High resolution voltammetric and field-effect transistor readout of carbon fiber microelectrode biosensors.

Rapid and sensitive pH measurements with increased spatiotemporal resolution are imperative to probe neurochemical signals and illuminate brain function. We interfaced carbon fiber microelectrode (CFME) sensors with both fast scan cyclic voltammetry (FSCV) and field-effect transistor (FET) transducers for dynamic pH measurements. The electrochemical oxidation and reduction of functional groups on the surface of CFMEs affect their response over a physiologically relevant pH range. When measured with FET transducers, the sensitivity of the measurements over the measured pH range was found to be (101 ± 18) mV, which exceeded the Nernstian value of 59 mV by approximately 70%. Finally, we validated the functionality of CFMEs as pH sensors with FSCV ex vivo in rat brain coronal slices with exogenously applied solutions of varying pH values indicating that potential in vivo study is feasible.

Improvement of Biophysical Properties and Affinity of a Human Anti-L1CAM Therapeutic Antibody through Antibody Engineering Based on Computational Methods.

The biophysical properties of therapeutic antibodies influence their manufacturability, efficacy, and safety. To develop an anti-cancer antibody, we previously generated a human monoclonal antibody (Ab417) that specifically binds to L1 cell adhesion molecule with a high affinity, and we validated its anti-tumor activity and mechanism of action in human cholangiocarcinoma xenograft models. In the present study, we aimed to improve the biophysical properties of Ab417. We designed 20 variants of Ab417 with reduced aggregation propensity, less potential post-translational modification (PTM) motifs, and the lowest predicted immunogenicity using computational methods. Next, we constructed these variants to analyze their expression levels and antigen-binding activities. One variant (Ab612)-which contains six substitutions for reduced surface hydrophobicity, removal of PTM, and change to the germline residue-exhibited an increased expression level and antigen-binding activity compared to Ab417. In further studies, compared to Ab417, Ab612 showed improved biophysical properties, including reduced aggregation propensity, increased stability, higher purification yield, lower pI, higher affinity, and greater in vivo anti-tumor efficacy. Additionally, we generated a highly productive and stable research cell bank (RCB) and scaled up the production process to 50 L, yielding 6.6 g/L of Ab612. The RCB will be used for preclinical development of Ab612.

An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage.

Targeting the molecular pathways underlying the cardiotoxicity associated with thoracic irradiation and doxorubicin (Dox) could reduce the morbidity and mortality associated with these anticancer treatments. Here, we find that vascular endothelial cells (ECs) with persistent DNA damage induced by irradiation and Dox treatment exhibit a fibrotic phenotype (endothelial-mesenchymal transition, EndMT) correlating with the colocalization of L1CAM and persistent DNA damage foci. We demonstrate that treatment with the anti-L1CAM antibody Ab417 decreases L1CAM overexpression and nuclear translocation and persistent DNA damage foci. We show that in whole-heart-irradiated mice, EC-specific p53 deletion increases vascular fibrosis and the colocalization of L1CAM and DNA damage foci, while Ab417 attenuates these effects. We also demonstrate that Ab417 prevents cardiac dysfunction-related decrease in fractional shortening and prolongs survival after whole-heart irradiation or Dox treatment. We show that cardiomyopathy patient-derived cardiovascular ECs with persistent DNA damage show upregulated L1CAM and EndMT, indicating clinical applicability of Ab417. We conclude that controlling vascular DNA damage by inhibiting nuclear L1CAM translocation might effectively prevent anticancer therapy-associated cardiotoxicity.

Optimal field-effect transistor operation for high-resolution biochemical measurements.

Field-effect transistors (FETs) are powerful tools for sensitive measurements of numerous biomarkers (e.g., proteins, nucleic acids, and antigen) and gaseous species. Most research studies in this field focused on building discrete devices with high performance. We show that instrumentation that is commonly used in multiple areas of physics and engineering can greatly improve the performance of measurement systems that embed FET-based transducers for biological applications. We review the state-of-the-art instrumentation in the field as applied to sensing with FETs. We show how high-performance dual-gate 2D FETs that we recently developed, when operated using closed-loop proportional-integral-derivative control, can drastically improve both the sensitivity and resolution. We further show that this closed-loop control approach can be extended to commonly used single-gate silicon FETs. The generalizability of the results will allow their application to virtually any previously developed FET-based sensor. Finally, we provide insight into further optimization and performance benefits that can be extracted by using the closed-loop feedback approach for applications in biosensing.

Generation, Diversity Determination, and Application to Antibody Selection of a Human Naïve Fab Library.

We constructed a large naïve human Fab library (3 × 1010 colonies) from the lymphocytes of 809 human donors, assessed available diversities of the heavy-chain variable (VH) and κ light-chain variable (VK) domain repertoires, and validated the library by selecting Fabs against 10 therapeutically relevant antigens by phage display. We obtained a database of unique 7,373 VH and 41,804 VK sequences by 454 pyrosequencing, and analyzed the repertoires. The distribution of VH and VK subfamilies and germline genes in our antibody repertoires slightly differed from those in earlier published natural antibody libraries. The frequency of somatic hypermutations (SHMs) in heavy-chain complementarity determining region (HCDR)1 and HCDR2 are higher compared with the natural IgM repertoire. Analysis of position-specific SHMs in CDRs indicates that asparagine, threonine, arginine, aspartate and phenylalanine are the most frequent non-germline residues on the antibody-antigen interface and are converted mostly from the germline residues, which are highly represented in germline SHM hotspots. The amino acid composition and length-dependent changes in amino acid frequencies of HCDR3 are similar to those in previous reports, except that frequencies of aspartate and phenylalanine are a little higher in our repertoire. Taken together, the results show that this antibody library shares common features of natural antibody repertoires and also has unique features. The antibody library will be useful in the generation of human antibodies against diverse antigens, and the information about the diversity of natural antibody repertoires will be valuable in the future design of synthetic human antibody libraries with high functional diversity.

Combination of anti-L1 cell adhesion molecule antibody and gemcitabine or cisplatin improves the therapeutic response of intrahepatic cholangiocarcinoma.

Cholangiocarcinoma has a poor prognosis and is refractory to conventional chemotherapy and radiation therapy. Improving survival of patients with advanced cholangiocarcinoma urgently requires the development of new effective targeted therapies in combination with chemotherapy. We previously developed a human monoclonal antibody (mAb) Ab417 that binds to both the human and mouse L1 cell adhesion molecule (L1CAM) with high affinities. In the present study, we observed that Ab417 exhibited tumor targeting ability in biodistribution studies and dose-dependent tumor growth inhibition in an intrahepatic cholangiocarcinoma (Choi-CK) xenograft mouse model. Regarding the mechanism of action, Ab417 was internalized into the tumor cells and thereby down-regulated membrane L1CAM, and inhibited tumor growth by reducing tumor cell proliferation in vivo. Gemcitabine inhibited the tumor growth in a dose-dependent manner in the Choi-CK xenograft model. However, cisplatin inhibited the tumor growth moderately and not in a dose-dependent way, suggesting that the tumors may have developed resistance to apoptosis induced by cisplatin. Combined treatment with Ab417 and gemcitabine or cisplatin exerted enhanced tumor growth inhibition compared to treatment with antibody or drug alone. The results suggest that Ab417 in combination with chemotherapy may have potential as a new therapeutic regimen for cholangiocarcinoma. Our study is the first to show an enhanced therapeutic effect of a therapeutic antibody targeting L1CAM in combination with chemotherapy in cholangiocarcinoma models.

Use of hybrid composite particles prepared using alkoxysilane-functionalized amphiphilic polymer precursors for simultaneous removal of various pollutants from water.

In this study, we present new inorganic-organic hybrid particles and their possible application as an adsorbent for simultaneous removal of hydrophobic and hydrophilic pollutants from water. These hybrid particles were prepared using tailor-made alkoxysilane-functionalized amphiphilic polymer precursors (M-APAS), which have amphiphilic polymers and reactive alkoxysilane groups attached to the same backbone. Through a single conventional sol-gel process, the polymerization of M-APAS and the chemical conjugation of M-APAS onto silica nanoparticles was simultaneous, resulting in the formation of hybrid particles (M-APAS-SiO2) comprised of hyperbranch-like amphiphilic polymers bonded onto silica nanoparticles with a relatively high grafting efficiency. A test for the adsorption of water-soluble dye (organe-16) and water insoluble dye (solvent blue-35) onto the hybrid particles was performed to evaluate the possibility of adsorbing hydrophilic and hydrophobic compound within the same particle. The hybrid particle was also evaluated as an adsorbent for the removal of contaminated water containing various pollutants by wastewater treatment test. The hybrid particle could remove phenolic compounds from wastewater and the azo dye reactive orange-16 from aqueous solutions, and it was easily separated from the treated wastewater because of the different densities involved. These results demonstrate that the hybrid particles are a promising sorbent for hydrophilic and/or hydrophobic pollutants in water.

Generation, characterization and preclinical studies of a human anti-L1CAM monoclonal antibody that cross-reacts with rodent L1CAM.

L1 cell adhesion molecule (L1CAM) is aberrantly expressed in malignant tumors and plays important roles in tumor progression. Thus, L1CAM could serve as a therapeutic target and anti-L1CAM antibodies may have potential as anticancer agents. However, L1CAM is expressed in neural cells and the druggability of anti-L1AM antibody must be validated at the earliest stages of preclinical study. Here, we generated a human monoclonal antibody that is cross-reactive with mouse L1CAM and evaluated its pharmacokinetic properties and anti-tumor efficacy in rodent models. First, we selected an antibody (Ab4) that binds human and mouse L1CAM from the human naïve Fab library using phage display, then increased its affinity 45-fold through mutation of 3 residues in the complementarity-determining regions (CDRs) to generate Ab4M. Next, the affinity of Ab4M was increased 1.8-fold by yeast display of single-chain variable fragment containing randomly mutated light chain CDR3 to generate Ab417. The affinities (KD) of Ab417 for human and mouse L1CAM were 0.24 nM and 79.16 pM, respectively. Ab417 specifically bound the Ig5 domain of L1CAM and did not exhibit off-target activity, but bound to the peripheral nerves embedded in normal human tissues as expected in immunohistochemical analysis. In a pharmacokinetics study, the mean half-life of Ab417 was 114.49 h when a single dose (10 mg/kg) was intravenously injected into SD rats. Ab417 significantly inhibited tumor growth in a human cholangiocarcinoma xenograft nude mouse model and did not induce any adverse effect in in vivo studies. Thus, Ab417 may have potential as an anticancer agent.