Antibody Fab Research Articles

Advances in chaperone-assisted RNA crystallography using synthetic antibodies

RNA molecules play essential roles in many biological functions, from gene expression regulation, cellular growth, and metabolism to catalysis. They frequently fold into three-dimensional structures to perform their functions. Therefore, determining RNA structure represents a key step for understanding the structure-function relationships and developing RNA-targeted therapeutics. X-ray crystallography remains a method of choice for determining high-resolution RNA structures, but it has been challenging due to difficulties associated with RNA crystallization and phasing. Several natural and synthetic RNA binding proteins have been used to facilitate RNA crystallography. Having unique properties to help crystal packing and phasing, synthetic antibody fragments, specifically the Fabs, have emerged as promising RNA crystallization chaperones, and so far, over a dozen of RNA structures have been solved using this strategy. Nevertheless, multiple steps in this approach need to be improved, including the recombinant expression of these anti-RNA Fabs, to warrant the full potential of these synthetic Fabs as RNA crystallization chaperones. This review highlights the nuts and bolts and recent advances in the chaperone-assisted RNA crystallography approach, specifically emphasizing the Fab antibody fragments as RNA crystallization chaperones.

Cannabis detection with solid sensors and paper-based immunoassays by conjugating antibodies to nanocellulose

Highly sensitive and specific diagnostics for cannabis usage are essential for rapid on-site screening for illicit drug usage. To improve the sensitivity of THC immunoassays, a proper immobilization of the sensing elements on the sensor substrate is critical. In this work, we demonstrated the utilization of EDC/NHS coupling chemistry with nanocellulose to obtain efficient anchor layers for the immobilization of anti-immune complex antibodies on surfaces. In our approach, the high surface-to-volume ratio, OH-group-rich surface, and high hygroscopicity of TOCNF enable efficient surface functionalization and enhance water permeation inside the nanocellulose network structure, offering a hydrophilic spacer for the sensing antibodies. THC detection was shown in both SPR (surface plasmon resonance technique) and paper-based sensing systems. In SPR, antibody immobilization and the related interactions with the target molecule complex with 1–10 μg/mL THC were followed in-situ in aqueous environment, revealing robust attachment of the antibody to the nanocellulose layer and preserved bioactivity. Additionally, quantitative THC detection was enabled on paper substrate by colorimetric means by employing labeled anti-THC Fab antibody fragments as detection antibodies. THC detection efficiency of covalently linked biointerface was superior compared to the performance of physically linked biointerface. The chemical conjugation of anti-IC to nanocellulose allowed efficient binding, whereas supramolecular conjugation led to insufficient binding, highlighting the relevance of the developed nanocellulose-based anchor layer.

Assessing and Engineering Antibody Stability Using Experimental and Computational Methods.

Engineering increased stability into antibodies can improve their developability. While a range of properties need to be optimized, thermal stability and aggregation are two key factors that affect the antibody yield, purity, and specificity throughout the development and manufacturing pipeline. Therefore, an ideal goal would be to apply protein engineering methods early-on, such as in parallel to affinity maturation, to screen out potential drug molecules with the desired conformational and colloidal stability. This chapter introduces our methods to computationally characterize an antibody Fab fragment, propose stabilizing variants, and then experimentally verify these predictions.

Anti-c-MET Fab-Grb2-Gab1 Fusion Protein-Mediated Interference of c-MET Signaling Pathway Induces Methuosis in Tumor Cells.

Bio-macromolecules have potential applications in cancer treatment due to their high selectivity and efficiency in hitting therapeutic targets. However, poor cell membrane permeability has limited their broad-spectrum application in cancer treatment. The current study developed highly internalizable anti-c-MET antibody Fab fusion proteins with intracellular epitope peptide chimera to achieve the dual intervention from the extracellular to intracellular targets in tumor therapy. In vitro experiments demonstrated that the fusion proteins could interfere with the disease-associated intracellular signaling pathways and inhibit the uncontrolled proliferation of tumor cells. Importantly, investigation of the underlying mechanism revealed that these protein chimeras could induce vacuolation in treated cells, thus interfering with the normal extension and arrangement of microtubules as well as the mitosis, leading to the induction of methuosis-mediated cell death. Furthermore, in vivo tumor models indicated that certain doses of fusion proteins could inhibit the A549 xenograft tumors in NOD SCID mice. This study thus provides new ideas for the intracellular delivery of bio-macromolecules and the dual intervention against tumor cell signaling pathways.

Molecular imaging of experimental atherosclerosis using anti-malondialdehyde-modified low-density lipoprotein humanised antibody fragment targeted nanoparticles

Abstract Introduction Oxidative modification of low-density lipoprotein (LDL), for example by malondialdehyde (MDA) adduction with subsequent uptake by macrophages to form foam cells and later the plaque necrotic core, is a key initiating event in atherogenesis. Accordingly, a larger lipid necrotic core is a key plaque vulnerability factor, predisposing plaques to rupture and subsequent thrombosis and development of an acute coronary syndrome. Thus, MDA-LDL is an attractive focus for the molecular targeting of atherosclerotic plaques. Purpose To develop antibody fragment-targeted nanoparticles that can be utilised for both the molecular imaging and therapeutics of vulnerable atherosclerotic plaques. Methods LO1 is an IgG3k natural monoclonal murine antibody that reacts with MDA-LDL. Humanised LO1Fab fragments have been engineered to reduce immunogenicity and improve lesion penetration. These humanised LO1Fab fragments were used to functionalise fluorescent poly(lactic-co-glycolic acid) (PLGA) - polyethylene glycol (PEG) nanoparticles. Nanoparticle in vitro function was assessed, prior to fluorescence molecular tomography (FMT) co-registered with micro-CT, four-hours after iv injection in atherosclerotic LDL-receptor−/− mice fed a high-fat diet for 40-weeks. Results Humanised LO1Fab fragment conjugated fluorescent PLGA-PEG nanoparticles were formulated with 210nm size and polydispersity index (variability of nanoparticle size around the average) of <0.2. Antibody conjugation efficiency was 30%. In vitro function was confirmed on ELISA versus the blank untargeted nanoparticles with MDA-LDL on solid phase, detecting nanoparticle presence via the conjugated LO1Fab, PEG corona or fluorescence. Fluorescence microscopy on stained aortic root cryosections from atherosclerotic mice confirmed binding to fatty lesions. Construct in vivo in half-life was 90-minutes for both the targeted and untargeted nanoparticles in a two-phase model in LDL-receptor−/− mice, based on fluorescence analysis of serial tail vein blood samples. There was greater uptake in the region-of-interest (heart and aortic arch vessels) in mice injected with LO1Fab-conjugated nanoparticles versus untargeted nanoparticles (mean ± standard deviation) (64.7±22.9 versus 25.2±26.5pmol of Cy5; n=7; p=0.02). Ex vivo analysis fluorescence reflectance imaging and quantitative FMT of the extracted aortae confirmed these findings (1.0±0.3 versus 0.5±0.2pmol of Cy5; n=7; p=0.002; Figure 1). Conclusions Humanised antibody Fab fragment fluorescent nanoparticles have been developed that successfully target MDA-LDL and localise to atherosclerotic plaques in murine experimental atherosclerosis. These targeted nanoparticles have the potential to amplify fluorescent signal for imaging and carry a therapeutic cargo for targeted drug delivery direct to atherosclerotic plaques. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Wellcome Trust Clinical Research Fellowship

Simultaneous crosslinking of CD20 and CD38 receptors by drug-free macromolecular therapeutics enhances B cell apoptosis in vitro and in vivo.

Drug-Free Macromolecular Therapeutics (DFMT) is a new paradigm in macromolecular therapeutics that induces apoptosis in target cells by crosslinking receptors without the need of low molecular weight drugs. Programmed cell death is initiated via a biomimetic receptor crosslinking strategy using a two-step approach: i) recognition of cell surface antigen by a morpholino oligonucleotide-modified antibody Fab' fragment (Fab'-MORF1), ii) followed by crosslinking with a multivalent effector motif - human serum albumin (HSA) grafted with multiple complementary morpholino oligonucleotides (HSA-(MORF2)x). This approach is effective in vitro, in vivo, and ex vivo on cells from patients diagnosed with various B cell malignancies. We have previously demonstrated DFMT can be applied to crosslink CD20 and CD38 receptors to successfully initiate apoptosis. Herein, we show simultaneous engagement, and subsequent crosslinking of both targets ("heteroreceptor crosslinking"), can further enhance the apoptosis induction capacity of this system. To accomplish this, we incubated Raji (CD20+; CD38+) cells simultaneously with anti-CD20 and anti-CD38 Fab'-MORF1 conjugates, followed by addition of the macromolecular crosslinker, HSA-(MORF2)x to co-cluster the bound receptors. Fab' fragments from Rituximab and Obinutuzumab were employed in the synthesis of anti-CD20 bispecific engagers (Fab'RTX-MORF1 and Fab'OBN-MORF1), whereas Fab' fragments from Daratumumab and Isatuximab (Fab'DARA-MORF1 and Fab'ISA-MORF1) targeted CD38. All heteroreceptor crosslinking DFMT combinations demonstrated potent apoptosis induction and exhibited synergistic effects as determined by Chou-Talalay combination index studies (CI<1). In vitro fluorescence resonance energy transfer (FRET) experiments confirmed the co-clustering of the two receptors on the cell surface in response to the combination treatment. The source of this synergistic therapeutic effect was further explored by evaluating the effect of combination DFMT on key apoptosis signaling events such as mitochondrial depolarization, caspase activation, lysosomal enlargement, and homotypic cell adhesion. Finally, a xenograft mouse model of CD20+/CD38+ Non Hodgkin lymphoma was employed to demonstrate in vivo the enhanced efficacy of the heteroreceptor-crosslinking DFMT design versus single-target systems.

Activated Charcoal Hemadsorption in Cerbera odollam Poisoning

Cerbera odollam belongs to the poisonous Apocynaceae family, grows in coastal salt swamps, creeks and along river banks found in South India. The poisonous part of the plant is the kernel containing the toxin cerberin which is a cardiac glycoside that produces symptoms and signs similar to acute digoxin poisoning. The treatment is mainly symptomatic as well as administration of digoxin immune fab antibodies. Digoxin immune fab antibodies bind with cardiac glycosides to reduce their active concentration. However, there could be various barriers in developing countries in regard to the administration of DigiFab antibodies such as availability and affordability. Activated charcoal, a cheaper alternative, reduces gastrointestinal absorption and enterohepatic recirculation. Although its role in C. odollam poisoning remains unclear, there have been various trials where multidose activated charcoal has been beneficial in glycoside poisoning. We present a case report of a young female with an alleged history of consumption of an unknown plant compound, identified as C. odollam presenting with acute-onset vomiting, altered mental status and laboratory parameters showed hyperkalemia and thrombocytopenia. Electrocardiogram (ECG) showed sinus bradycardia followed by inverted tick mark sign. Serum digoxin levels were raised (4.2 ng/ml). The patient was stabilized with supportive measures and hemadsorption with 300 g of cellulose-coated activated charcoal, which led to the reversal of the ECG and laboratory parameters to normal, leading to the complete recovery of the patient.

A case series of oleander poisoning: challenges faced by emergency physicians in developing countries

Objective: Through the reporting of this case series, we aim to establish whether a conservative approach, through managing arrhythmias and vital signs, can be reliably used as a treatment modality for oleander poisoning in developing countries.&#x0D; &#x0D; Methods: This study is a case series of 11 patients who presented with oleander poisoning and were conservatively managed in the absence of standard antidote.&#x0D; &#x0D; Results: All 11 patients treated with conservative approach survived. Conservative approach included use of atropine for management of symptomatic bradycardia followed by Dopamine infusion, correction of serum potassium and magnesium levels, standby defibrillation, and transvenous pacing.&#x0D; &#x0D; Conclusion: The absence of reliable dosage of poison ingested, the lack of facilities for serum digoxin estimation, and the unavailability of digoxin fab antibodies pose challenges for the management of patients with oleander poisoning. Patients can, however, be managed conservatively following the Advanced Cardiac Life Support (ACLS) algorithm in a setting that lacks the standard treatment of this poison.

Anti-Human CD9 Fab Fragment Antibody Blocks the Extracellular Vesicle-Mediated Increase in Malignancy of Colon Cancer Cells.

Intercellular communication between cancer cells themselves or with healthy cells in the tumor microenvironment and/or pre-metastatic sites plays an important role in cancer progression and metastasis. In addition to ligand–receptor signaling complexes, extracellular vesicles (EVs) are emerging as novel mediators of intercellular communication both in tissue homeostasis and in diseases such as cancer. EV-mediated transfer of molecular activities impacting morphological features and cell motility from highly metastatic SW620 cells to non-metastatic SW480 cells is a good in vitro example to illustrate the increased malignancy of colorectal cancer leading to its transformation and aggressive behavior. In an attempt to intercept the intercellular communication promoted by EVs, we recently developed a monovalent Fab fragment antibody directed against human CD9 tetraspanin and showed its effectiveness in blocking the internalization of melanoma cell-derived EVs and the nuclear transfer of their cargo proteins into recipient cells. Here, we employed the SW480/SW620 model to investigate the anti-cancer potential of the anti-CD9 Fab antibody. We first demonstrated that most EVs derived from SW620 cells contain CD9, making them potential targets. We then found that the anti-CD9 Fab antibody, but not the corresponding divalent antibody, prevented internalization of EVs from SW620 cells into SW480 cells, thereby inhibiting their phenotypic transformation, i.e., the change from a mesenchymal-like morphology to a rounded amoeboid-like shape with membrane blebbing, and thus preventing increased cell migration. Intercepting EV-mediated intercellular communication in the tumor niche with an anti-CD9 Fab antibody, combined with direct targeting of cancer cells, could lead to the development of new anti-cancer therapeutic strategies.

D-VITylation: Harnessing the biology of vitamin D to improve the pharmacokinetic properties of peptides and small proteins

Peptides have great potential to be potent and specific therapeutics, yet their small size leads to rapid glomerular filtration, which severely limits therapeutic applications. Although conjugation of small proteins to large polymers typically results in longer residence times, these conjugates often have a significant loss of biological activity due to steric hindrance. Here, we improve the pharmacokinetics (PK) of peptide therapeutics by harnessing the biology of vitamin D. Attachment of a small vitamin D-based molecule (D-VITylation) protects the conjugated peptide or protein from renal clearance by virtue of reversible binding to the serum-circulating vitamin D binding protein (DBP), without compromising bioactivity. Varying the conjugation site on vitamin D affects the binding to DBP, with higher affinity corresponding to a longer plasma half-life. We also demonstrate the important contribution of the peptide to the overall PK, likely due to alternative clearance mechanisms such as protease degradation and receptor-mediated cellular uptake. With a Fab antibody fragment, for which these alternate clearance mechanisms are not significant, D-VITylation increases the half-life of elimination from 14 to 61 h in rats. The PK profile in minipigs and projected lifetime in humans suggest that D-VITylation is a viable strategy to achieve once-weekly dosing of peptide therapeutics in humans.

Investigation of SARS-CoV-2 nucleocapsid protein interaction with a specific antibody by combined spectroscopic ellipsometry and quartz crystal microbalance with dissipation

Detailed evaluations of the antigen and antibody interaction rate and strength of the immune complex formed are very important for medical and bioanalytical applications. These data are crucial for the development of sensitive and fast immunosensors suitable for continuous measurements. Therefore, combined spectroscopic ellipsometry (SE) and quartz crystal microbalance with dissipation (QCM-D) technique (SE/QCM-D) was used for the evaluation: (i)of covalent immobilization of SARS-CoV-2 nucleocapsid protein (SCoV2-N) on QCM-D sensor disc modified by self-assembled monolayer based on 11-mercaptoundecanoic acid and (ii)interaction of immobilized SCoV2-N with specific polyclonal anti-SCoV2-N antibodies followed by immune complex formation process. The results show that the SCoV2-N monolayer is rigid due to the low energy dissipation registered during the QCM-D measurement. In contrast, the anti-SCoV2-N layer produced after interaction with the immobilized SCoV2-N formed a soft and viscous layer. It was determined, that the sparse distribution of SCoV2-N on the surface affected the spatial arrangement of the antibody during the formation of immune complexes. The hinge-mediated flexibility of the antibody Fab fragments allows them to reach the more distantly located SCoV2-N and establish a bivalent binding between proteins in the formed SCoV2-N/anti-SCoV2-N complex. It was noted that the SE/QCM-D method can provide more precise quantitative information about the flexibility and conformational changes of antibody during the formation of the immune complex on the surface over time.

Structural and functional analysis of a potent human neutralizing antibody against enterovirus A71.

Enterovirus A71 (EV-A71) causes major outbreaks of hand, foot, and mouth disease (HFMD) in many countries, most frequently affecting children, and a small proportion of cases may lead to death. Currently, no vaccine is available in most endemic regions, and no licenced treatments for EV-A71 infection are available. Here, we characterize a human monoclonal antibody (HuMAb), E1, by screening a Fab antibody phage library derived from patients who recovered from EV-A71 infection. E1 exhibits strong neutralizing activity against EV-A71 virus in cells. The cryo-electron microscopy (cryo-EM) structures of the EV-A71 virion in complex with E1 Fab fragments demonstrated that E1 recognized an epitope formed by residues in the BC and HI loops of VP1. In a mouse model, E1 effectively protected against lethal EV-A71 challenge in both prophylactic and therapeutic treatment. In particular, E1 significantly reduces virus titers and muscle damage. E1 might represent a potential adjunct to EV-A71 treatment.Supporting InformationThe supporting information is available online at 10.1007/s11427-021-2095-0. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

Antibody homotypic interactions are encoded by germline light chain complementarity determining region 2

SignificanceWeak transient interactions are fundamental to immune responses, enabling avidity-driven triggers for pathogen neutralization and cellular regulation. In contrast to obligate binding interactions that can be directly investigated structurally, the low or transitory abundance of weak interactions make them difficult to identify and characterize. This study leverages receptor agonism systems that are sensitive to oligomerization to investigate transient homotypic interfaces between antibody Fab regions. Our results show that self-association determinants are encoded naturally by the antibody germline through light chain complementarity determining region 2 (CDRL2), and these determinants can be engineered into antibodies to enhance their therapeutic properties. Insights into avidity-driven interactions create opportunities for optimization, and accordingly this work expands the engineering toolbox for antibody-based drugs.

Structural Insights into Phospholipase Cε

PLC (phospholipase C) enzymes hydrolyze phosphatidylinositol lipids to generate inositol phosphates (IPx) and diacylglycerol (DAG). These second messengers stimulate intracellular calcium release and protein kinase C activation. PLCε is the most recently identified member of the PLC family, and is activated downstream of G protein‐coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) through direct interactions with G proteins. In the cardiovascular system, PLCε is necessary for normal cardiac contractility, and its dysregulation contributes to cardiac hypertrophy and heart failure. PLCε shares a highly conserved core that is observed in other PLC subfamilies, and contains a unique N‐terminal CDC25 domain and two C‐terminal Ras association (RA) domains. However, structural insights into the full‐length protein, or catalytically active fragments that contain two or more regulatory domains, have been lacking. Using a combination of structural and functional studies, we previously showed that the PH domain and first two EF hands are dynamic in solution, providing an explanation as to why high‐resolution structures have been elusive. We are now pursuing high‐resolution structural studies of larger PLCε variants and the full‐length enzyme using single particle cryo‐electron microscopy (cryo‐EM). To facilitate this process, we are utilizing domain‐specific fab antibodies as fiduciary markers and to stabilize this conformationally heterogeneous protein. Understanding the structure of PLCε will be critical in identifying sites that can be targeted by small molecule chemical probes, ultimately validating this enzyme as a therapeutic target in cardiovascular disease.

Dynamic features of tapasin as revealed by structures of two tapasin/Fab complexes

Abstract Intracellular loading of major histocompatibility complex class I (MHC-I) molecules occurs within the peptide loading complex and is accomplished through the concerted action of several proteins, including the chaperone/catalyst tapasin. In the absence of tapasin, peptide loading is compromised, resulting in decreased cell surface expression of some, though not all, MHC-I allelomorphs. To gain a mechanistic understanding of tapasin’s function we have produced recombinant Fab fragments of two anti-tapasin antibodies, PaSta1 and PaSta2, and investigated their binding to human recombinant soluble tapasin by surface plasmon resonance methods and X-ray crystallography. Both antibody Fabs bind with nanomolar affinities characterized by slow off rates. PaSta1 and PaSta2 recognize distinct epitopes as tapasin captured on a surface immobilized with one PaSta Fab binds the other PaSta Fab with nanomolar affinity. However, tapasin captured on a PaSta2 Fab surface is not able to bind peptide/HLA-B*44:05/beta-2 microglobulin complexes while tapasin captured on a PaSta1 Fab surface can, indicating that PaSta2 sterically competes for the MHC-I binding site. This finding was echoed in the structural characterization of PaSta Fab-tapasin complexes by X-ray crystallography. PaSta1 binds to an extended N-terminal region of tapasin, a site accessible even in the tapasin/ERp57 complex. By contrast, PaSta2 binds at the junction of the N-terminal and C-terminal domains of tapasin, where tapasin may interact with HLA-I molecules. Comparison of conformations of tapasin in complex with PaSta1, PaSta2, HLA-B*44:05, and ERp57 reveals the dynamic nature of tapasin, a versatile chaperone and catalyst. Supported by the intramural research program of the NIAID, NIH

Modular Cloning of MUC1 Recombinant Antibodies by Assembly of Synthetic Domain Genes

Antibody immunotherapy has emerged as a constructive way to fight cancer. New immunotherapies require the identification of a tumor‐specific antigen. The protein mucin 1 (MUC1) has long been recognized as an important specific antigen and a potential immunotherapy target. In healthy human tissues, MUC1 is responsible for protecting the epithelial lining. However, in cancer cells, MUC1 displays truncated glycosylation, revealing tumor‐specific epitopes. Recombinant antibodies are preferred for therapeutic development, and in some cases, it is desirable to produce different antibody formats, including intact IgG or Fab fragments. Using the MUC1 specific antibody 4H5 as a model, we have developed a modular cloning approach that can rapidly produce different antibody modalities to facilitate the rapid production of both IgG and Fab antibody formats. The 4H5 variable domains were PCR amplified to contain 20 base‐pair (bp) overlaps with the pcDNA3.1 vector on the 5’ end, and 20 bp overlaps to either the human CH1 or Ck domains on the 3’end of the genes. To produce the Fab, a His‐tag and a 20 base‐pair (bp) overlap to the vector was added to the 3’ end of a human CH1 domain gene sequence. For IgG production, human CH1‐CH2‐CH3 domains were PCR amplified, and the same vector overlap sequence was added to the 3’ end of the sequence. Finally, a 20 bp overlap to the vector was added to the 3’ end of a human Ck domain. The PCR products were purified, and the constructs were assembled using the NEBuilder®HiFi DNA Assembly Cloning Kit. DNA sequencing confirmed the successful assembly of the coding sequence, and the 4H5 Fab fragments and IgG were produced by transient transfection in CHO cells. The antibodies (Fab and IgG) were purified by affinity chromatography and binding to recombinant MUC1 antigens confirmed by ELISA. The cloning method presented here simultaneously produces multiple antibody formats. This approach will permit rapid assembly and screening of different antibody formats and isotypes for therapeutic antibody discovery and analysis.

Direct Affinity Purification of Long-Acting PASylated Proteins with Therapeutic Potential Using L-Prolinamide for Mild Elution.

Both insufficient plasma half‐life (circulation for only few hours or less) and laborious downstream purification can be bottleneck for biological drug development. We report a novel strategy for the efficient and gentle affinity purification of pharmacologically relevant proteins modified by PASylation for prolonged action in vivo. We previously described antibodies specific for Pro/Ala‐rich sequences (PAS) covering a range of binding characteristics. Our present approach relies on a chromatography matrix functionalized with a low‐affinity PAS‐specific antibody Fab fragment for specific adsorption of the PASylated protein from a macromolecular mixture. With the complete absence of hydrophobic/aromatic or ionic groups in the PAS sequence epitope, binding is mediated by Van der Waals contacts and distinct hydrogen bonds only. Surprisingly, selective competitive elution is achieved by application of the highly soluble and biologically inactive imino acid derivative L‐prolinamide. Based on the specific but strongly dynamic biomolecular interaction, our procedure allows the direct one‐step purification of PASylated proteins from a cell extract or culture supernatant while avoiding harsh elution conditions as they are often needed for conventional affinity chromatography.

Direkte Affinitätsreinigung lang wirksamer PASylierter Proteine mit therapeutischem Anwendungspotential unter Nutzung von L‐Prolinamid zur milden Elution

AbstractSowohl unzureichende Plasma‐Halbwertszeit (Zirkulation für nur wenige Stunden oder kürzer) als auch ein aufwendiger Reinigungsprozeß können Flaschenhals sein für die biologische Wirkstoffentwicklung. Wir berichten über eine neuartige Strategie zur effizienten und schonenden Afinitätsreinigung pharmakologisch relevanter Proteine, die durch PASylierung hinsichtlich verlängerter Aktivität in vivo modifiziert wurden. Zuvor hatten wir Antikörper mit Spezifität für Pro/Ala‐reiche Sequenzen (PAS) beschrieben, die ein Spektrum an Bindungseigenschaften abdecken. Unser Ansatz hier beruht auf einer Chromatographiematrix, die mit einem niedrigaffinen PAS‐spezifischen Fab‐Fragment zum Zweck der spezifischen Adsorption des PASylierten Proteins aus einer makromolekularen Mischung funktionalisiert ist. Aufgrund vollständiger Abwesenheit hydrophober/aromatischer oder ionischer Gruppen in dem PAS‐Sequenzepitop wird die Bindung allein durch Van‐der‐Waals‐Kontakte und charakteristische Wasserstoffbrückenbindungen vermittelt. Überraschenderweise kann die selektive kompetitive Elution durch Anwendung des hochlöslichen und biologisch inaktiven Iminosäurederivats L‐Prolinamid erreicht werden. Basierend auf der spezifischen, aber hochdynamischen biomolekularen Wechselwirkung gestattet unser Verfahren die direkte Einschrittreinigung PASylierter Proteine aus einem Zellextrakt oder Kulturüberstand, wobei grobe Elutionsbedingungen, wie sie häufig für die konventionelle Affinitätschromatographie erforderlich sind, vermieden werden.

Improved Characteristics of RANKL Immuno-PET Imaging Using Radiolabeled Antibody Fab Fragments.

Purpose: RANKL expression in the tumor microenvironment has been identified as a biomarker of immune suppression, negating the effect of some cancer immunotherapies. Previously we had developed a radiotracer based on the FDA-approved RANKL-specific antibody denosumab, [89Zr]Zr-DFO-denosumab, enabling successful immuno-PET imaging. Radiolabeled denosumab, however, showed long blood circulation and delayed tumor uptake, potentially limiting its applications. Here we aimed to develop a smaller radiolabeled denosumab fragment, [64Cu]Cu-NOTA-denos-Fab, that would ideally show faster tumor accumulation and better diffusion into the tumor for the visualization of RANKL. Experimental design: Fab fragments were prepared from denosumab using papain and conjugated to a NOTA chelator for radiolabeling with 64Cu. The bioconjugates were characterized in vitro using SDS-PAGE analysis, and the binding affinity was assessed using a radiotracer cell binding assay. Small animal PET imaging evaluated tumor targeting and biodistribution in transduced RANKL-ME-180 xenografts. Results: The radiolabeling yield of [64Cu]Cu-NOTA-denos-Fab was 58 ± 9.2%, with a specific activity of 0.79 ± 0.11 MBq/µg (n = 3). A radiotracer binding assay proved specific targeting of RANKL in vitro. PET imaging showed fast blood clearance and high tumor accumulation as early as 1 h p.i. (2.14 ± 0.21% ID/mL), which peaked at 5 h p.i. (2.72 ± 0.61% ID/mL). In contrast, [64Cu]Cu-NOTA-denosumab reached its highest tumor uptake at 24 h p.i. (6.88 ± 1.12% ID/mL). [64Cu]Cu-NOTA-denos-Fab specifically targeted human RANKL in transduced ME-180 xenografts compared with the blocking group and negative ME-180 xenograft model. Histological analysis confirmed RANKL expression in RANKL-ME-180 xenografts. Conclusions: Here, we report on a novel RANKL PET imaging agent, [64Cu]Cu-NOTA-denos-Fab, that allows for fast tumor imaging with improved imaging contrast when compared with its antibody counterpart, showing promise as a potential PET RANKL imaging tool for future clinical applications.

Safety and Efficacy of Systemic Anti-Scg3 Therapy to Treat Oxygen-Induced Retinopathy.

To circumvent possible systemic side effects, anti-angiogenic drugs targeting vascular endothelial growth factor (VEGF) for ocular neovascular diseases in adults are approved only for intravitreal administration. However, intravitreal injection itself can elicit injection-related adverse effects, and premature eyes of infants with retinopathy of prematurity (ROP) may be particularly susceptible to intravitreal injection. Therefore, an unmet clinical need is to develop safe systemic anti-angiogenic therapies for ROP. We recently reported that secretogranin III (Scg3) is a disease-restricted angiogenic factor and that systemic anti-Scg3 mAb alleviates ROP in animal models with minimal side effects on developing eyes and organs. The aim of this study is to investigate the safety and efficacy of a humanized anti-Scg3 antibody via systemic administration. We analyzed the safety and efficacy of a humanized anti-Scg3 antibody Fab fragment (hFab) delivered by intraperitoneal injection in oxygen-induced retinopathy (OIR) mice, a surrogate model of ROP. The results showed that systemic anti-Scg3 hFab effectively alleviated pathological retinal neovascularization in OIR mice with similar efficacy to the anti-VEGF drug aflibercept. Systemic aflibercept conferred significant adverse side effects in neonatal mice, including reduced body weight, abnormalities in retinal and renal development, and retarded physiological neovascularization, whereas systemic anti-Scg3 hFab elicited no such side effects. The findings suggest that systemic anti-Scg3 hFab is a safe and effective therapy for OIR and support further development for ROP treatment.