Hum-αD11 preserves the <i>in vitro</i> binding properties of the parental mAb αD11.

<p>(a) ELISA assay with mNGF coating (5 µg/ml); serial dilutions of parental mAb αD11, chimeric IgG1 αD11 and IgG1 hum-αD11, Protein A-Sepharose purified from transiently cotransfected CHO cells supernatants. (b) Binding curves of a range of concentrations (0.29–37.5 nM) of hNGF to immobilized parental mAb αD11 (immobilization level 1280.0 RU). (c) Binding curves of a range of concentrations (0.39–25.0 nM) of Fab hum-αD11 to immobilized hNGF (immobilization level 100.3 RU).</p

Data_Sheet_1_Cerebrospinal fluid level of proNGF as potential diagnostic biomarker in patients with frontotemporal dementia.docx

IntroductionFrontotemporal dementia (FTD) is an extremely heterogeneous and complex neurodegenerative disease, exhibiting different phenotypes, genetic backgrounds, and pathological states. Due to these characteristics, and to the fact that clinical symptoms overlap with those of other neurodegenerative diseases or psychiatric disorders, the diagnosis based only on the clinical evaluation is very difficult. The currently used biomarkers help in the clinical diagnosis, but are insufficient and do not cover all the clinical needs.MethodsBy the means of a new immunoassay, we have measured and analyzed the proNGF levels in 43 cerebrospinal fluids (CSF) from FTD patients, and compared the results to those obtained in CSF from 84 Alzheimer’s disease (AD), 15 subjective memory complaints (SMC) and 13 control subjects.ResultsA statistically significant difference between proNGF levels in FTD compared to AD, SMC and controls subjects was found. The statistical models reveal that proNGF determination increases the accuracy of FTD diagnosis, if added to the clinically validated CSF biomarkers.DiscussionThese results suggest that proNGF could be included in a panel of biomarkers to improve the FTD diagnosis.</p

Hum-αD11 retains the biological activity of the parental mAb αD11 <i>in vitro</i>.

<p>(a–d) mNGF induced differentiation of PC-12 cells. Neurite outgrowth inhibition assay: photomicrographs of PC-12 cells, treated with (a) mNGF 100 ng/ml alone or preincubated (b) with mAb αD11 (5 µg/ml) or (c) with IgG1 hum-αD11 (5 µg/ml), concentrated from stable cotransfected CHO cells supernatants. (d) Negative control: untreated PC-12 cells. (e) TrkA phosphorylation inhibition assay in 3T3 TrkA cells. 3T3 TrkA cells were incubated with the indicated combinations of mNGF, parental mAb αD11, IgG1 hum-αD11 (purified by Protein A-Sepharose) and the irrelevant mAb SV5 as a negative control. Cell lysates were separated on a 10% SDS gel and phosphorylated TrkA detected using anti-phospho-Y490 TrkA Ab. The ubiquitous band of tubulin served as gel loading control.</p

Selection of acceptor human FWRs for mAb αD11 humanization.

<p>Deviations of parental Fab αD11 from each of the selected human and humanized antibody, taking into account the sequence alignment and structural comparison (calculated considering both the overall sequence homology and identity percentage and the percentage of sequence homology and identity restricted to FWRs).</p

Primary structure comparison.

<p>Sequence alignment of V_κ (A) and V_H (B), respectively of parental Fab αD11 (PDB_ID: 1ZAN) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032212#pone.0032212-Covaceuszach1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032212#pone.0032212-Covaceuszach2" target="_blank">[27]</a> with the selected template for humanization (PDB_ID: 1JPS) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032212#pone.0032212-Faelber1" target="_blank">[38]</a> and hum-αD11, obtained by CDRs grafting (highlighted in yellow) on PDB_ID: 1JPS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032212#pone.0032212-Faelber1" target="_blank">[38]</a> FWRs (highlighted in magenta) with the retro-mutations (highlighted in green) and the mutation (highlighted in cyan) . The six CDRs are underlined and the residues belonging to the Vernier zones are colored in red. The residues numbering is according to Kabat <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032212#pone.0032212-Kabat1" target="_blank">[39]</a>.</p

<i>In vivo</i> analgesic activity of parental mAb αD11 in inflammatory and neuropathic pain models.

<p>(a) Analgesic effects of αD11 antibody administration on the late phase (15–40 min) in the course of the formalin test. Treatment consisted in saline (negative control) or antibody injection (single doses: 12.5 µg of mock mouse mAb or two different molecular formats of αD11, <i>i.e.</i> mAb or Fab) performed (in the same paw as for formalin) 45 min before formalin injection and testing. Statistical analysis was performed on each phase (ANOVA and Fisher's Test for comparison of each couple of groups). Each experimental group included at least 8 animals. (b) Analgesic effects of αD11 antibody in the short lasting protocol of neuropathic pain model: mAb αD11 significantly increased the value of ipsilateral/contralateral index (ratio between the threshold forces measured for the two hind paws, the one ipsilateral to surgery and the contralateral one. Mean value ± s.e.), starting from day 4 to day 14, one week after the last antibody injection. Control mice were injected with either mock mouse IgG, (1.4 mg/Kg) or saline solution (sal). ANOVA test for repeated measures resulted in statistical significance for treatment (p<0.0001), time (p<0.0001) and the interaction between the two factors (treatment×time) (p<0.0001). (c) Analgesic efficacy of mAb αD11 (one dose: 2 mg/kg) in the long lasting protocol of neuropathic pain model. MAb αD11 increased the ipsilateral/contralateral index, starting either from day 5. The analgesic effect, which disappeared around days 17–19, increases again to reach a plateau between day 27 and day 31, identifying a late phase in the action of mAb αD11 (long-term effect). ANOVA test for repeated measures resulted in statistical significance for treatment (p<0.005), time (p<0.005) and the interaction between two factors (treatment×time) (p<0.005).</p

SPR analysis.

<p>Summary of the derived kinetic and equilibrium binding constants of parental IgG αD11 and Fab hum-αD11 towards hNGF.</p

Phi-X derived and Phred score derived error rate distribution.

<p>A) Phred score error rate distribution for the hscFv1 library of the merged reads. Error rate increases with sequencing cycles. B) Control Phi-X derived error rate distribution for the hscFv1 library of the merged reads. Error rate is more prominent in the early sequencing cycles (spikes), with a small increase at the end of each read. The error distribution does not match the Phred score distribution and the shape differs as well. C) Scatter plot of the correlation of Q-score and log₂(% Mismatches) in Phi-x control spike-in library. Each point represents the mean value from a single flow cell tile at a given sequencing read number, encoded by colour (red to blue: R1 cycle 1 to 350; R2 cycle 1 to 250; colour flex point is set at cycle 38). The Q score in the first 40 reads fails to be predictive of mismatch rate. Similar results were obtained for hscFv2 and hVH libraries.</p

Length distribution of human VH nanobody library sequences.

<p>Barplot of the length distribution of human VH nanobody library sequences coloured by reading frame.</p

Diagram of sequenced adaptor-antibody-adaptor constructs.

<p>A) scFv library (gray), comprising heavy (VH) and light chain (VL) Complementary Determining Regions (CDR), was ligated to adapters (light green and pink) harbouring Illumina P5 and P7 flowcell hybridization sequences (green and red). B) VH nanobody library (gray), comprising heavy chain (VH) Complementary Determining Regions (CDR), was ligated to adapters (light green and pink) harbouring Illumina P5 and P7 flowcell hybridization sequences (green and red). The forward read (R1) uses SBS3 sequencing primer (Illumina), while the reverse read (R2) uses SBS12 primer (Illumina). iS1 and iS2 = index/shifter sequences.</p