Bacterial production and biophysical characterization of a hard-to-fold scFv against myeloid leukemia cell surface marker, IL-1RAP.

BACKGROUND: Interleukin-1 receptor accessory protein (IL-1RAP) is one of the most promising therapeutic targets proposed for myeloid leukemia. Antibodies (Abs) specific to IL-1RAP could be valuable tools for targeted therapy of this lethal malignancy. This study is about the preparation of a difficult-to-produce single-chain variable fragment (scFv) construct against the membrane-bound isoform of human IL-1RAP using Escherichia coli (E. coli). METHODS: Different approaches were examined for refolding and characterization of the scFv. Binding activities of antibody fragments were comparatively evaluated using cell-based enzyme-linked immunosorbent assay (ELISA). Homogeneity and secondary structure of selected scFv preparation were analyzed using analytical size exclusion chromatography (SEC) and circular dichroism (CD) spectroscopy, respectively. The activity of the selected preparation was evaluated after long-term storage, repeated freeze-thaw cycles, or following incubation with normal and leukemic serum. RESULTS: Strategies for soluble expression of the scFv failed. Even with the help of Trx, ≥ 98% of proteins were expressed as inclusion bodies (IBs). Among three different refolding methods, the highest recovery rate was obtained from the dilution method (11.2%). Trx-tag substantially enhanced the expression level (18%, considering the molecular weight (MW) differences), recovery rate (˃1.6-fold), and binding activity (˃2.6-fold increase in absorbance450nm). The produced scFv exhibited expected secondary structure as well as acceptable bio-functionality, homogeneity, and stability. CONCLUSION: We were able to produce  21 mg/L culture functional and stable anti-IL-1RAP scFv via recovering IBs by pulse dilution procedure. The produced scFv as a useful targeting agent could be used in scheming new therapeutics or diagnostics for myeloid malignancies.

Targeted integration in CHO cells using CRIS-PITCh/Bxb1 recombinase-mediated cassette exchange hybrid system.

Recombinant Chinese hamster ovary (CHO) cell line development for complex biotherapeutic production is conventionally based on the random integration (RI) approach. Due to the lack of control over the integration site and copy number, RI-generated cell pools are always coupled with rigorous screening to find clones that satisfy requirements for production titers, quality, and stability. Targeted integration into a well-defined genomic site has been suggested as a possible strategy to mitigate the drawbacks associated with RI. In this work, we employed the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system in combination with the Bxb1 recombinase-mediated cassette exchange (RMCE) system to generate an isogenic transgene-expressing cell line. We successfully utilized the CRIS-PITCh system to target a 2.6 kb Bxb1 landing pad with homology arms as short as 30 bp into the upstream region of the S100A gene cluster, achieving a targeting efficiency of 10.4%. The platform cell line (PCL) with a single copy of the landing pad was then employed for the Bxb1-mediated landing pad exchange with an EGFP encoding cassette to prove its functionality. Finally, to accomplish the main goal of our cell line development method, the PCL was applied for the expression of a secretory glycoprotein, human recombinant soluble angiotensin-converting enzyme 2 (hrsACE2). Taken together, on-target, single-copy, and stable expression of the transgene over long-term cultivation demonstrated our CRIS-PITCh/RMCE hybrid approach might possibly improve the cell line development process in terms of timeline, specificity, and stability. KEY POINTS: • CRIS-PITCh system is an efficient method for single copy targeted integration of the landing pad and generation of platform cell line • Upstream region of the S100A gene cluster of CHO-K1 is retargetable by recombinase-mediated cassette exchange (RMCE) approach and provides a stable expression of the transgene • CRIS-PITCh/Bxb1 RMCE hybrid system has the potential to overcome some limitations of the random integration approach and accelerate the cell line development timeline.

Palliative care in amyotrophic lateral sclerosis clinics: A survey of NEALS consortium membership.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative condition managed with a multidisciplinary approach. Palliative care is important for this approach, but there is a lack of recommendations for the role and involvement of palliative medicine (PM) specialists in multidisciplinary ALS care. METHODS: This questionnaire-based survey assessed the state of palliative care in selective sites that are a part of Northeast Amyotrophic Lateral Sclerosis Consortium (NEALS). RESULTS: Twenty-seven percent of the sites included palliative care specialists as a part of their ALS clinics and they were introduced to the patients and family by ALS specialists (75.94%) usually at an advanced stage of ALS. DISCUSSION: Our study when compared with other study results having a similar aim showed that there is a variability in the practice of palliative care specialists in ALS clinics. Having evidence-based guidelines will help in the management of ALS patients more effectively.

Optimization of culture conditions for high-level expression of soluble and active tumor necrosis factor-α in E. coli.

Anti-TNF inhibitors exert their therapeutic effect by inhibition of the excessive amounts of TNF-α within the body. Recombinant TNF-α should be produced in a soluble refolded form to investigate the effectiveness and efficiency of anti-TNF-α compounds. In this research, the designed cassette was subcloned in the pET28a expression vector and expressed in E. coli BL21 (DE3). The identity of the protein was confirmed through SDS-PAGE and Western blotting. After optimizing expression conditions, protein purification was performed using native Ni-NTA affinity chromatography. The biological activity of the soluble recombinant TNF-α was investigated using MTT assay. Also, the affinity of an anti-TNF-α agent, Altebrel, was investigated against the expressed protein through ELISA. Optimization of TNF-α expression conditions represented that the highest expression could be achieved at 37 °C using 0.5 mM IPTG 6 h post-induction. The recombinant protein represented an inhibitory effect on the L929 murine fibroblast cell line and was successfully detected by Altebrel in ELISA. Binding kinetics were also studied using Cimzia as an anti-TNF-α molecule and 7.2 E-13M was calculated as the equilibrium dissociation constant value (KD). The significant expression level of the recombinant protein in the soluble form, its high purity, and assessment of its biological activity showed that the expressed protein could be used in tests of ELISA and MTT to assess the activity of anti-TNF-α agents.

DARPin Ec1-LMWP protein scaffold in targeted delivery of siRNA molecules through EpCAM cancer stem cell marker.

This study is to investigate the binding ability of Designed Ankyrin Repeat Proteins type Ec1that was fused to Low Molecular Weight Protamine (DARPin Ec1-LMWP) protein scaffold to the Epithelial Cell Adhesion Molecule (EpCAM) Cancer Stem Cell (CSC) marker and its efficiency in targeted delivery of small interfering RNA (siRNA) molecules into the studied cells. Gene fragment encoding the DARPIn Ec1-LMWP fusion protein was subcloned into pET28a expression vector following molecular docking studies performed to examine the affinity of the fusion protein towards EpCAM marker. The binding of the siRNA to the expressed fusion protein was tested through native PAGE. The toxicity of the fusion protein was tested by MTT assay. Attachment of the complex to the EpCAM marker was investigated by flow cytometry and delivery of siRNA into the cells was assessed by fluorescence microscopy. The expressed 21.6 kDa DARPin Ec1-LMWP fusion protein was purified and showed no cytotoxicity on tested cells. Arginine rich LMWP was efficiently bounded to the negatively charged siRNA molecule. Successful attachment of the fusion protein:siRNA complex to the EpCAM marker and its internalization into MCF-7 breast cancer cell line were confirmed. Here for the first time the recombinant DARPin Ec1-LMWP protein scaffold was designed and tested for targeting EpCAM surface marker and successful internalization of the siRNA into MCF-7 cells.

A comparative study of the bispecific monoclonal antibody, blinatumomab expression in CHO cells and E. coli.

The "bispecifics" market improved over the past decade due to the development of many technological platforms including bispecific T cell engagers (BiTEs). The approval of blinatumomab, the most advanced bispecific T-cell engager (BiTE) in clinical trials, can be a significant milestone in the development of bispecific antibodies. Both Chinese hamster ovary (CHO) cells and E. coli strain are considered as the most widely used hosts for the large-scale production of therapeutic monoclonal antibodies. Since both of the economic and qualitative aspects of protein production are important in industry, selection of a suitable protein expression system is very critical. The BsAb gene was cloned into the expression vectors FC550A-1, pcDNA3.1 (+), and PET22b and 6 × His-tagged BsAb then purified on a Ni-NTA chromatography column. Both SDS-PAGE and Western blotting analysis of the purified protein demonstrated that blinatumomab was successfully expressed as a 55 kDa in both expression systems. The antigen-binding properties of blinatumomab were compared in the mammalian system versus Escherichia coli. The results showed that the purified antibody from a mammalian expression system has better binding activity than the one from E. coli host.

Ulnar neuropathy with prominent proximal Martin-Gruber anastomosis.

Martin-Gruber anastomosis (MGA) is the most common nerve anastomosis in the upper extremities and it crosses from the median nerve to the ulnar nerve. Proximal MGA is an under recognized anastomosis between the ulnar and median nerves at or above the elbow and should not be missed during nerve conduction studies. We presented two patients with ulnar neuropathy mimicking findings including numbness and tingling of the 4th and 5th digits and mild weakness of intrinsic hand muscles. However, both cases had an apparently remarkable conduction block between the below- and above-elbow sites that was disproportionate to their clinical findings. To explain this discrepancy, a large MGA was detected with stimulation of the median nerve at the elbow. Thus, proximal MGA should be considered in ulnar neuropathy at the elbow when apparent conduction block or/and discrepancy between clinical and electrodiagnostic findings is found.

Expression, Purification, and Biological Evaluation of XTEN-GCSF in a Neutropenic Rat Model.

Granulocyte colony-stimulating factor (GCSF) stimulates the proliferation of neutrophils but it has low serum half-life. Therefore, the present study was done to investigate the effect of XTENylation on biological activity, pharmacokinetics, and pharmacodynamics of GCSF in a neutropenic rat model. XTEN tag was genetically fused to the N-terminal region of GCSF-encoding gene fragment and subcloned into pET28a expression vector. The cytoplasmic expressed recombinant protein was characterized through intrinsic fluorescence spectroscopy (IFS), dynamic light scattering (DLS), and size exclusion chromatography (SEC). In vitro biological activity of the XTEN-GCSF protein was evaluated on NFS60 cell line. Hematopoietic properties and pharmacokinetics were also investigated in a neutropenic rat model. An approximately 140 kDa recombinant protein was detected on SDS-PAGE. Dynamic light scattering and size exclusion chromatography confirmed the increase in hydrodynamic diameter of GCSF molecule after XTENylation. GCSF derivatives showed efficacy in proliferation of NFS60 cell line among which the XTEN-GCSF represented the lowest EC50 value (100.6 pg/ml). Pharmacokinetic studies on neutropenic rats revealed that XTEN polymer could significantly increase protein serum half-life in comparison with the commercially available GCSF molecules. PEGylated and XTENylated GCSF proteins were more effective in stimulation of neutrophils compared to the GCSF molecule alone. XTENylation of GCSF represented promising results in in vitro and in vivo studies. This approach can be a potential alternative to PEGylation strategies for increasing serum half-life of protein.

A novel nanobody-based immunocytokine of a mutant interleukin-2 as a potential cancer therapeutic.

The immunotherapeutic application of interleukin-2 (IL-2) in cancer treatment is limited by its off-target effects on different cell populations and insufficient activation of anti-tumor effector cells at the site of the tumor upon tolerated doses. Targeting IL-2 to the tumor microenvironment by generating antibody-cytokine fusion proteins (immunocytokine) would be a promising approach to increase efficacy without associated toxicity. In this study, a novel nanobody-based immunocytokine is developed by the fusion of a mutant (m) IL-2 with a decreased affinity toward CD25 to an anti-vascular endothelial growth factor receptor-2 (VEGFR2) specific nanobody, denoted as VGRmIL2-IC. The antigen binding, cell proliferation, IFN-γ-secretion, and cytotoxicity of this new immunocytokine are evaluated and compared to mIL-2 alone. Furthermore, the pharmacokinetic properties are analyzed. Flow cytometry analysis shows that the VGRmIL2-IC molecule can selectively target VEGFR2-positive cells. The results reveal that the immunocytokine is comparable to mIL-2 alone in the stimulation of Primary Peripheral Blood Mononuclear Cells (PBMCs) and cytotoxicity in in vitro conditions. In vivo studies demonstrate improved pharmacokinetic properties of VGRmIL2-IC in comparison to the wild or mutant IL-2 proteins. The results presented here suggest VGRmIL2-IC could be considered a candidate for the treatment of VEGFR2-positive tumors.

Recombinant anti-human epidermal growth factor receptor type 2 single-chain variable fragment-alpha-luffin fusion protein as a putative immunotoxin against human epidermal growth factor receptor type 2-positive breast cancer cells: an experimental research.

Background: Breast cancer is one of the most frequent causes of cancer death in women. The application of immunotoxins to target overexpressed biomarkers on the surface of cancer cells and delivery of the toxin molecules into these cells has attracted too much attention during the last decade. Objectives: This study was conducted to investigate the possible in-vitro cytotoxic and apoptotic activity of previously designed recombinant immunotoxin compromising anti-HER2 single-chain variable fragment (scFv) and alpha-luffin protein in human epidermal growth factor receptor type 2 (HER2)-positive and HER2-negative breast cancer cell lines. Materials and methods: The previously designed recombinant immunotoxin and alpha-luffin protein were expressed in E. coli host cells and purified using Ni-affinity chromatography. The cytotoxicity of the proteins was tested through MTT and apoptosis studies on HER2-positive and HER2-negative breast cancer cell lines. Results: Treatment of SKBR3 and MDA-MB-468 cells with the immunotoxin caused differential cytotoxicity and apoptotic events. Flow cytometry analysis indicated that the immunotoxin could arrest SKBR3 cells at the G0/G1 phase and induce apoptosis and cell death which were not observed in HER2-negative MDA-MB-468 cells. Annexin V/PI staining revealed late apoptotic events in SKBR3 cells treated with the immunotoxin which was different from the early apoptosis induced by the alpha-luffin protein alone. Conclusions: This immunotoxin could be a promising tool in developing new targeted therapeutic agents against HER2-positive cancer cells. Animal experiments are needed before making firmed conclusions.

White matter alterations differ in primary lateral sclerosis and amyotrophic lateral sclerosis.

Primary lateral sclerosis is a sporadic disorder characterized by slowly progressive corticospinal dysfunction. Primary lateral sclerosis differs from amyotrophic lateral sclerosis by its lack of lower motor neuron signs and long survival. Few pathological studies have been carried out on patients with primary lateral sclerosis, and the relationship between primary lateral sclerosis and amyotrophic lateral sclerosis remains uncertain. To detect in vivo structural differences between the two disorders, diffusion tensor imaging of white matter tracts was carried out in 19 patients with primary lateral sclerosis, 18 patients with amyotrophic lateral sclerosis and 19 age-matched controls. Fibre tracking was used to reconstruct the intracranial portion of the corticospinal tract and three regions of the corpus callosum: the genu, splenium and callosal fibres connecting the motor cortices. Both patient groups had reduced fractional anisotropy, a measure associated with axonal organization, and increased mean diffusivity of the reconstructed corticospinal and callosal motor fibres compared with controls, without changes in the genu or splenium. Voxelwise comparison of the whole brain white matter using tract-based spatial statistics confirmed the differences between patients and controls in the diffusion properties of the corticospinal tracts and motor fibres of the callosum. This analysis further revealed differences in the regional distribution of white matter alterations between the patient groups. In patients with amyotrophic lateral sclerosis, the greatest reduction in fractional anisotropy occurred in the distal portions of the intracranial corticospinal tract, consistent with a distal axonal degeneration. In patients with primary lateral sclerosis, the greatest loss of fractional anisotropy and mean diffusivity occurred in the subcortical white matter underlying the motor cortex, with reduced volume, suggesting tissue loss. Clinical measures of upper motor neuron dysfunction correlated with reductions in fractional anisotropy in the corticospinal tract in patients with amyotrophic lateral sclerosis and increased mean diffusivity and volume loss of the corticospinal tract in patients with primary lateral sclerosis. Changes in the diffusion properties of the motor fibres of the corpus callosum were strongly correlated with changes in corticospinal fibres in patients, but not in controls. These findings indicate that degeneration is not selective for corticospinal neurons, but affects callosal neurons within the motor cortex in motor neuron disorders.

Extension of human GCSF serum half-life by the fusion of albumin binding domain.

Granulocyte colony stimulating factor (GCSF) can decrease mortality of patients undergo chemotherapy through increasing neutrophil counts. Many strategies have been developed to improve its blood circulating time. Albumin binding domain (ABD) was genetically fused to N-terminal end of GCSF encoding sequence and expressed as cytoplasmic inclusion bodies within Escherichia coli. Biological activity of ABD-GCSF protein was assessed by proliferation assay on NFS-60 cells. Physicochemical properties were analyzed through size exclusion chromatography, circular dichroism, intrinsic fluorescence spectroscopy and dynamic light scattering. Pharmacodynamics and pharmacokinetic properties were also investigated in a neutropenic rat model. CD and IFS spectra revealed that ABD fusion to GCSF did not significantly affect the secondary and tertiary structures of the molecule. DLS and SEC results indicated the absence of aggregation formation. EC50 value of the ABD-GCSF in proliferation of NFS-60 cells was 75.76 pg/ml after 72 h in comparison with control GCSF molecules (Filgrastim: 73.1 pg/ml and PEG-Filgrastim: 44.6 pg/ml). Animal studies of ABD-GCSF represented improved serum half-life (9.3 ± 0.7 h) and consequently reduced renal clearance (16.1 ± 1.4 ml/h.kg) in comparison with Filgrastim (1.7 ± 0.1 h). Enhanced neutrophils count following administration of ABD-GCSF was comparable with Filgrastim and weaker than PEG-Filgrastim treated rats. In vitro and in vivo results suggested the ABD fusion as a potential approach for improving GCSF properties.

ALS-Like Disorder in Three HIV-Positive Patients: Case Series.

There appears to be a relationship between retroviruses such as HIV and the development of an ALS-like syndrome. Few cases have been reported; however, there exists evidence of a higher frequency of motor neuron disease in HIV-infected patients, as well as potential slowing and reversibility of disease course with combination antiretroviral therapy. We conducted a retrospective chart review of patients presenting to the George Washington University ALS Clinic from September 2006 to June 2018 to identify patients with HIV receiving HAART who were subsequently diagnosed with ALS or an ALS-like disorder. Our goals were to describe our patients' disease course and compare them to general characteristics of ALS. We report three cases of HIV-positive individuals, all male, who were subsequently diagnosed with ALS. Each presented with symptoms of limb onset ALS with involvement of upper and lower motor neurons and whose disease originated at the cervical level. All three had been diagnosed with HIV prior to presentation and were presumably compliant with antiretroviral therapy throughout. Our patients demonstrated effective control of their HIV infection. Each experienced relatively slow progression of motor impairment compared to general ALS characteristics. Our study offers a distinct profile of HIV-positive patients compliant with HAART subsequently diagnosed with an ALS-like disorder. Further study should aim to uncover pathophysiological similarities between motor neuron disease both in the presence and absence of retroviral infection and to develop effective medical therapy for each.

Targeted integration into pseudo attP sites of CHO cells using CRISPR/Cas9.

Chinese hamster ovary (CHO) cells are regarded as a prominent host for manufacturing therapeutic proteins. Although conventional strategies for generating recombinant proteins in CHO cells depend on the random integration of a gene of interest (GOI), these established techniques occasionally result in genetically heterogeneous cell lines, which causes diminished expression of the recombinant proteins in the long run. Production instability can be reduced by SSI and creates stable cell lines with a consistent expression of the GOI. In this experiment, we demonstrate the targeted incorporation of a reporter cassette in two PhiC31 pseudo attP sites of CHO cells exploiting the homology-directed repair (HDR) generated by the CRISPR/Cas9 platform. Genes encoding GFP and puromycin resistance marker were precisely inserted into these loci via CRISPR/Cas9. Stable cell lines were suitably produced following antibiotic selection. Junction PCR and fluorescence assay determined targeted integration and expression homogeneity of the reporter cassette, respectively. Taken together, our results indicate the possibility of these two PhiC31 pseudo attP sites as the target sites for site-specific integration of a transgene mediated by CRISPR/Cas9. Furthermore, higher knock-in efficiency and expression homogeneity was observed in the pseudo attP site associated with chromosome 6 compared to the pseudo attP site from chromosome 3.

Production of Soluble and Functional Anti-TNF-α Fab' Fragment in Cytoplasm of E. coli: Investigating the Effect of Process Conditions on Cellular Biomass and Protein Yield Using Response Surface Methodology.

With the increasing dominance of monoclonal antibodies (mAbs) in the biopharmaceutical industry and smaller antibody fragments bringing notable advantages over full-length antibodies, it is of considerable significance to choose the most suitable production system. Although mammalian expression system has been the preferred choice in recent years for mAbs production, E. coli could be the favorable host for non-glycosylated small antibody fragments due to the emergence of new engineered E. coli strains capable of forming disulfide-bonds in their cytoplasm.In this study, non-glycosylated anti-TNF-α Fab' moiety of Certolizumab pegol, produced by periplasmic expression in E. coli in previous studies, was produced in the cytoplasm of E. coli SHuffle strain. The results indicated that it is biologically functional by testing the antigen-binding activity via indirect ELISA and inhibition of TNF-α induced cytotoxicity using MTT test. Major factors affecting protein production and, optimized culture conditions were examined by analyzing growth characteristics and patterns of expression in 24 h of post-induction cultivation and, optimization of culture conditions by response surface methodology considering temperature, time of induction and concentration of inducer in small (tube) and shake-flask scale. Based on the results, temperature had the most significant influence on functional protein yield while exerting different impacts in small and shake-flask scales, which indicated that cultivation volume is also an important factor that should be taken into account in optimization process. Furthermore, richness of medium and slower cellular growth rate improved specific cellular yield of functional protein by having a positive effect on the solubility of Fab' antibody.

Production of an Antibody Fragment (scFv) Targeting PcrV Protein of Pseudomonas aeruginosa in Fed-Batch Cultivation Mode

Background: Pseudomonas aeruginosa is one of the opportunistic pathogens causing frequent hospital-acquired life-threatening infections in mechanically ventilated patients. The most significant virulence factor of P. aeruginosa is type III secretion system (T3SS). PcrV is an important structural protein of the T3SS. Methods: In the current investigation, a recombinant single-chain fragment variable (scFv) mAb against the PcrV protein was expressed in EnBase® (fed-batch) cultivation mode. The pETiteTM N-His SUMO Kan vector, including anti-PcrV scFv gene, was transformed into Escherichia coli (BL21) cells. The expression and solubility of anti-PcrV scFv protein were investigated at two different temperatures (25 °C and 30 °C) and at different induction times (4, 6, 8, 12, and 24 hours). Results: : Increased efficiency was achieved by EnBase® compared to Luria­Bertani broth; owing to the slow release of glucose, the maximum level of solubility and total protein expression was observed in EnBase® cultivation system at 30 °C and 24 h post induction. Furthermore, IC50 for anti-PcrV scFv protein was determined to be approximately 7 µg/mL. Conclusion: : Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+).

Characterization of a novel mCH3 conjugated anti-PcrV scFv molecule.

Pseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. The stereochemical properties of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular interactions towards Pseudomonas aeruginosa PcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within the E. coli BL21-DE3 strain. After Ni-NTA affinity chromatography, the biological activity of the proteins in inhibition of PA induced hemolysis as well as cellular cytotoxicity was assessed. In silico analysis revealed the satisfactory stereochemical quality of the models as well as common residues in their interface with PcrV. The structural differences of proteins through circular dichroism spectroscopy were confirmed by NMR analysis. Both proteins indicated inhibition of ExoU positive PA strains in hemolysis of red blood cells compared to ExoU negative strains as well as cytotoxicity effect on lung epithelial cells. The ELISA test showed the longer serum stability of the YFL001 molecule than YFL002. The results were encouraging to further evaluation of these two scFv molecules in animal models.

Psychiatric aspects of amyotrophic lateral sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is a progressive disorder characterized by degeneration of motor neurons. Given the severe nature of ALS, many believed that patients would suffer from a high level of depression and a low quality of life. However, research into the psychological health of patients with ALS has shown that this is not the case. This article reviews the state of current knowledge as it pertains to the psychological health of ALS patients in four broad areas: quality of life, personality characteristics, emotional reactions, and end-of-life choices.

Improvement of Certolizumab Fab' properties by PASylation technology.

Certolizumab pegol is a Fab' antibody fragment for treatment of rheumatoid arthritis and Crohn's disease which is conjugated to a 40 kDa PEG molecule in order to increase the protein half-life. PEGylation may have disadvantages including immunogenicity, hypersensitivity, vacuolation, decreased binding affinity and biological activity of the protein. To overcome these problems, PASylation has been developed as a new approach. The nucleotide sequence encoding 400 amino acid PAS residues was genetically fused to the corresponding nucleotide sequences of both chains of certolizumab. Then, the bioactivity as well as physicochemical and pharmacokinetic properties of the recombinant PASylated expressed protein was assayed. Circular dichroism spectroscopy demonstrated that the random coil structure of PAS sequences did not change the secondary structure of the PASylated Fab' molecule. It was observed that PASylation influenced the properties of the Fab' molecule by which the hydrodynamic radius and neutralization activity were increased. Also, the antigen binding and binding kinetic parameters improved in comparison to the PEGylated Fab' antibody. Pharmacokinetic studies also showed prolonged terminal half-life and improved pharmacokinetic parameters in PASylated recombinant protein in comparison to the PEGylated and Fab' control molecules. The results reconfirmed the efficiency of PASylation approach as a potential alternative method in increasing the half-life of pharmaceutical proteins.

Clinical Effects of the Self-administered Subcutaneous Complement Inhibitor Zilucoplan in Patients With Moderate to Severe Generalized Myasthenia Gravis: Results of a Phase 2 Randomized, Double-Blind, Placebo-Controlled, Multicenter Clinical Trial.

Importance: Many patients with generalized myasthenia gravis (gMG) have substantial clinical disability, persistent disease burden, and adverse effects attributable to chronic immunosuppression. Therefore, there is a significant need for targeted, well-tolerated therapies with the potential to improve disease control and enhance quality of life. Objective: To evaluate the clinical effects of zilucoplan, a subcutaneously (SC) self-administered macrocyclic peptide inhibitor of complement component 5, in a broad population of patients with moderate to severe gMG. Design, Setting, and Participants: This randomized, double-blind, placebo-controlled phase 2 clinical trial at 25 study sites across North America recruited participants between December 2017 and August 2018. Fifty-seven patients were screened, of whom 12 did not meet inclusion criteria and 1 was lost to follow-up after randomization but before receiving study drug, resulting in a total of 44 acetylcholine receptor autoantibody (AChR-Ab)-positive patients with gMG with baseline Quantitative Myasthenia Gravis (QMG) scores of at least 12, regardless of treatment history. Interventions: Patients were randomized 1:1:1 to a daily SC self-injection of placebo, 0.1-mg/kg zilucoplan, or 0.3-mg/kg zilucoplan for 12 weeks. Main Outcomes and Measures: The primary and key secondary end points were the change from baseline to week 12 in QMG and MG Activities of Daily Living scores, respectively. Significance testing was prespecified at a 1-sided α of .10. Safety and tolerability were also assessed. Results: The study of 44 patients was well balanced across the 3 treatment arms with respect to key demographic and disease-specific variables. The mean age of patients across all 3 treatment groups ranged from 45.5 to 54.6 years and most patients were white (average proportions across 3 treatment groups: 78.6%-86.7%). Clinically meaningful and statistically significant improvements in primary and key secondary efficacy end points were observed. Zilucoplan at a dose of 0.3 mg/kg SC daily resulted in a mean reduction from baseline of 6.0 points in the QMG score (placebo-corrected change, -2.8; P = .05) and 3.4 points in the MG Activities of Daily Living score (placebo-corrected change, -2.3; P = .04). Clinically meaningful and statistically significant improvements were also observed in other secondary end points, the MG Composite and MG Quality-of-Life scores. Outcomes for the 0.1-mg/kg SC daily dose were also statistically significant but slower in onset and less pronounced than with the 0.3-mg/kg dose. Rescue therapy (intravenous immunoglobulin or plasma exchange) was required in 3 of 15, 1 of 15, and 0 of 14 participants in the placebo, 0.1-mg/kg zilucoplan, and 0.3-mg/kg zilucoplan arms, respectively. Zilucoplan was observed to have a favorable safety and tolerability profile. Conclusions and Relevance: Zilucoplan yielded rapid, meaningful, and sustained improvements over 12 weeks in a broad population of patients with moderate to severe AChR-Ab-positive gMG. Near-complete complement inhibition appeared superior to submaximal inhibition. The observed safety and tolerability profile of zilucoplan was favorable. Trial Registration: ClinicalTrials.gov Identifier: NCT03315130.