Automated Scoring of Tablet-Administered Expressive Language Tests.

Speech and language impairments are common pediatric conditions, with as many as 10% of children experiencing one or both at some point during development. Expressive language disorders in particular often go undiagnosed, underscoring the immediate need for assessments of expressive language that can be administered and scored reliably and objectively. In this paper, we present a set of highly accurate computational models for automatically scoring several common expressive language tasks. In our assessment framework, instructions and stimuli are presented to the child on a tablet computer, which records the child's responses in real time, while a clinician controls the pace and presentation of the tasks using a second tablet. The recorded responses for four distinct expressive language tasks (expressive vocabulary, word structure, recalling sentences, and formulated sentences) are then scored using traditional paper-and-pencil scoring and using machine learning methods relying on a deep neural network-based language representation model. All four tasks can be scored automatically from both clean and verbatim speech transcripts with very high accuracy at the item level (83-99%). In addition, these automated scores correlate strongly and significantly (ρ = 0.76-0.99, p < 0.001) with manual item-level, raw, and scaled scores. These results point to the utility and potential of automated computationally-driven methods of both administering and scoring expressive language tasks for pediatric developmental language evaluation.

Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFß1 antibody.

Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFß1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFß1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFß1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFß1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies.

Engineering Aglycosylated IgG Variants with Wild-Type or Improved Binding Affinity to Human Fc Gamma RIIA and Fc Gamma RIIIAs.

The binding of human IgG1 to human Fc gamma receptors (hFcγRs) is highly sensitive to the presence of a single N-linked glycosylation site at asparagine 297 of the Fc, with deglycosylation resulting in a complete loss of hFcγR binding. Previously, we demonstrated that aglycosylated human IgG1 Fc variants can engage the human FcγRII class of the low-affinity hFcγRs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFcγR engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with FcγRIIIA, as well as the human Fc gamma RII subset. We also assess the biophysical properties and serum half-life of the aglycosylated IgG variants to measure stability. Aglycosylated constructs N297D/S298T (DTT)-K326I/A327Y/L328G (IYG) and N297D/S298A-IYG optimally drove tumor cell phagocytosis. A mathematical model of phagocytosis suggests that hFcγRI and hFcγRIIIA dimers were the main drivers of phagocytosis. In vivo tumor control of B16F10 lung metastases further confirmed the variant DTT-IYG to be the best at restoring wild-type-like properties in prevention of lung metastases. While deuterium incorporation was similar across most of the protein, several peptides within the CH2 domain of DTT-IYG showed differential deuterium uptake in the peptide region of the FG loop as compared to the aglycosylated N297Q. Thus, in this study, we have found an aglycosylated variant that may effectively substitute for wild-type Fc. These aglycosylated variants have the potential to allow therapeutic antibodies to be produced in virtually any expression system and still maintain effector function.

Structures of a pan-specific antagonist antibody complexed to different isoforms of TGFß reveal structural plasticity of antibody-antigen interactions.

Various important biological pathways are modulated by TGFß isoforms; as such they are potential targets for therapeutic intervention. Fresolimumab, also known as GC1008, is a pan-TGFß neutralizing antibody that has been tested clinically for several indications including an ongoing trial for focal segmental glomerulosclerosis. The structure of the antigen-binding fragment of fresolimumab (GC1008 Fab) in complex with TGFß3 has been reported previously, but the structural capacity of fresolimumab to accommodate tight interactions with TGFß1 and TGFß2 was insufficiently understood. We report the crystal structure of the single-chain variable fragment of fresolimumab (GC1008 scFv) in complex with target TGFß1 to a resolution of 3.00 Å and the crystal structure of GC1008 Fab in complex with TGFß2 to 2.83 Å. The structures provide further insight into the details of TGFß recognition by fresolimumab, give a clear indication of the determinants of fresolimumab pan-specificity and provide potential starting points for the development of isoform-specific antibodies using a fresolimumab scaffold.

Site-specific antibody-drug conjugation through glycoengineering.

Antibody-drug conjugates (ADCs) have been proven clinically to be more effective anti-cancer agents than native antibodies. However, the classical conjugation chemistries to prepare ADCs by targeting primary amines or hinge disulfides have a number of shortcomings including heterogeneous product profiles and linkage instability. We have developed a novel site-specific conjugation method by targeting the native glycosylation site on antibodies as an approach to address these limitations. The native glycans on Asn-297 of antibodies were enzymatically remodeled in vitro using galactosyl and sialyltransferases to introduce terminal sialic acids. Periodate oxidation of these sialic acids yielded aldehyde groups which were subsequently used to conjugate aminooxy functionalized cytotoxic agents via oxime ligation. The process has been successfully demonstrated with three antibodies including trastuzumab and two cytotoxic agents. Hydrophobic interaction chromatography and LC-MS analyses revealed the incorporation of ~1.6 cytotoxic agents per antibody molecule, approximating the number of sialic acid residues. These glyco-conjugated ADCs exhibited target-dependent antiproliferative activity toward antigen-positive tumor cells and significantly greater antitumor efficacy than naked antibody in a Her2-positive tumor xenograft model. These findings suggest that enzymatic remodeling combined with oxime ligation of the native glycans of antibodies offers an attractive approach to generate ADCs with well-defined product profiles. The site-specific conjugation approach presented here provides a viable alternative to other methods, which involve a need to either re-engineer the antibody sequence or develop a highly controlled chemical process to ensure reproducible drug loading.

Recognition of nonverbal communication of emotion after traumatic brain injury.

BACKGROUND: Individuals who have had a traumatic brain injury (TBI) often have difficulty processing nonverbal communication (Ekman, 1976) The published research in this area has focused on a TBI patient's ability to recognize facial expression, vocal intonation, and postural expression (Croker, 2005; Hopkins, Dywan & Segalowitz, 2002). OBJECTIVE: This study compared the non-verbal processing skills of brain-injured patients versus non-injured controls in all three domains. METHODS: The stimuli were photographs of facial and postural expressions and audio recordings of intonational expressions. RESULTS: The results indicated that persons with TBI have particular difficulty recognizing non-verbal communication resulting from vocal intonations. CONCLUSIONS: The TBI patients had difficulty processing tonality, therefore, it is reasonable to suggest that clinicians, friends, and family members should emphasize the explicit verbal content of spoken language when speaking to a person with TBI.

Carbohydrate-mediated polyethylene glycol conjugation of TSH improves its pharmacological properties.

Thyrogen (thyrotropin alfa for injection), recombinant human TSH (rhTSH), has been successfully used to enhance diagnostic radioiodine scanning and thyroglobulin testing in the follow-up of patients with thyroid cancer and as an adjunctive treatment for radioiodine thyroid remnant ablation. However, the short half-life of rhTSH in the circulation requires a multidose regimen. We developed novel sialic acid-mediated and galactose-mediated conjugation chemistries for targeting polyethylene glycol (PEG) to the three N-linked glycosylation sites on the protein, to prolong plasma half-life by eliminating kidney filtration and potential carbohydrate-mediated clearance. Conjugates of different PEG sizes and copy numbers were screened for reaction yield, TSH receptor binding, and murine phamacokinetics/pharmacodynamics studies. The best performing of these products, a 40-kDa mono-PEGylated sialic acid-mediated conjugate, exhibited a 3.5-fold longer duration of action than rhTSH in rats, as a 5-fold lower affinity was more than compensated by a 23-fold extension of circulation half-life. Biochemical characterization confirmed conjugation through the sialic acids. Correlation of PEG distribution on the three N-linked glycosylation sites and the PEG effect on receptor binding supported the previously reported structure-function relationship of rhTSH glycosylation. This long-acting rhTSH has the potential to significantly improve patient convenience and provider flexibility while reducing potential side effects associated with a sudden elevation of serum TSH.

Site-specific PEGylation of human thyroid stimulating hormone to prolong duration of action.

Recombinant human thyroid stimulating hormone (rhTSH or Thyrogen) has been approved for thyroid cancer diagnostics and treatment under a multidose regimen due to its short circulating half-life. To reduce dosing frequency, PEGylation strategies were explored to increase the duration of action of rhTSH. Lysine and N-terminal PEGylation resulted in heterogeneous product profiles with 40% or lower reaction yields of monoPEGylated products. Eleven cysteine mutants were designed based on a structure model of the TSH-TSH receptor (TSHR) complex to create unique conjugation sites on both α and ß subunits for site-specific conjugation. Sequential screening of mutant expression level, oligomerization tendency, and conjugation efficiency resulted in the identification of the αG22C rhTSH mutant for stable expression and scale-up PEGylation. The introduced cysteine in the αG22C rhTSH mutant was partially blocked when isolated from conditioned media and could only be effectively PEGylated after mild reduction with cysteine. This produced a higher reaction yield, ~85%, for the monoPEGylated product. Although the mutation had no effect on receptor binding, PEGylation of αG22C rhTSH led to a PEG size-dependent decrease in receptor binding. Nevertheless, the 40 kDa PEG αG22C rhTSH showed a prolonged duration of action compared to rhTSH in a rat pharmacodynamics model. Reverse-phase HPLC and N-terminal sequencing experiments confirmed site-specific modification at the engineered Cys 22 position on the α-subunit. This work is another demonstration of successful PEGylation of a cysteine-knot protein by an engineered cysteine mutation.

High-affinity VEGF antagonists by oligomerization of a minimal sequence VEGF-binding domain.

Vascular endothelial growth factor (VEGF) neutralizing antagonists including antibodies or receptor extracellular domain Fc fusions have been applied clinically to control angiogenesis in cancer, wet age-related macular degeneration, and edema. We report here the generation of high-affinity VEGF-binding domains by chemical linkage of the second domain of the VEGF receptor Flt-1 (D2) in several configurations. Recombinant D2 was expressed with a 13 a.a. C-terminal tag, including a C-terminal cysteine to enable its dimerization by disulfide bond formation or by attachment to divalent PEGs and oligomerization by coupling to multivalent PEGs. Disulfide-linked dimers produced by Cu(2+) oxidation of the free-thiol form of the protein demonstrated picomolar affinity for VEGF in solution, comparable to that of a D2-Fc fusion (sFLT01) and ~50-fold higher than monomeric D2, suggesting the 26 a.a. tag length between the two D2 domains permits simultaneous interaction of both faces of the VEGF homodimer. Extending the separation between the D2 domains by short PEG spacers from 0.35 kD to 5 kD produced a modest ~2-fold increase in affinity over the disulfide, thus defining the optimal distance between the two D2 domains for maximum affinity. By surface plasmon resonance (SPR), a larger (~5-fold) increase in affinity was observed by conjugation of the D2 monomer to the termini of 4-arm PEG, and yielding a product with a larger hydrodynamic radius than sFLT01. The higher affinity displayed by these D2 PEG tetramers than either D2 dimer or sFLT01 was largely a consequence of a slower rate of dissociation, suggesting the simultaneous binding by these tetramers to neighboring surface-bound VEGF. Finally, disulfide-linked D2 dimers showed a greater resistance to autocatalytic fragmentation than sFLT01 under elevated temperature stress, indicating such minimum-sequence constructs may be better suited for sustained-release formulations. Therefore, these constructs represent novel Fc-independent VEGF antagonists with ultrahigh affinity, high stability, and a range of hydrodynamic radii for application to multiple therapeutic targets.

X-ray and biochemical analysis of N370S mutant human acid ß-glucosidase.

Gaucher disease is caused by mutations in the enzyme acid ß-glucosidase (GCase), the most common of which is the substitution of serine for asparagine at residue 370 (N370S). To characterize the nature of this mutation, we expressed human N370S GCase in insect cells and compared the x-ray structure and biochemical properties of the purified protein with that of the recombinant human GCase (imiglucerase, Cerezyme®). The x-ray structure of N370S mutant acid ß-glucosidase at acidic and neutral pH values indicates that the overall folding of the N370S mutant is identical to that of recombinant GCase. Subtle differences were observed in the conformation of a flexible loop at the active site and in the hydrogen bonding ability of aromatic residues on this loop with residue 370 and the catalytic residues Glu-235 and Glu-340. Circular dichroism spectroscopy showed a pH-dependent change in the environment of tryptophan residues in imiglucerase that is absent in N370S GCase. The mutant protein was catalytically deficient with reduced V(max) and increased K(m) values for the substrate p-nitrophenyl-ß-D-glucopyranoside and reduced sensitivity to competitive inhibitors. N370S GCase was more stable to thermal denaturation and had an increased lysosomal half-life compared with imiglucerase following uptake into macrophages. The competitive inhibitor N-(n-nonyl)deoxynojirimycin increased lysosomal levels of both N370S and imiglucerase 2-3-fold by reducing lysosomal degradation. Overall, these data indicate that the N370S mutation results in a normally folded but less flexible protein with reduced catalytic activity compared with imiglucerase.