Expression of a Tagless Single-Chain Variable Fragment (scFv) of Anti-TNF-α by a Salt Inducible System and its Purification and Characterization

Background: Anti-TNF-α scFv is gaining acceptance as an effective drug for various diseases such as rheumatoid arthritis and Crohn’s disease that involve elevated levels of TNF-α. The single-chain variable fragment (scFv) consists of variable regions of heavy and light chains of monoclonal antibodies (mAb). Due to its smaller size, it curbs the mAb’s auto-antibody effects and their limitation of penetration into the tissues during the neutralization of TNF-α. Objective: In this work, a cDNA coding for anti-TNF-α scFv was successfully cloned into a pRSET-B vector and efficiently expressed in an E. coli strain GJ1158, a salt inducible system that uses sodium chloride instead of IPTG as an inducer. Methods: The protein was expressed in the form of inclusion bodies (IB), solubilized using urea, and refolded by pulse dilution. Further, the amino acid sequence coverage of scFv was confirmed by ESI-Q-TOF MS/MS and MALDI-TOF. Further studies on scaling up the production of scFv and its application of scFv are being carried out Results: The soluble fraction of anti-TNF-α scFv was then purified in a single chromatographic step using CM-Sephadex chromatography, a weak cation exchanger with a yield of 10.3 mg/L. The molecular weight of the scFv was found to be ∼28 kDa by SDS PAGE, and its presence was confirmed by western blot analysis and mass spectrometry. Conclusion: Anti-TNF-α scFv has been successfully purified in a salt inducible system GJ1158. As per the best of our knowledge, this is the first report of purification of Anti-TNF-α scFv in a salt inducible system from soluble fractions as well as inclusion bodies.

Therapeutic anti-PD-L1 antibody affects ESCRT-mediated functions in cells expressing oncogenic EGFR.

PD-L1 is a ligand for immune checkpoint receptor PD1. Anti-PD-L1 antibody is an effective therapy for a variety of solid tumors, although a durable response is only achieved in a subset of patients. For unknown reasons, EGFR-mutant tumors respond poorly to checkpoint blockade. Applying quantitative cell biological methods to study PD-L1 biology in lung cancer cells, we establish that growth factors acutely regulate PD-L1 trafficking between the plasma membrane and the interior of cells. Changes in plasma membrane PD-L1 levels will impact PD1 engagement on T cells, thereby influencing PD-L1's immune suppressive activity. To discover potential cell-intrinsic functions of PD-L1, we used APEX2 biotinylation to generate a high-resolution map of the PD-L1 proximal proteome. ESCRT pathway proteins were enriched in PD-L1's proximal proteome, and two ESCRT-dependent functions, turnover of mutant EGFR and biogenesis of extracellular vesicles, were affected by anti-PD-L1 treatment, suggesting a link between PD-L1 and ESCRT function. Proteins that control cytoskeletal dynamics were also enriched in the PD-L1 proteome, and anti-PD-L1 treatment reduced cell migration, identifying migration as a PD-L1 associated function. PD-L1 knockout mimics the effects of the antibody treatment, suggesting anti-PD-L1 antibody effects are loss of function(s). The effects of anti-PD-L1 on the ESCRT-dependent functions and cell migration were restricted to cells harboring oncogenic EGFR mutations. Wildtype and KRAS mutant cells lines were unaffected. Our study reveals new cell-intrinsic roles for PD-L1 in EGFR mutant cells, activities that might contribute to the resistance of EGFR mutant tumors to PD-L1 checkpoint blockade.

COVID-19 Convalescent Plasma Is More Than Neutralizing Antibodies: A Narrative Review of Potential Beneficial and Detrimental Co-Factors

COVID-19 convalescent plasma (CCP) is currently under investigation for both treatment and post-exposure prophylaxis. The active component of CCP mediating improved outcome is commonly reported as specific antibodies, particularly neutralizing antibodies, with clinical efficacy characterized according to the level or antibody affinity. In this review, we highlight the potential role of additional factors in CCP that can be either beneficial (e.g., AT-III, alpha-1 AT, ACE2+ extracellular vesicles) or detrimental (e.g., anti-ADAMTS13, anti-MDA5 or anti-interferon autoantibodies, pro-coagulant extracellular vesicles). Variations in these factors in CCP may contribute to varied outcomes in patients with COVID-19 and undergoing CCP therapy. We advise careful, retrospective investigation of such co-factors in randomized clinical trials that use fresh frozen plasma in control arms. Nevertheless, it might be difficult to establish a causal link between these components and outcome, given that CCP is generally safe and neutralizing antibody effects may predominate.

Modeling the population effects of epitope specific escape mutations in SARS-CoV-2 to guide vaccination strategies

Escape mutations (EM) to SARS-Cov-2 have been detected and are spreading. Vaccines may need adjustment to respond to these or future mutations. We designed a population level model integrating both waning immunity and EM. We also designed a set of criteria for elaborating and fitting this model to cross-neutralization and other data in a manner that minimizes vaccine decision errors. We formulated four model variations. These define criteria for which prior infections provide immunity that can be escaped. They also specify different sequences where one EM follows another. At all reasonable parameter values, these model variations led to patterns where: 1) EM were rare in the first epidemic, 2) rebound epidemics after the first epidemic were accelerated more by increasing drifting than by increasing waning (with some exceptions), 3) the long term endemic level of infection was determined mostly by waning rates with small effects of the drifting parameter, 4) EM caused loss of vaccine effectiveness and under some conditions, vaccines induced EM that caused higher levels of infection with vaccines than without them. The differences and similarities across the four models suggest paths for developing models specifying the epitopes where EM act. This model is a base on which to construct epitope specific evolutionary models using new high-throughput assay data from population samples to guide vaccine decisions.HighlightsThis model is the first to integrate both antigenic drifting from escape mutations and immunity waning in continuous time.Tiny amounts of only waning or only escape mutation drifting have small or no effects. Together, they have large effects.There are no or few escape mutations during the first epidemic peak and no effect of drifting parameters on the size of that wave.After the first epidemic peak, escape mutations accumulate rapidly. They increase with increases in waning rates and with increases in the drifting rate. Escape mutations then amplify other escape mutations since these raise the frequency of reinfections.Escape mutations can completely negate the effects of vaccines and even lead to more infections with vaccination than without, especially at very low waning rates.The model generates population level cross-neutralization patterns that enable the model to be fitted to population level serological data.The model can be modified to use laboratory data that determine the epitope specific effects of mutations on ACE2 attachment strength or escape from antibody effects.The model, although currently unable to predict the effects of escape mutations in the real world, opens up a path that can guide model incorporation of molecularly studied escape mutations and improve predictive value. We describe that path.Model analysis indicates that vaccine trials and serological surveys are needed now to detect the effects of epitope specific escape mutations that could cause the loss of vaccine efficacy.

Placental transfer of NMDAR antibodies causes reversible alterations in mice

ObjectiveTo determine whether maternofetal transfer of NMDA receptor (NMDAR) antibodies has pathogenic effects on the fetus and offspring, we developed a model of placental transfer of antibodies.MethodsPregnant C57BL/6J mice were administered via tail vein patients' or controls' immunoglobulin G (IgG) on days 14–16 of gestation, when the placenta is able to transport IgG and the immature fetal blood-brain barrier is less restrictive to IgG crossing. Immunohistochemical and DiOlistic (gene gun delivery of fluorescent dye) staining, confocal microscopy, standardized developmental and behavioral tasks, and hippocampal long-term potentiation were used to determine the antibody effects.ResultsIn brains of fetuses, patients' IgG, but not controls' IgG, bound to NMDAR, causing a decrease in NMDAR clusters and cortical plate thickness. No increase in neonatal mortality was observed, but offspring exposed in utero to patients' IgG had reduced levels of cell-surface and synaptic NMDAR, increased dendritic arborization, decreased density of mature (mushroom-shaped) spines, microglial activation, and thinning of brain cortical layers II–IV with cellular compaction. These animals also had a delay in innate reflexes and eye opening and during follow-up showed depressive-like behavior, deficits in nest building, poor motor coordination, and impaired social-spatial memory and hippocampal plasticity. Remarkably, all these paradigms progressively improved (becoming similar to those of controls) during follow-up until adulthood.ConclusionsIn this model, placental transfer of patients' NMDAR antibodies caused severe but reversible synaptic and neurodevelopmental alterations. Reversible antibody effects may contribute to the infrequent and limited number of complications described in children of patients who develop anti-NMDAR encephalitis during pregnancy.

Allosteric modulation of NMDA receptors prevents the antibody effects of patients with anti-NMDAR encephalitis

Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is an immune-mediated disease characterized by a complex neuropsychiatric syndrome in association with an antibody-mediated decrease of NMDAR. About 85% of patients respond to immunotherapy (and removal of an associated tumour if it applies), but it often takes several months or more than 1 year for patients to recover. There are no complementary treatments, beyond immunotherapy, to accelerate this recovery. Previous studies showed that SGE-301, a synthetic analogue of 24(S)-hydroxycholesterol, which is a potent and selective positive allosteric modulator of NMDAR, reverted the memory deficit caused by phencyclidine (a non-competitive antagonist of NMDAR), and prevented the NMDAR dysfunction caused by patients’ NMDAR antibodies in cultured neurons. An advantage of SGE-301 is that it is optimized for systemic delivery such that plasma and brain exposures are sufficient to modulate NMDAR activity. Here, we used SGE-301 to confirm that in cultured neurons it prevented the antibody-mediated reduction of receptors, and then we applied it to a previously reported mouse model of passive cerebroventricular transfer of patient’s CSF antibodies. Four groups were established: mice receiving continuous (14-day) infusion of patients’ or controls’ CSF, treated with daily subcutaneous administration of SGE-301 or vehicle (no drug). The effects on memory were examined with the novel object location test at different time points, and the effects on synaptic levels of NMDAR (assessed with confocal microscopy) and plasticity (long-term potentiation) were examined in the hippocampus on Day 18, which in this model corresponds to the last day of maximal clinical and synaptic alterations. As expected, mice infused with patient’s CSF antibodies, but not those infused with controls’ CSF, and treated with vehicle developed severe memory deficit without locomotor alteration, accompanied by a decrease of NMDAR clusters and impairment of long-term potentiation. All antibody-mediated pathogenic effects (memory, synaptic NMDAR, long-term potentiation) were prevented in the animals treated with SGE-301, despite this compound not antagonizing antibody binding. Additional investigations on the potential mechanisms related to these SGE-301 effects showed that (i) in cultured neurons SGE-301 prolonged the decay time of NMDAR-dependent spontaneous excitatory postsynaptic currents suggesting a prolonged open time of the channel; and (ii) it significantly decreased, without fully preventing, the internalization of antibody-bound receptors suggesting that additional, yet unclear mechanisms, contribute in keeping unchanged the surface NMDAR density. Overall, these findings suggest that SGE-301, or similar NMDAR modulators, could potentially serve as complementary treatment for anti-NMDAR encephalitis and deserve future investigations.

DPPX antibody–associated encephalitis

Objective:To report the main syndrome of dipeptidyl-peptidase–like protein 6 (DPPX) antibody–associated encephalitis, immunoglobulin G (IgG) subclass, and the antibody effects on DPPX/Kv4.2 potassium channels.Methods:A retrospective analysis of new patients and cases reported since 2013 was performed. IgG subclass and effects of antibodies on cultured neurons were determined with described techniques.Results:Nine new patients were identified (median age 57 years, range 36–69 years). All developed severe prodromal weight loss or diarrhea followed by cognitive dysfunction (9), memory deficits (5), CNS hyperexcitability (8; hyperekplexia, myoclonus, tremor, or seizures), or brainstem or cerebellar dysfunction (7). The peak of the disease was reached 8 months (range 1–54 months) after onset. All patients had both IgG4 and IgG1 DPPX antibodies. In cultured neurons, the antibodies caused a decrease of DPPX clusters and Kv4.2 protein that was reversible on removal of the antibodies. Considering the current series and previously reported cases (total 39), 67% developed the triad: weight loss (median 20 kg; range 8–53 kg)/gastrointestinal symptoms, cognitive-mental dysfunction, and CNS hyperexcitability. Outcome was available from 35 patients (8 not treated with immunotherapy): 60% had substantial or moderate improvement, 23% had no improvement (most of them not treated), and 17% died. Relapses occurred in 8 of 35 patients (23%) and were responsive to immunotherapy.Conclusions:DPPX antibodies are predominantly IgG1 and IgG4 and associate with cognitive-mental deficits and symptoms of CNS hyperexcitability that are usually preceded by diarrhea, other gastrointestinal symptoms, and weight loss. The disorder is responsive to immunotherapy, and this is supported by the reversibility of the antibody effects in cultured neurons.