformational destabilization and refolding
of the holeāhole homodimer Fc. Compared with the holeāhole
homodimer under different storage conditions, the bispecific heterodimer,
guided by the knob-into-hole assembly, proved to be a stable conformation
with homogeneous distribution, confirming its high quality as a desired
therapeutic. Functional studies by antigen binding and neonatal Fc
receptor (FcRn) binding correlated very well with the structural characterization.
Comprehensive interpretation of the results has provided a better
understanding of both the homodimer variant and the bispecific molecule