Probing the anti-Aß42 aggregation and protective effects of prenylated xanthone against Aß42-induced toxicity in transgenic Caenorhabditis elegans model.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid-ß (Aß) protein aggregates, leading to synaptic dysfunction and neuronal cell death. In this study, we used a comprehensive approach encompassing in vitro assays, computational analyses, and an in vivo Caenorhabditis elegans model to evaluate the inhibitory effects of various xanthones, focusing on Garcinone D (GD), on Aß42 oligomer formation. Dot blot analysis revealed concentration-dependent responses among xanthones, with GD consistently inhibiting Aß42 oligomer formation at low concentrations (0.1 and 0.5 µM, inhibitions of 84.66 ± 2.25% and 85.06 ± 6.57%, respectively). Molecular docking and dynamics simulations provided insights into the molecular interactions between xanthones and Aß42, highlighting the disruption of key residues involved in Aß42 aggregation. The neuroprotective potential of GD was established using transgenic C. elegans GMC101, with substantial delays in paralysis reported at higher concentrations. Our findings show that GD is a potent suppressor of Aß42 oligomer formation, suggesting its potential as a therapeutic candidate for AD. The concentration-dependent effects observed in both in vitro and in vivo models underscore the need for nuanced dose-response assessments. These findings contribute novel insights into the therapeutic landscape of xanthones against AD, emphasizing the multifaceted potential of GD for further translational endeavors in neurodegenerative disorder research.

Phase transforming in situ gels for sustained and controlled transmucosal drug delivery via the intravaginal route.

Direct, reliable, controlled, and sustained drug delivery to female reproductive tract (FRT) remains elusive, with conventional dosage forms falling way short of the mark, leading to premature leakage, erratic drug delivery, and loss of compliance. Historically, the intravaginal route remains underserved by the pharmaceutical sector. To comprehensively address this, we turned our focus to phase-transforming sol-gels, using poloxamers, a thermosensitive polymer and, doxycycline (as hyclate salt, DOXH) as our model agent given its potential use in sexually transmitted infections (STIs). We further enhanced mucoadhesiveness through screening of differing viscosity grade hydroxypropyl methyl celluloses (HPMCs). The optimised sol-gels remained gelled at body temperature (<37 °C) and were prepared in buffer aligned to vaginal cavity pH and osmolality. Lead formulations were progressed based on their ability to retain key rheological properties, and acidic pH in the presence of simulated vaginal fluid (SVF). From a shelf-life perspective, DOXH stability, gelation temperature (Tsol-gel), and pH to three months (2-8 °C) was attained. In summary, the meticulously engineered, phase-transforming sol-gels provided sustained mucoretention despite dilution by vaginal fluid, paving the way for localised antimicrobial drug delivery at concentrations that potentially far exceed the minimum inhibitory concentration (MIC) for target STI-causing bacteria of the FRT.

Flow cytometric reporter assays provide robust functional analysis of signaling complexes.

Conventional assays to probe signaling protein interactions and function involve measurement of luciferase reporter expression within the bulk cell population, with lack of control over target-protein expression level. To address this issue, we have developed a rapid and robust flow cytometric assay for analysis of signaling protein function. A fluorescent reporter and fluorescent tagging of the target protein enables simultaneous assessment of protein expression and signaling within individual cells. We have applied our technique to the analysis of variants of the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) and its adapter protein MyD88, using a NF-кB-responsive promoter driving mScarlet-I expression. The assay enables exclusion of nontransfected cells and overexpressing cells that signal spontaneously. Additionally, our assay allows the identification of protein variants that fail to express. We found that the assays were highly sensitive, with cells expressing an appropriate level of GFP-MyD88 showing approximately 200-fold induction of mScarlet-I by lipopolysaccharide, and we can detect subtle protein concentration-dependent effects of mutations. Importantly, the assay is adaptable to various signaling pathways.

Production of afucosylated antibodies in CHO cells by coexpression of an anti-FUT8 intrabody.

Some effector functions prompted by immunoglobulin G (IgG) antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC), strongly depend on the N-glycans linked to asparagine 297 of the Fc region of the protein. A single α-(1,6)-fucosyltransferase (FUT8) is responsible for catalyzing the addition of an α-1,6-linked fucose residue to the first GlcNAc residue of the N-linked glycans. Antibodies missing this core fucose show a significantly enhanced ADCC and increased antitumor activity, which could help reduce therapeutic dose requirement, potentially translating into reduced safety concerns and manufacturing costs. Several approaches have been developed to modify glycans and improve the biological functions of antibodies. Here, we demonstrate that expression of a membrane-associated anti-FUT8 intrabody engineered to reside in the endoplasmic reticulum and Golgi apparatus can efficiently reduce FUT8 activity and therefore the core-fucosylation of the Fc N-glycan of an antibody. IgG1-producing CHO cells expressing the intrabody secrete antibodies with reduced core fucosylation as demonstrated by lectin blot analysis and UPLC-HILIC glycan analysis. Cells engineered to inhibit directly and specifically alpha-(1,6)-fucosyltransferase activity allows for the production of g/L levels of IgGs with strongly enhanced ADCC effector function, for which the level of fucosylation can be selected. The quick and efficient method described here should have broad practical applicability for the development of next-generation therapeutic antibodies with enhanced effector functions.

Structure-based dual affinity optimization of a SARS-CoV-1/2 cross-reactive single-domain antibody.

The SARS coronavirus 2 (SARS-CoV-2) spike (S) protein binding to the human ACE2 receptor is the molecular event that initiates viral entry into host cells and leads to infection and virus replication. There is a need for agents blocking viral entry into host cells that are cross-reactive with emerging virus variants. VHH-72 is an anti-SARS-CoV-1 single-domain antibody that also exhibits cross-specificity with SARS-CoV-2 but with decreased binding affinity. Here we applied a structure-based approach to affinity-mature VHH-72 for the SARS-CoV-2 spike protein while retaining the original affinity for SARS-CoV-1. This was achieved by employing the computational platform ADAPT in a constrained dual-affinity optimization mode as a means of broadening specificity. Select mutants designed by ADAPT were formatted as fusions with a human IgG1-Fc fragment. These mutants demonstrated improved binding to the SARS-CoV-2 spike protein due to decreased dissociation rates. Functional testing for virus neutralization revealed improvements relative to the parental VHH72-Fc up to 10-fold using a SARS-CoV-2 pseudotyped lentivirus and 20-fold against the SARS-CoV-2 authentic live virus (Wuhan variant). Binding and neutralization improvements were maintained for some other SARS-CoV-2 variants currently in circulation. These improved VHH-72 mutants are predicted to establish novel interactions with the S antigen. They will be useful, alone or as fusions with other functional modules, in the global quest for treatments of COVID-19 infections.

A simple liquid extraction for simultaneous determination of 12 opioid ligands in plasma by LC-MS/MS.

Opioids are commonly used as analgesics to relieve chronic pain and have high abuse potential. Due to their strong potency and trace concentration in plasma, a robust analytical method is necessary for quantification in forensic and pharmacology fields. Hence, this study developed and validated a simple, rapid, and robust method for the simultaneous determination of 12 opioids and metabolites which were available legally by prescription or abused for non-medical purposes, in plasma samples by simple liquid extraction and high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We compared the extraction recovery of our sample pre-treatment to two other sample pre-treatments (namely QuEChERS and simplified QuEChERS) and showed that the method used in our study gave the highest recoveries. The method validation followed the European Medicines Agency guidelines, including selectivity, carryover, accuracy and precision, dilution integrity, matrix effect and freeze/thaw stability. This method's accuracy ranged from 85% to 115% with a precision less than 15%, within the acceptable range of the validation protocol. The lower limit of quantification of the method ranged between 0.05 µg L-1 and 0.38 µg L-1 among 12 opioids/metabolites. Stability was assessed, with all opioids observed as relatively stable at 0.5 µg L-1 and 5 µg L-1 levels under -20 °C and 25 °C storage conditions. In summary, the developed method has the potential to achieve simultaneous analysis for monitoring opioids in forensic and pain management regimens using a simple sample pre-treatment.

Application of Sol-Gels for Treatment of Gynaecological Conditions-Physiological Perspectives and Emerging Concepts in Intravaginal Drug Delivery.

Approaches for effective and sustained drug delivery to the female reproductive tract (FRT) for treating a range of gynaecological conditions remain limited. The development of versatile delivery platforms, such as soluble gels (sol-gels) coupled with applicators/devices, holds considerable therapeutic potential for gynaecological conditions. Sol-gel systems, which undergo solution-to-gel transition, triggered by physiological conditions such as changes in temperature, pH, or ion composition, offer advantages of both solution- and gel-based drug formulations. Furthermore, they have potential to be used as a suitable drug delivery vehicle for other novel drug formulations, including micro- and nano-particulate systems, enabling the delivery of drug molecules of diverse physicochemical character. We provide an anatomical and physiological perspective of the significant challenges and opportunities in attaining optimal drug delivery to the upper and lower FRT. Discussion then focuses on attributes of sol-gels that can vastly improve the treatment of gynaecological conditions. The review concludes by showcasing recent advances in vaginal formulation design, and proposes novel formulation strategies enabling the infusion of a wide range of therapeutics into sol-gels, paving the way for patient-friendly treatment regimens for acute and chronic FRT-related conditions such as bacterial/viral infection control (e.g., STDs), contraception, hormone replacement therapy (HRT), infertility, and cancer.

Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX.

The architectural complexity and heterogeneity of the tumor microenvironment (TME) remains a substantial obstacle in the successful treatment of cancer. Hypoxia, caused by insufficient oxygen supply, and acidosis, resulting from the expulsion of acidic metabolites, are prominent features of the TME. To mitigate the consequences of the hostile TME, cancer cells metabolically rewire themselves and express a series of specific transporters and enzymes instrumental to this adaptation. One of these proteins is carbonic anhydrase (CA)IX, a zinc-containing extracellular membrane bound enzyme that has been shown to play a critical role in the maintenance of a neutral intracellular pH (pHi), allowing tumor cells to survive and thrive in these harsh conditions. Although CAIX has been considered a promising cancer target, only two antibody-based therapeutics have been clinically tested so far. To fill this gap, we generated a series of novel monoclonal antibodies (mAbs) that specifically recognize the extracellular domain (ECD) of human CAIX. Here we describe the biophysical and functional properties of a set of antibodies against the CAIX ECD domain and their applicability as: 1) suitable for development as an antibody-drug-conjugate, 2) an inhibitor of CAIX enzyme activity, or 3) an imaging/detection antibody. The results presented here demonstrate the potential of these specific hCAIX mAbs for further development as novel cancer therapeutic and/or diagnostic tools.

Hydrogen-deuterium exchange mass spectrometry reveals three unique binding responses of mAbs directed to the catalytic domain of hCAIX.

Human carbonic anhydrase (hCAIX), an extracellular enzyme that catalyzes the reversible hydration of CO2, is often overexpressed in solid tumors. This enzyme is instrumental in maintaining the survival of cancer cells in a hypoxic and acidic tumor microenvironment. Absent in most normal tissues, hCAIX is a promising therapeutic target for detection and treatment of solid tumors. Screening of a library of anti-hCAIX monoclonal antibodies (mAbs) previously identified three therapeutic candidates (mAb c2C7, m4A2 and m9B6) with distinct biophysical and functional characteristics. Selective binding to the catalytic domain was confirmed by yeast surface display and isothermal calorimetry, and deeper insight into the dynamic binding profiles of these mAbs upon binding were highlighted by bottom-up hydrogen-deuterium exchange mass spectrometry (HDX-MS). Here, a conformational and allosterically silent epitope was identified for the antibody-drug conjugate candidate c2C7. Unique binding profiles are described for both inhibitory antibodies, m4A2 and m9B6. M4A2 reduces the ability of the enzyme to hydrate CO2 by steric gating at the entrance of the catalytic cavity. Conversely, m9B6 disrupts the secondary structure that is necessary for substrate binding and hydration. The synergy of these two inhibitory mechanisms is demonstrated in in vitro activity assays and HDX-MS. Finally, the ability of m4A2 to modulate extracellular pH and intracellular metabolism is reported. By highlighting three unique modes by which hCAIX can be targeted, this study demonstrates both the utility of HDX-MS as an important tool in the characterization of anti-cancer biotherapeutics, and the underlying value of CAIX as a therapeutic target.

Interaction of Opioids with TLR4-Mechanisms and Ramifications.

The innate immune receptor toll-like receptor 4 (TLR4) is known as a sensor for the gram-negative bacterial cell wall component lipopolysaccharide (LPS). TLR4 activation leads to a strong pro-inflammatory response in macrophages; however, it is also recognised to play a key role in cancer. Recent studies of the opioid receptor (OR)-independent actions of opioids have identified that TLR4 can respond to opioids. Opioids are reported to weakly activate TLR4, but to significantly inhibit LPS-induced TLR4 activation. The action of opioids at TLR4 is suggested to be non-stereoselective, this is because OR-inactive (+)-isomers of opioids have been shown to activate or to inhibit TLR4 signalling, although there is some controversy in the literature. While some opioids can bind to the lipopolysaccharide (LPS)-binding cleft of the Myeloid Differentiation factor 2 (MD-2) co-receptor, pharmacological characterisation of the inhibition of opioids on LPS activation of TLR4 indicates a noncompetitive mechanism. In addition to a direct interaction at the receptor, opioids affect NF-κB activation downstream of both TLR4 and opioid receptors and modulate TLR4 expression, leading to a range of in vivo outcomes. Here, we review the literature reporting the activity of opioids at TLR4, its proposed mechanism(s), and the complex functional consequences of this interaction.

New Insights on Tramadol and Immunomodulation.

PURPOSE OF REVIEW: Opioids are administered to cancer patients although concerns have been raised that they may promote tumour growth or metastasis owing to their ability to suppress anti-cancer immunity. Tramadol has been reported to preserve or promote the immune response and may therefore be preferred to other opioids in cancer patients. We reviewed the literature documenting the immunomodulatory effects of tramadol. RECENT FINDINGS: Recent clinical evidence appears to confirm that tramadol possesses anti-inflammatory properties, and preserves some signalling cascades of the immune system relevant to anti-cancer defence. Tramadol is reported to promote or preserve immunity including natural killer cell activity which is important in anti-cancer defences.

Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response.

Caveolae-associated protein 3 (cavin3) is inactivated in most cancers. We characterized how cavin3 affects the cellular proteome using genome-edited cells together with label-free quantitative proteomics. These studies revealed a prominent role for cavin3 in DNA repair, with BRCA1 and BRCA1 A-complex components being downregulated on cavin3 deletion. Cellular and cell-free expression assays revealed a direct interaction between BRCA1 and cavin3 that occurs when cavin3 is released from caveolae that are disassembled in response to UV and mechanical stress. Overexpression and RNAi-depletion revealed that cavin3 sensitized various cancer cells to UV-induced apoptosis. Supporting a role in DNA repair, cavin3-deficient cells were sensitive to PARP inhibition, where concomitant depletion of 53BP1 restored BRCA1-dependent sensitivity to PARP inhibition. We conclude that cavin3 functions together with BRCA1 in multiple cancer-related pathways. The loss of cavin3 function may provide tumor cell survival by attenuating apoptotic sensitivity and hindering DNA repair under chronic stress conditions.

When cells become cancerous they often stop making certain proteins. This includes a protein known as cavin3 which resides in bulb-shaped pits of the membrane that surrounds the cell called caveolae. These structures work like stress detectors, picking up changes in the membrane and releasing proteins, such as cavin3, into the cell's interior. Past studies suggest that cavin3 might interact with a protein called BRCA1 that suppresses the formation of tumors. Cells with mutations in the gene for BRCA1 struggle to fix damage in their DNA, and have to rely on other repair proteins, such as PARPs (short for poly (ADP-ribose) polymerases). Blocking PARP proteins with drugs can kill cancer cells with problems in their BRCA1 proteins. However, it was unclear what role cavin3 plays in this mechanism. To investigate this, McMahon et al. exposed cells grown in the laboratory to DNA-damaging UV light to stimulate the release of cavin3 from caveolae. This revealed that cavin3 interacts with BRCA1 when cells are under stress, and helps stabilize the protein so it can perform DNA repairs. Cells without cavin3 showed decreased levels of the BRCA1 protein, but compensated for the loss of BRCA1 by increasing the levels of their PARP proteins. These cells also had increased DNA damage following treatment with drugs that block PARPs, similar to cancer cells carrying mutations in the gene for BRCA1. These findings suggest that cavin3 helps BRCA1 to suppress the formation of tumors, and therefore should be considered when developing new anti-cancer treatments.

Anticancer activities of dietary benzyl isothiocyanate: A comprehensive review.

Benzyl isothiocyanate (BITC) is one of the common isothiocyanates found in cruciferous vegetables such as broccoli, cabbage or watercress. Preclinical studies report of its effectiveness in the prevention and treatment against several cancers. This review aims to report and discuss findings on anticancer activities of BITC and its modes of action against 14 types of cancer. A literature search was conducted using the keywords "BITC" and "anticancer" from PubMed, Google Scholar and CINAHL Plus to obtain relevant research articles. This review highlights the anticancer efficacy of BITC through modulation of various signaling pathways involved in apoptosis, cell proliferation, cell cycle arrest, metastasis, angiogenesis, autophagy and the effects of BITC in combination with other drugs. With the available pharmacology evidence, we conclude that further studies are needed to validate its effectiveness in humans for further development and translation into prophylaxis or therapy by promoting optimal therapeutic effects and minimizing toxicity in cancer treatment.

High intraluminal pressure promotes vascular inflammation via caveolin-1.

The aetiology and progression of hypertension involves various endogenous systems, such as the renin angiotensin system, the sympathetic nervous system, and endothelial dysfunction. Recent data suggest that vascular inflammation may also play a key role in the pathogenesis of hypertension. This study sought to determine whether high intraluminal pressure results in vascular inflammation. Leukocyte adhesion was assessed in rat carotid arteries exposed to 1 h of high intraluminal pressure. The effect of intraluminal pressure on signaling mechanisms including reactive oxygen species production (ROS), arginase expression, and NFĸB translocation was monitored. 1 h exposure to high intraluminal pressure (120 mmHg) resulted in increased leukocyte adhesion and inflammatory gene expression in rat carotid arteries. High intraluminal pressure also resulted in a downstream signaling cascade of ROS production, arginase expression, and NFĸB translocation. This process was found to be angiotensin II-independent and mediated by the mechanosensor caveolae, as caveolin-1 (Cav1)-deficient endothelial cells and mice were protected from pressure-induced vascular inflammatory signaling and leukocyte adhesion. Cav1 deficiency also resulted in a reduction in pressure-induced glomerular macrophage infiltration in vivo. These findings demonstrate Cav1 is an important mechanosensor in pressure-induced vascular and renal inflammation.

Opioid Receptor-Mediated and Non-Opioid Receptor-Mediated Roles of Opioids in Tumour Growth and Metastasis.

Opioids are administered to cancer patients in the period surrounding tumour excision, and in the management of cancer-associated pain. The effects of opioids on tumour growth and metastasis, and their consequences on disease outcome, continue to be the object of polarised, discrepant literature. It is becoming clear that opioids contribute a range of direct and indirect effects to the biology of solid tumours, to the anticancer immune response, inflammation, angiogenesis and importantly, to the tumour-promoting effects of pain. A common misconception in the literature is that the effect of opioid agonists equates the effect of the mu-opioid receptor, the major target of the analgesic effect of this class of drugs. We review the evidence on opioid receptor expression in cancer, opioid receptor polymorphisms and cancer outcome, the effect of opioid antagonists, especially the peripheral antagonist methylnaltrexone, and lastly, the evidence available of a role for opioids through non-opioid receptor mediated actions.

Caveola-forming proteins and prostate cancer.

Caveolae are specialised and dynamic plasma membrane subdomains, involved in many cellular functions including endocytosis, signal transduction, mechanosensing and lipid storage, trafficking, and metabolism. Two protein families are indispensable for caveola formation and function, namely caveolins and cavins. Mutations of genes encoding these caveolar proteins cause serious pathological conditions such as cardiomyopathies, skeletal muscle diseases, and lipodystrophies. Deregulation of caveola-forming protein expression is associated with many types of cancers including prostate cancer. The distinct function of secretion of the prostatic fluid, and the unique metabolic phenotype of prostate cells relying on lipid metabolism as a main bioenergetic pathway further suggest a significant role of caveolae and caveolar proteins in prostate malignancy. Accumulating in vitro, in vivo, and clinical evidence showed the association of caveolin-1 with prostate cancer grade, stage, metastasis, and drug resistance. In contrast, cavin-1 was found to exhibit tumour suppressive roles. Studies on prostate cancer were the first to show the distinct function of the caveolar proteins depending on their localisation within the caveolar compartment or as cytoplasmic or secreted proteins. In this review, we summarise the roles of caveola-forming proteins in prostate cancer and the potential of exploiting them as therapeutic targets or biological markers.

Matrix protease production, epithelial-to-mesenchymal transition marker expression and invasion of glioblastoma cells in response to osmotic or hydrostatic pressure.

Both hydrostatic and osmotic pressures are altered in the tumour microenvironment. Glioblastoma (GBM) is a brain tumour with high invasiveness and poor prognosis. We hypothesized that physical and osmotic forces regulate glioblastoma (GBM) invasiveness. The osmotic pressure of GBM cell culture medium was adjusted using sodium chloride or water. Alternatively, cells were subjected to increased hydrostatic force. The proteolytic profile and epithelial-mesenchymal transition (EMT) were investigated using zymography and real-time qPCR. The EMT markers assessed were Snail-1, Snail-2, N-cadherin, Twist and vimentin. Invasion was investigated in vitro using extracellular matrix-coated Transwell inserts. In response to osmotic and mechanical pressure, GBM cell lines U87 and U251 and patient-derived neural oncospheres upregulated the expression of urokinase-type plasminogen activator (uPA) and/or matrix metalloproteinases (MMPs) as well as some of the EMT markers tested. The adherent cell lines invaded more when placed in media of increased osmolality. Therefore, GBM respond to osmotic or mechanical pressure by increasing matrix degrading enzyme production, and adopting a phenotype reminiscent of EMT. Better understanding the molecular and cellular mechanisms by which increased pressure promotes GBM invasiveness may help to develop innovative therapeutic approaches.

A role for caveola-forming proteins caveolin-1 and CAVIN1 in the pro-invasive response of glioblastoma to osmotic and hydrostatic pressure.

In solid tumours, elevated interstitial fluid pressure (osmotic and hydrostatic pressure) is a barrier to drug delivery and correlates with poor prognosis. Glioblastoma (GBM) further experience compressive force when growing within a space limited by the skull. Caveolae are proposed to play mechanosensing roles, and caveola-forming proteins are overexpressed in GBM. We asked whether caveolae mediate the GBM response to osmotic pressure. We evaluated in vitro the influence of spontaneous or experimental down-regulation of caveola-forming proteins (caveolin-1, CAVIN1) on the proteolytic profile and invasiveness of GBM cells in response to osmotic pressure. In response to osmotic pressure, GBM cell lines expressing caveola-forming proteins up-regulated plasminogen activator (uPA) and/or matrix metalloproteinases (MMPs), some EMT markers and increased their in vitro invasion potential. Down-regulation of caveola-forming proteins impaired this response and prevented hyperosmolarity-induced mRNA expression of the water channel aquaporin 1. CRISPR ablation of caveola-forming proteins further lowered expression of matrix proteases and EMT markers in response to hydrostatic pressure, as a model of mechanical force. GBM respond to pressure by increasing matrix-degrading enzyme production, mesenchymal phenotype and invasion. Caveola-forming proteins mediate, at least in part, the pro-invasive response of GBM to pressure. This may represent a novel target in GBM treatment.

Structure-based engineering of pH-dependent antibody binding for selective targeting of solid-tumor microenvironment.

Recent development of monoclonal antibodies as mainstream anticancer agents demands further optimization of their safety for use in humans. Potent targeting and/or effector activities on normal tissues is an obvious toxicity concern. Optimization of specific tumor targeting could be achieved by taking advantage of the extracellular acidity of solid tumors relative to normal tissues. Here, we applied a structure-based computational approach to engineer anti-human epidermal growth factor receptor 2 (Her2) antibodies with selective binding in the acidic tumor microenvironment. We used an affinity maturation platform in which dual-pH histidine-scanning mutagenesis was implemented for pH selectivity optimization. Testing of a small set of designs for binding to the recombinant Her2 ectodomain led to the identification of antigen-binding fragment (Fab) variants with the desired pH-dependent binding behavior. Binding selectivity toward acidic pH was improved by as much as 25-fold relative to the parental bH1-Fab. In vitro experiments on cells expressing intact Her2 confirmed that designed variants formatted as IgG1/k full-size antibodies have high affinity and inhibit the growth of tumor spheroids at a level comparable to that of the benchmark anti-Her2 antibody trastuzumab (Herceptin®) at acidic pH, whereas these effects were significantly reduced at physiological pH. In contrast, both Herceptin and the parental bH1 antibody exhibited strong cell binding and growth inhibition irrespective of pH. This work demonstrates the feasibility of computational optimization of antibodies for selective targeting of the acidic environment such as that found in many solid tumors.

Corrigendum: Morphine Binds Creatine Kinase B and Inhibits Its Activity.

[This corrects the article DOI: 10.3389/fncel.2018.00464.].