Strategies to develop highly drug-tolerant cell-based neutralizing antibody assay: neutralizing antidrug antibodies extraction and drug depletion.

Aim: Drug interference poses great analytical challenges for cell-based neutralizing antidrug antibodies (NAb) assay. The work aimed to improve assay drug tolerance through biotin-drug extraction with acid dissociation method optimization and developing new approach. Results: The NAb extraction with biotin-drug extraction with acid dissociation approach has been optimized by reducing biotinylated drug leaching and improving NAb elution efficiency, resulting in drug tolerance of up to 160 µg/ml. To circumvent the low acid elution efficiency of NAb from drug, a novel drug depletion approach was developed, which combined acid dissociation and drug targeted crosslinked capture, achieved drug tolerance up to 400 µg/ml. At last, a strategy workflow for sample pretreatment approach selection and optimization was established for improving drug tolerance of NAb assay. Conclusion: We demonstrated that reduced biotinylated drug leaching and the high NAb elution efficiency was critical for improving assay drug tolerance. Drug depletion offers an alternative approach to overcome low NAb elution efficiency.

Placental transfer of 125 iodinated humanized immunoglobulin G2Δa in the cynomolgus monkey.

Antibody-like biopharmaceuticals cross the placenta by utilizing existing transport pathways (e.g., FcRn receptor). There are limited data evaluating this transfer during organogenesis in any species. Understanding placental transfer of antibody-like biopharmaceuticals can help to predict risk of developmental toxicity across species, including humans. To complement previously published placental transfer data in the rat with humanized IgGΔ2 (hIgG2), the timing and magnitude of transfer in the cynomolgus monkey and embryo/fetal biodistribution of maternally administered 125 I-radiolabeled hIgG2 was quantified on gestation days (GD) 35, 40, 50, 70, and 140 using gamma counting and whole body autoradiography 24 hr following intravenous injection. Chorioallantoic placental tissues were collected at all time points for Western Blot analysis with anti-FcRn antibody. Maternally administered 125 I-hIgG2 was found in embryo/fetal tissues at all time points, including organogenesis. Embryo/fetal plasma 125 I-hIgG2 concentration increased during gestation, but only slightly up to GD 70 in embryo/fetal tissues, with hIgG2 tissue concentrations generally similar between GD70 and 140. The embryo/fetal:maternal 125 I-hIgG2 plasma concentration ratio was approximately 2.3 fold higher on GD 140, in comparison to ratios on GD 40. Importantly, placental FcRn protein expression was confirmed at all timepoints. These data demonstrate placental transfer of hIgG2 in a nonhuman primate model, and at levels comparable to the rat model, raising the potential for adverse developmental outcomes by direct antibody binding to biological targets.

Addressing soluble target interference in the development of a functional assay for the detection of neutralizing antibodies against a BCMA-CD3 bispecific antibody.

Proper evaluation of immunogenicity during clinical development of biotherapeutics is a major challenge to bioanalytical scientists, in part due to matrix interference in anti-drug antibody (ADA) and neutralizing antibody (NAB) assays. If not addressed, matrix interference could confound the immunogenicity assessment of a given biotherapeutic in clinical development. To support clinical development of a B cell maturation antigen (BCMA)-CD3 bispecific antibody, a cell-based NAB assay was developed as part of a tiered approach to evaluating the immunogenicity of the drug. The assay endpoint (T cell activation) was chosen based on its strong association with the mechanism of action of the drug. The BCMA-CD3 bispecific antibody activates T cells through simultaneous binding of CD3 on T cells and BCMA on target cells. In this system, T cell activation was assessed through the measurement of luciferase activity in an engineered Jurkat cell line. In the presence of NAB, the degree of T cell activation measured by the amount of luciferase activity can be reduced. During method development, soluble BCMA (sBCMA) interference in the NAB assay was apparent. The binding of sBCMA to the anti-BCMA domain of the bispecific drug led to reduced T cell activation, which caused false positive results in NAB testing. To mitigate this interference, several strategies to eliminate sBCMA were investigated. Among the procedures tested, a bead-based approach proved most effective in depleting sBCMA, while maintaining robust assay performance and achieving fit-for-purpose sensitivity. Using this sample pretreatment procedure, the NAB assay tolerated sBCMA up to 2 µg/mL, or approximately four times the estimated median sBCMA concentration in serum samples from patients with active multiple myeloma.

Establishing in vitro in vivo correlations to screen monoclonal antibodies for physicochemical properties related to favorable human pharmacokinetics.

Implementation of in vitro assays that correlate with in vivo human pharmacokinetics (PK) would provide desirable preclinical tools for the early selection of therapeutic monoclonal antibody (mAb) candidates with minimal non-target-related PK risk. Use of these tools minimizes the likelihood that mAbs with unfavorable PK would be advanced into costly preclinical and clinical development. In total, 42 mAbs varying in isotype and soluble versus membrane targets were tested in in vitro and in vivo studies. MAb physicochemical properties were assessed by measuring non-specific interactions (DNA- and insulin-binding ELISA), self-association (affinity-capture self-interaction nanoparticle spectroscopy) and binding to matrix-immobilized human FcRn (surface plasmon resonance and column chromatography). The range of scores obtained from each in vitro assay trended well with in vivo clearance (CL) using both human FcRn transgenic (Tg32) mouse allometrically projected human CL and observed human CL, where mAbs with high in vitro scores resulted in rapid CL in vivo. Establishing a threshold value for mAb CL in human of 0.32 mL/hr/kg enabled refinement of thresholds for each in vitro assay parameter, and using a combinatorial triage approach enabled the successful differentiation of mAbs at high risk for rapid CL (unfavorable PK) from those with low risk (favorable PK), which allowed mAbs requiring further characterization to be identified. Correlating in vitro parameters with in vivo human CL resulted in a set of in vitro tools for use in early testing that would enable selection of mAbs with the greatest likelihood of success in the clinic, allowing costly late-stage failures related to an inadequate exposure profile, toxicity or lack of efficacy to be avoided.

Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies.

Therapeutic antibodies continue to develop as an emerging drug class, with a need for preclinical tools to better predict in vivo characteristics. Transgenic mice expressing human neonatal Fc receptor (hFcRn) have potential as a preclinical pharmacokinetic (PK) model to project human PK of monoclonal antibodies (mAbs). Using a panel of 27 mAbs with a broad PK range, we sought to characterize and establish utility of this preclinical animal model and provide guidance for its application in drug development of mAbs. This set of mAbs was administered to both hemizygous and homozygous hFcRn transgenic mice (Tg32) at a single intravenous dose, and PK parameters were derived. Higher hFcRn protein tissue expression was confirmed by liquid chromatography-high resolution tandem mass spectrometry in Tg32 homozygous versus hemizygous mice. Clearance (CL) was calculated using non-compartmental analysis and correlations were assessed to historical data in wild-type mouse, non-human primate (NHP), and human. Results show that mAb CL in hFcRn Tg32 homozygous mouse correlate with human (r(2) = 0.83, r = 0.91, p < 0.01) better than NHP (r(2) = 0.67, r = 0.82, p < 0.01) for this dataset. Applying simple allometric scaling using an empirically derived best-fit exponent of 0.93 enabled the prediction of human CL from the Tg32 homozygous mouse within 2-fold error for 100% of mAbs tested. Implementing the Tg32 homozygous mouse model in discovery and preclinical drug development to predict human CL may result in an overall decreased usage of monkeys for PK studies, enhancement of the early selection of lead molecules, and ultimately a decrease in the time for a drug candidate to reach the clinic.

Novel molecularly imprinted polymers with carbon nanotube as matrix for selective solid-phase extraction of emodin from kiwi fruit root.

In this paper, we present a novel surface imprinting technique for the preparation of molecularly imprinted polymers/multi-walled carbon nanotubes (MIPs/MWNTs) for extraction of emodin from kiwi fruit root. The MIPs/MWNTs were characterised by scanning electron microscopy (SEM) and Fourier transform-infrared spectroscopy (FT-IR). The properties involving adsorption dynamics, static adsorption, and selective recognition capacity were evaluated. The MIPs/MWNTs exhibited good site accessibility in which it only took 60 min to achieve adsorption equilibrium and highly selective recognition for the template emodin. Furthermore, the performance of the MIPs/MWNTs as solid phase extraction (SPE) material was investigated in detail. The proposed MIPs/MWNTs-SPE procedure for emodin exhibited satisfactory recoveries ranging from 89.2% to 93.8% for real samples. It was used for the purification and enrichment of emodin from kiwi fruit root successfully.

Novel magnetic bovine serum albumin imprinted polymers with a matrix of carbon nanotubes, and their application to protein separation.

Novel magnetic multi-walled carbon nanotubes@Fe(3)O(4) molecularly imprinted polymers (MWNTs@Fe(3)O(4)-MIPs) intended for bovine serum albumin (BSA) recognition were successfully developed. The MWNTs@Fe(3)O(4)-MIPs were characterized with scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT-IR). Scanning electron microscopy images showed that the Fe(3)O(4) nanoparticles (diameter: 50-60 nm) were coated with a layer of MIPs with an average thickness of 25-30 nm. The magnetic material was easily dispersed and retrieved through the application of an external magnetic field. Adsorption experiments showed that the estimated maximum amount of BSA that could be adsorbed onto the MWNTs@Fe(3)O(4)-MIPs was 52.8 mg/g, and the time taken to reach equilibrium was about 40 min. Meanwhile, the MWNTs@Fe(3)O(4)-MIPs exhibited excellent selectivity towards (i.e., recognition of) BSA. The feasibility of the use of the MWNTs@Fe(3)O(4)-MIPs as a solid-phase extraction (SPE) sorbent was evaluated, and the results showed that the MWNTs@Fe(3)O(4)-MIPs were able to separate the template protein BSA from a binary protein solution. The proposed sorbent based on MWNTs@Fe(3)O(4)-MIPs for BSA separation exhibited satisfactory recoveries ranging from 92.0% to 97.3% in real samples. It was also successfully used for the purification of BSA from bovine calf serum.

Novel molecularly imprinted polymers based on multi-walled carbon nanotubes with binary functional monomer for the solid-phase extraction of erythromycin from chicken muscle.

A new surface imprinting technique was reported to synthesize multi-walled carbon nanotubes-molecularly imprinted polymers (MWNTs-MIPs) using erythromycin as the template, acryloyl-ß-cyclodextrin (acryloyl-ß-CD) and methacrylic acid (MAA) as the binary functional monomers. The MWNTs-MIPs were characterized by transmission electron microscopy (TEM), scanning electron micrograph (SEM) and Fourier transform-infrared spectroscopy (FT-IR). Adsorption experiments indicated the MWNTs-MIPs prepared with acryloyl-ß-CD and MAA have high selective for erythromycin. The feasibility of the MWNTs-MIPs as solid-phase extraction (SPE) sorbent was evaluated, and the results showed that it can selectively extract erythromycin from chicken muscle samples with the recoveries ranging from 85.3% to 95.8%. The molecularly imprinted solid-phase extraction (MISPE) method could be applied for preconcentration and purification of erythromycin from chicken muscle samples.

Retinal pigment epithelial cells synthesize laminins, including laminin 5, and adhere to them through alpha3- and alpha6-containing integrins.

PURPOSE: Retinal diseases are often accompanied by changes in the structure of the multilayered extracellular matrix underlying the retina, Bruch's membrane (BrM). These structural revisions potentially lead to alterations in retinal pigment epithelium (RPE) adhesion, likely via modification of interactions with extracellular matrix (ECM) proteins including laminins in BrM. The purpose of this study was to identify specific laminins in BrM and their receptors in RPE cells. METHODS: The laminin composition of BrM was determined using biochemical, molecular biological, and immunohistochemical techniques of rat, bovine, and human tissue and cell lines. An adhesion assay was used to test RPE attachment to laminins and the receptors used for this attachment. RESULTS: BrM contained laminin chains that could form laminin heterotrimers including laminins 1, 5, 10, and 11. RPE cells synthesized these laminin chains in vitro. Therefore, RPE cells may synthesize BrM laminins. The RPE cells preferentially adhered to potential BrM laminins. Although the cells adhered to the BrM component collagen IV, these cells preferentially adhered to laminins. Of the laminins tested, the RPE cells adhered preferentially to laminin 5. The cells interacted with these laminins via specific integrins and attained a different morphology on each laminin. In particular, the RPE cells rapidly attached and flattened on laminin 5. CONCLUSIONS: BrM contains specific laminins, and RPE cells express integrin receptors for those laminins. The interaction of these specific laminins and integrins most likely leads to differential behavior of RPE cells.

Ubiquitin-proteasome pathway function is required for lens cell proliferation and differentiation.

PURPOSE: The ubiquitin proteasome pathway is involved in the regulation of many cellular processes, such as cell cycle control, signal transduction, transcription, and removal of obsolete proteins. The objective of this work was to investigate roles for this proteolytic pathway in controlling the differentiation of lens epithelial cells into lens fibers. METHODS: bFGF-induced cell proliferation was monitored in rat lens epithelial explants by bromodeoxyuridine (BrdU) incorporation. Indicators of lens differentiation included expression of crystallins, lens major intrinsic protein 26 (MIP26), CP49, and filensin and morphologic changes such as cell multilayering and elongation or loss of nuclei. Clasto-lactacystin-beta-lactone, the proteasome-specific inhibitor, was used to study the role of the proteasome in controlling the proliferation and differentiation processes. RESULTS: Explants treated with bFGF initially underwent enhanced proliferation, as indicated by BrdU incorporation and multilayering of the epithelial cells. By 4 days of bFGF treatment, most cells withdrew from the cell cycle, as indicated by diminished BrdU incorporation. After 7 days of treatment with bFGF, lens epithelial explants displayed characteristics of lens fibers, including higher ratios of crystallins to other cytoplasmic proteins and expression of large quantities of MIP26, CP49, and filensin. Adding the proteasome inhibitor to the medium simultaneously with bFGF (day 0) or at day 4 prohibited or delayed bFGF-induced cell proliferation and differentiation. This was indicated by reduced BrdU incorporation and decreased expression of beta- and gamma-crystallins, MIP26, CP49, and filensin. Proteasome inhibition also significantly decreased the number of layers and the sizes of differentiating fibers. CONCLUSIONS: These data show that proteasome activity is required not only for lens cell proliferation but also required for the transition from the epithelial phenotype to the fiber phenotype.

The extracellular matrix component WIF-1 is expressed during, and can modulate, retinal development.

We have shown previously that components of the extracellular matrix (ECM) modulate neuronal development. Here, we searched for additional ECM elements that might play roles in retinal histogenesis and identified a secreted glycoprotein that is heavily expressed in the retina. This molecule, named by others Wnt Inhibitory Factor-1 (WIF-1), is expressed during and after the period of rod photoreceptor morphogenesis in the mouse. We show that a potential WIF-1 ligand, Wnt4, as well as a potential Wnt4 receptor, fzd4, and a potential Wnt4 coreceptor, LRP6, are expressed in the region of, and at the time of, rod photoreceptor genesis. WIF-1 and Wnt4 are coexpressed during retinal development and bind to each other; therefore, they are likely to interact during rod production. WIF-1 protein inhibits rod production, and anti-WIF-1 antibodies increase rod production; in contrast, Wnt4 promotes rod production. Together, these data suggest that WIF-1 and Wnt4, both components of the ECM, regulate mammalian photoreceptor development.