Fab Fragment Of Monoclonal Antibody Research Articles

Unraveling the inhibitory potential of fatty acids from Cola lepidota seed against monoclonal antibody Fab fragment (9F8) (3VG0) leptin antagonism and restoration of ‘satiety’ in obesity condition: insight from quantum chemical analysis, pharmacokinetics, and molecular docking

Abstract Obesity represents a significant global health issue that continues to escalate in prevalence. Interestingly, there is a less explored connection between obesity and compromised leptin function. Prior studies have highlighted the limited availability of drugs to address this issue hence, the relentless struggle against obesity persists and the need to develop new therapeutic strategies becomes necessary. In the present study, fatty acids from the seed of Cola lepidota were utilized to prevent antibody Fab fragment (9F8) (3VG0), an antagonist of leptin from binding to the leptin pocket of the human obesity receptor (ObR) thereby restoring ‘satiety’. This study is the first to investigate the effect of plant derived fatty acids from C. lepidota seed for the purpose of reversing leptin resistance in obesity condition. Our research employed experimental GCMS extraction technique and theoretical FT-IR and UV–vis analysis and compared result with those reported in literature. All computational methodologies were carried out within the framework of density functional theory (DFT) at the B3LYP/6-311++G(d,p) level of theory while molecular docking and pharmacokinetics studies were employed to investigate the biological activities and druglikeness of the compounds. Result shows that linoleic acid (LA), methylhexadecanoate (HXD), ocatadecanoic acid methyl ester (ODA) and Bis(2-ethylhexyl) phthalate (BISP) recorded energy gaps of 2.8216 eV, 7.4230 eV, 7.4244 eV, and 5.5849 eV respectively, suggesting that LA is the most reactive while BISP is the most stable as they recorded lowest and highest energy gaps respectively. The dipole moment (μ) result shows that LA recorded the highest dipole moment at 6.1119 Debye (D) indicating that it has the highest polarizability capacity. The order of polarizability is LA > BISP > HXD > ODA. The visualized electron localization function result shows that the red regions are electron rich, followed by yellow region then green and finally blue region. Electron density was distributed within the O and H atoms of the molecules indicating strong electronegativity nature of oxygen and hydrogen atoms of the compounds. LA, ODA and HXD absorbed light at the vacuum level UV region while BISP absorbed light at the UV visible region. The compounds exhibited C–H and C–O stretching vibrations except for ODA that lacks the C–O functional group. The compounds exhibited biological activities with the target receptor protein (leptin antagonist) with bis(-2ethylhexyl) phthalate (BISP) having a docking score of −4.4 kcal/mol and containing the highest number of favorable hydrogen bond interactions with LYS41, PRO42, GLN44, GLY43 residues along the polypeptide L chain and PRO173 along the polypeptide H chain of the receptor. These interactions predominantly induced conformational changes in the amino acid sequence of the protein, thereby disrupting its three-dimensional structure and mitigating the antagonistic effects at the leptin binding domain (LBD) of the human obesity receptor (ObR), thus, effectively reversing leptin resistance in obese condition. Importantly, the pharmacokinetics revealed favorable drug-like properties with no toxicity effects with respect to hepatotoxicity, immunotoxicity, cytotoxicity, mutagenicity, carcinogenicity and did not also penetrate the blood-brain barrier (BBB) or exhibit clearance delays. The therapeutic strategy presented in this study is highly thoughtful and capable of recording huge success in obesity management, thereby reducing the burden of obesity on other chronic diseases. Therefore, these compounds have positioned themselves as promising agents in leptin resistance reversal and obesity management, warranting significant interest as potential drug candidates.

The origins of nonideality exhibited by monoclonal antibodies and Fab fragments in human serum.

The development of therapeutic antibodies remains challenging, time-consuming, and expensive. A key contributing factor is a lack of understanding of how proteins are affected by complex biological environments such as serum and plasma. Nonideality due to attractive or repulsive interactions with cosolutes can alter the stability, aggregation propensity, and binding interactions of proteins in solution. Fluorescence correlation spectroscopy (FCS) can be used to measure apparent second virial coefficient (B2,app ) values for therapeutic and model monoclonal antibodies (mAbs) that capture the nature and strength of interactions with cosolutes directly in undiluted serum and similar complex biological media. Here, we use FCS-derived B2,app measurements to identify the components of human serum responsible for nonideal interactions with mAbs and Fab fragments. Most mAbs exhibit neutral or slightly attractive interactions with intact serum. Generally, mAbs display repulsive interactions with albumin and mildly attractive interactions with IgGs in the context of whole serum. Crucially, however, these attractive interactions are much stronger with pooled IgGs isolated from other serum components, indicating that the effects of serum nonideality can only be understood by studying the intact medium (rather than isolated components). Moreover, Fab fragments universally exhibited more attractive interactions than their parental mAbs, potentially rendering them more susceptible to nonideality-driven perturbations. FCS-based B2,app measurements have the potential to advance our understanding of how physiological environments impact protein-based therapeutics in general. Furthermore, incorporating such assays into preclinical biologics development may help de-risk molecules and make for a faster and more efficient development process.

Development and characterization of anti-IL-5 monoclonal antibody Fab fragment for blocking IL-5/IL-5Rα binding

Interleukin-5 (IL-5) is a homodimeric cytokine that is a crucial regulator of the proliferation, activation, and maturation of eosinophils. Anti-IL-5 monoclonal antibodies, which block the binding of IL-5 to the IL-5 receptor subunit alpha (IL-5Rα), have been successfully used to treat eosinophilic (EOS) asthma. The currently marketed monoclonal antibody drugs require repeated injections for administration, which seriously affect patient compliance and high systemic exposure for injectable drug delivery. Here we successfully screened and developed the Fab (fragment of antigen binding), which is 1/3rd the molecular weight of IgG, favoring inhalation-mediated delivery to the lungs, making it more effective for asthma treatment. The 20A12-Fab-H12L3 can bind to IL-5 with a binding constant of 1.236E-09 M while significantly inhibiting the IL-5/IL-5Rα complex formation. We found that the light chain amino acids (S46 and F71) significantly affected the antibody expression during humanization. The 20A12-Fab-H12L3 significantly inhibited the proliferation of TF-1 cells and blocked the IL-5 binding to the IL-5Rα-overexpressing human embryonic kidney (HEK)-293 cells in vitro. Therefore, based on the mutant IL-5 binding with Fab, we explained why antibodies blocked IL-5 binding to IL-5Rα. Thus, this study provided a candidate pharmaceutical antibody for inhalation drug delivery.

Amplified inhibition of atherosclerotic plaque-induced platelet activation by glenzocimab with dual antiplatelet therapy

BackgroundAspirin and platelet P2Y12 inhibitors, such as ticagrelor, suboptimally inhibit microvascular thrombosis during ST-elevation myocardial infarction. Glycoprotein (GP) IIb/IIIa inhibitors may further inhibit this but cause excessive bleeding. ObjectivesWe investigated whether combination of glenzocimab, a GPVI inhibitor, with aspirin and ticagrelor provides additional antithrombotic effects, as GPVI has a critical role in atherothrombosis but minimal involvement in hemostasis. MethodsWe investigated the effects of glenzocimab (monoclonal antibody Fab fragment) using blood from healthy donors and patients with acute coronary syndrome treated with aspirin and ticagrelor. Platelets were stimulated with multiple agonists, including atherosclerotic plaque, from patients undergoing carotid endarterectomy. ResultsAspirin and ticagrelor partially inhibited atherosclerotic plaque-induced platelet aggregation by 48% compared with control (34 ± 3 vs 65 ± 4 U; P < .001). Plaque-induced platelet aggregation, adhesion, secretion, and activation were critically dependent on GPVI activation. Glenzocimab alone reduced plaque-induced aggregation by 75% compared with control (16 ± 4 vs 65 ± 4 U; P < .001) and by >95% when combined with aspirin and ticagrelor (3 ± 1 vs 65 ± 4 U; P < .001). Glenzocimab reduced platelet aggregation, adhesion, and thrombin generation when added to blood of aspirin- and ticagrelor-treated patients with acute coronary syndrome. Glenzocimab shared several antithrombotic effects with the GPIIb/IIIa inhibitor eptifibatide with less effect on general hemostasis assessed by rotational thromboelastometry. In a murine intravital model of ST-elevation myocardial infarction, genetic depletion of GPVI reduced microvascular thrombosis. ConclusionAddition of glenzocimab to aspirin and ticagrelor enhances platelet inhibition via multiple mechanisms of atherothrombosis. Compared with a GPIIb/IIIa inhibitor, glenzocimab shares multiple antithrombotic effects, with less inhibition of mechanisms involved in general hemostasis.

Biochemical separation of Cetuximab-Fab from papain-digested antibody fragments and radiolabeling with 64Cu for potential use in radioimmunotheranostics

Engineered Fab fragments of monoclonal antibodies (mAbs) after radiolabeling with suitable radiometals have the potential to play a key role in personalized radioimmunotheranostics of cancer patients. In this study, we have generated Fab fragment of Cetuximab, a mAb targeting epidermal growth factor receptor (EGFR) expression and purified from the Fc and other fragments by ultrafiltration and affinity chromatography. The Cetuximab-Fab was conjugated with a suitable bifunctional chelator and radiolabeled with no-carrier-added (NCA) 64Cu produced via 64Zn (n, p) 64Cu reaction in a nuclear reactor. The radioimmunoconjugate obtained after size exclusion chromatographic separation possessed >95% radiochemical purity and it retained its integrity over at least three half-lives of the radiometal. Biodistribution studies was performed in fibrosarcoma tumor bearing Swiss mice, which demonstrated the explicit need for purification of the Cetuximab-Fab from Fc fragments. Enhanced and rapid tumor uptake with decent tumor-to-background ratio with prolonged retention was observed when radiolabeled purified Cetuximab-Fab was intravenously administered in animal models. Overall, this preclinical study established the pivotal role of separation science and technology to obtain the radioimmunoconjugate with requisite purity in order to demonstrate optimal pharmacokinetics and maximized treatment efficacy.

Modern trends in anti-VEGF therapy for age-related macular degeneration

Age-related macular degeneration (AMD) develops in people aged 50 years and older, its pathogenesis involves progressive destruction of the retinal pigment epithelium and Bruch's membrane. There are eight currently known anti-VEGF drugs for treating the neovascular form of AMD, four of them have already been registered and are used in clinical practice. The first registered drug was pegaptanib, which selectively blocks VEGF165. Subsequently, a molecule with a similar mechanism of action was developed and named ranibizumab, which is a humanized monoclonal Fab fragment; it was specifically designed for ophthalmology. Its advantage over pegaptanib was neutralization of all active VEGF-A isoforms. Aflibercept and conbercept are recombinant fusion proteins that act as soluble decoy receptors for VEGF family proteins. Phase III data from the VIEW 1 and 2 studies showed that intraocular injections (IVI) of aflibercept every 1 or 2 months for a year resulted in comparable functional outcomes to monthly IVI of ranibizumab for one year. The next molecule for anti-VEGF therapy that showed effectiveness was brolucizumab - a single-chain fragment of a humanized antibody that binds with high affinity to various VEGF-A isoforms. Simultaneously with studying brolucizumab, another study was conducted involving Abicipar pegol, but that drug showed a high rate of complications. The latest drug registered for the treatment of neovascular AMD is faricimab. The molecule of this drug is a humanized immunoglobulin G antibody that acts on two key points of angiogenesis: VEGF-A and angiopoietin-2 (Ang-2). Thus, the strategy for advancing anti-VEGF therapy lies in the development of molecules with greater efficiency (better effect on newly formed vessels leading to resorption of exudate in the retina, under the neuroepithelium and under the retinal pigment epithelium), which allows not just to preserve vision, but to also significantly improve it when there is no macular atrophy.

Glenzocimab: A GPVI (Glycoprotein VI)-Targeted Potential Antiplatelet Agent for the Treatment of Acute Ischemic Stroke.

It has previously been shown in several animal experiments that platelet GPVI (glycoprotein VI) contributes to thrombosis, particularly in ischemic stroke. Moreover, GPVI levels are upregulated in stroke patients. This review describes the therapeutic roles of anti-GPVI antibody in preclinical models of ischemic stroke and provides the current evidence for potential benefits of glenzocimab, a Fab fragment of humanized anti-GPVI monoclonal antibody, in stroke patients. Anti-GPVI antibody, JAQ1, significantly decreased infarct volume and improved neurological function in mice with transient middle cerebral artery occlusion, a model of ischemic stroke, with no or minor bleeding tendency. Intravenous injection of glenzocimab in nonhuman primates produced rapid inhibition of ex vivo platelet aggregation induced by collagen (a GPVI ligand). Complete platelet inhibition is observed at 30 minutes following administration without increasing the risk of bleeding. In humans, glenzocimab is well tolerated and produces dose-dependent antiplatelet activity. More importantly, glenzocimab (125-1000 mg) was safe when administered as soon as possible (<3 hours) following reperfusion with the r-tPA (recombinant tissue-type plasminogen activator) in patients with acute ischemic stroke. Although glenzocimab 1000 mg (a selected dose) did not demonstrate a significant improvement in overall clinical outcomes, it appeared to provide benefits in severe cases and in patients who required thrombectomy. This promising efficacy together with a good safety profile of glenzocimab warrant further investigation in phase III (ACTISAVE [Adaptive Efficacy and Safety Study of Glenzocimab Used as an Add-On Therapy on Top of Standard of Care in the 4.5 Hours Following an Acute Ischemic Stroke]) clinical study.

Nanobody-based microfluidic human Fc assay for preclinical plasma quantification of IgG1/1.1 and IgG1-Fc-conjugates

BackgroundTherapeutic antibodies and Fc-conjugates are becoming increasingly popular for disease management and accurate and sensitive pharmacokinetic measurements are critical in lead candidate selection in pre-clinical drug discovery. Methods and study designHuman Fc-specific intact monoclonal antibodies, polyclonal antibodies, Fab fragments, aptamers, affibodies and nanobodies were screened for potential as biotinylated capture moieties in a microfluidic assay. Test compounds were Bevacizumab, Rituximab, Infliximab as well as an in-house IgG1.1 and an IgG1-drug conjugate. ResultsCapture molecules were tested for specificity in plasma matrices from beagle dog, rat, mouse, pig, rhesus monkey and cynomolgus monkey. We find that the llama nanobody provides the best selectivity across across species. The assay usability were verified in cynomolgus monkey pharmacokinetic studies of in-house IgG1.1 and IgG1-fusion molecules. ConclusionThe presented generic nanobody-based assay may find relevance in preclinical testing of future human Fc-containing drug conjugates devoid of Fab fragments and intact monoclonal antibodies.

Overcoming purification challenges in antibody therapeutics manufacturing with subdomain-specific affinity solutions

Novel biotherapeutic antibody formats, such as bispecific monoclonal antibodies, Fab fragments, and Fc-fusion proteins have created new purification challenges in downstream processing. The use of bacterial surface proteins, such as Protein A, is not always the most efficient solution for the purification of these novel modalities. Affinity chromatography resins, specifically developed to bind antibody subdomain regions, can provide an alternative solution for a variety of antibody formats.

FcRH1: an activation coreceptor on human B cells

AbstractB-cell activation and differentiation is regulated through the coordinated function of a dynamic array of cell surface receptors. At different stages in their differentiation, human B cells may express one or more members of a large family of immunoglobulin Fc receptor homologs (FcRH) with regulatory potential. Among these newly identified transmembrane molecules, FcRH1 is unique in having 2 immunoreceptor tyrosine-based activation motif (ITAM)–like motifs in its intracellular domain. Here we used the Fab fragments of new monoclonal anti-FcRH1 antibodies and mRNA analysis to evaluate FcRH1 expression and function during B-cell differentiation. FcRH1 expression begins in pre-B cells, reaches peak levels on naive B cells, and is down-regulated after B cells are activated to begin to form germinal centers. This FcRH1 down-regulation coincides with dramatic enlargement of the pre-germinal center cells, cell cycle entry, and other overt signs of activation that include CD80 and CD86 up-regulation and immunoglobulin D (IgD) down-regulation. In vitro analysis indicates that ligation of FcRH1 leads to its tyrosine phosphorylation and to modest B-cell activation and proliferation. Concomitant FcRH1 ligation enhances B-cell antigen receptor (BCR)–induced Ca2+ mobilization and proliferation. FcRH1 thus has the potential to serve as an activating coreceptor on B cells.

Crystal Structure of a Bivalent Antibody Fab Fragment

We determined the crystal structure to 1.8 Å resolution of the Fab fragment of an affinity-matured human monoclonal antibody (HC84.26.5D) that recognizes the E2 envelope glycoprotein of hepatitis C virus (HCV). Unlike conventional Fabs, which are monovalent monomers, Fab HC84.26.5D assembles into a bivalent domain-swapped dimer in which the two VL/VH modules are separated by ~25 Å. In solution, Fab HC84.26.5D exists predominantly as a dimer (~80%) in equilibrium with the monomeric form of the Fab (~20%). Dimerization is mediated entirely by deletion of a single residue, VHSer113 (Kabat numbering), in the elbow region linking the VH and CH1 domains. In agreement with the crystal structure, dimeric Fab HC84.26.5D is able to bind two HCV E2 molecules in solution. This is only the second example of a domain-swapped Fab dimer from among >3000 Fab crystal structures determined to date. Moreover, the architecture of the doughnut-shaped Fab HC84.26.5D dimer is completely different from that of the previously reported Fab 2G12 dimer. We demonstrate that the highly identifiable shape of dimeric Fab HC84.26.5D makes it useful as a fiducial marker for single-particle cryoEM analysis of HCV E2. Bivalent domain-swapped Fab dimers engineered on the basis of HC84.26.5D may also serve as a means of doubling the effective size of conventional Fab–protein complexes for cryoEM.

Application of antibody phage display to identify potential antigenic neural precursor cell proteins

BackgroundThe discovery of neural precursor cells (NPCs) and the concomitant intensive research in the field offer regenerative medicine novel approaches, enabling it to tackle conditions, such as neurodegenerative diseases. Transplantation of NPCs is nowadays considered a cutting-edge treatment for these conditions and many related clinical trials have been already completed or are still ongoing. However, little is known about the antigenicity of NPCs, with most studies addressing the question whether their antigenicity could lead to rejection of the transplanted cells.ResultsIn this study we investigated the antigenic potential of syngeneic NPCs emulsion, upon subcutaneous (s.c.) administration to wild type C57BL/6 mice, following a standard immunization protocol. The whole IgG repertoire expressed upon immunization was cloned into a Fab phage display vector. From the created phage display library, Fab expressing clones interacting with NPCs lysate proteins were selected with the biopanning technique. The IgG Fab fragment from clone 65 proved to be reactive against antigens originating from NPCs lysates and/or whole brain lysate in diverse immunological assays.ConclusionsUsing a standard immunization protocol to administer NPCs antigens, and applying the Fab fragment phage display technique, we were able to isolate at least a monoclonal IgG Fab fragment, which interacts with different mouse brain proteins. It is not clear whether such antibodies are produced in the host organisms, following NPCs transplantation.

Structure and function of a malaria transmission blocking vaccine targeting Pfs230 and Pfs230-Pfs48/45 proteins

Proteins Pfs230 and Pfs48/45 are Plasmodium falciparum transmission-blocking (TB) vaccine candidates that form a membrane-bound protein complex on gametes. The biological role of Pfs230 or the Pfs230-Pfs48/45 complex remains poorly understood. Here, we present the crystal structure of recombinant Pfs230 domain 1 (Pfs230D1M), a 6-cysteine domain, in complex with the Fab fragment of a TB monoclonal antibody (mAb) 4F12. We observed the arrangement of Pfs230 on the surface of macrogametes differed from that on microgametes, and that Pfs230, with no known membrane anchor, may exist on the membrane surface in the absence of Pfs48/45. 4F12 appears to sterically interfere with Pfs230 function. Combining mAbs against different epitopes of Pfs230D1 or of Pfs230D1 and Pfs48/45, significantly increased TB activity. These studies elucidate a mechanism of action of the Pfs230D1 vaccine, model the functional activity induced by a polyclonal antibody response and support the development of TB vaccines targeting Pfs230D1 and Pfs230D1-Pfs48/45.

Expression of a short antibody heavy chain peptide effectively antagonizes adenosine 2A receptor in vitro and in vivo

ABSTRACT Background Adenosine 2A receptor (A2AR) is involved in many physiological and pathological functions and serves as an important drug target. Inhibition of A2AR may alleviate symptoms associated with a variety of neuropsychiatric disorders. However, the currently used A2AR antagonists have specificity limitations. Research design and methods A Fab fragment (Fab2838) of an A2AR mouse monoclonal antibody can specifically bind to A2AR to form a complex and inhibit the activity of its receptor. We constructed the vector AntiA2AR, a small-molecule peptide that binds to and inhibits A2AR based on Fab2838. Results Experiments in HEK293T cells showed that peptide AntiA2AR of 29 peptides was the most effective among the synthesized peptides in inhibiting the A2AR downstream signal cAMP/PKA/CREB. In neurons, the AntiA2AR reversed the calcium flow change induced by the A2AR agonist CGS21680 (1 μM). Furthermore, AntiA2AR expression in the mice striatum weakened the p-PKA/p-CREB signal, enhanced locomotor abilities and increased time spent in the center area in the home-cage observation experiment and increased anxiolytic behavior in the elevated-plus maze test. Conclusions Antagonistic peptide AntiA2AR can effectively block the A2AR signaling pathway. This provides a new strategy for the specific inhibition of A2AR and provides information needed for drug development.

Hydrogen/deuterium exchange mass spectrometry with improved electrochemical reduction enables comprehensive epitope mapping of a therapeutic antibody to the cysteine-knot containing vascular endothelial growth factor

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has become a popular method for analysis of the conformational dynamics and interactions of proteins. Disulfide-bonded proteins, however, present a challenge to HDX-MS as they require efficient disulfide bond reduction prior to enzymatic proteolysis. Electrochemical reduction (ER) provides an attractive solution to tackle disulfide-bonded proteins that are resistant to conventional chemical reduction during HDX-MS. However, ER-enabled HDX-MS has been limited by technical challenges including partial unwanted protein oxidation side-reactions, incompatibility with certain buffer components and most importantly, a lack of overall method robustness. In this study, we have sought to address these challenges. We perform a systematic screening of the compatibility of ER to buffers commonly used in HDX-MS samples by using a reliable and simple system suitability test (SST). Furthermore, we demonstrate the benefits of a new design of the electrochemical cell (EC) for ER-enabled HDX-MS, which include a) high repeatability and robustness over large sample batches without the need for electrode polishing and b) high reduction efficiency of disulfide-bonded proteins without unwanted oxidation side-reactions. We show the real-world applicability of the optimized ER-enabled HDX-MS workflow by performing an epitope mapping of a Fab fragment of a therapeutic monoclonal antibody (mAb) to the cysteine knot-containing vascular endothelial growth factor (VEGF). The results allow us to comprehensively map sites in VEGF involved in mAb binding. Overall, our findings show how ER and HDX-MS can be combined to enable analysis of the conformation and interactions of challenging disulfide-rich proteins.

Involvement of p38 Activation and Mitochondria in Death of Human Leukemia Cells Induced by an Agonistic Human Monoclonal Antibody Fab Specific to TRAIL Receptor 1.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death with minimal damage to normal cells; however, some cancer cells are resistant to TRAIL. TRAIL resistance may be overcome by agonistic antibodies to TRAIL receptors. In this study, we report the toxic effects of a novel recombinant agonistic human anti–TRAIL receptor 1 (DR4) monoclonal antibody Fab fragment, DR4-4, on various TRAIL-resistant and -sensitive cancer cell lines. The mechanisms of DR4-4 Fab–induced cell death in a human T cell leukemia cell line (Jurkat) were investigated using cell viability testing, immunoblotting, immunoassays, flow cytometry, and morphological observation. DR4-4 Fab–induced caspase-independent necrosis was observed to occur in Jurkat cells in association with p38 mitogen-activated protein kinase activation, cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein degradation, decreased mitochondrial membrane potential, and increased mitochondrial reactive oxygen species production. Increased cytotoxic effects of DR4-4 Fab were observed in combination with TRAIL or γ-irradiation. Our results indicate that the novel DR4-4 Fab might overcome TRAIL-resistance and induce death in leukemia cells via cellular mechanisms different from those activated by TRAIL. DR4-4 Fab may have application as a potential therapeutic antibody fragment in single or combination therapy for cancer.

111In-Labeled Glycoprotein Nonmetastatic b (GPNMB) Targeted Gemini Surfactant-Based Nanoparticles against Melanoma: In Vitro Characterization and in Vivo Evaluation in Melanoma Mouse Xenograft Model.

Melanoma is a devastating form of skin cancer with high tendency to metastasis. This work addresses the development of new targeted nanoparticles that can be used for single-photon emission computed tomography (SPECT) imaging of melanoma. Melanoma-specific glycoprotein nonmetastatic b (GPNMB) antigen targeted and nontargeted gemini nanoparticles were prepared, characterized, and radiolabeled with 111In. 111In-labeled nanoparticles were composed of gemini surfactant grafted with monoclonal antibody Fab fragment that targeted GPNMB. Specific uptake of GPNMB-Fab was studied in six melanoma cell lines using flow cytometry. In vitro cellular uptake and internalization were studied using flow cytometry, confocal laser scanning microscopy, and radiometric techniques. Specific uptake of anti-GPNMB targeted nanoparticles was observed in GPNMB expressing cells, which was higher than low expressing or control cells. In vitro studies showed that conjugation of GPNMB targeted nanoparticles led to enhanced intracellular uptake of the nanodelivery system, which is critical for drug delivery. In vivo distribution of the nanoparticles was studied by microSPECT/CT imaging and ex vivo biodistribution. Tumor uptake was significantly higher ( p < 0.05) in nontargeted nanoparticles (5.47 ± 0.46%IA/cc) compared to GPNMB targeted nanoparticles (1.87 ± 0.27% ID/cc), which might be attributed to the high spleen uptake of the targeted formulation. These findings demonstrated that the radiolabeled gemini nanoparticles are promising for image-guided radiotherapy of melanoma. Formulation optimization is needed to improved tumor uptake and in vivo intracellular delivery for radiotherapeutic applications.

Efficacy of an Inhaled IL-13 Antibody Fragment in a Model of Chronic Asthma.

IL-13 is an important cytokine implicated in the pathogenesis of allergic asthma and is an attractive target for an inhaled therapeutic. To investigate the efficacy of CDP7766, a nebulized inhaled anti-IL-13 monoclonal antibody Fab fragment, in a model of allergic asthma in cynomolgus macaques naturally sensitized to Ascaris suum. CDP7766 was nebulized using a vibrating-membrane nebulizer on the basis of eFlow technology. The aerosol generated was analyzed to determine the particle size profile and the biophysical and functional properties of CDP7766. Nebulized CDP7766 (0.1-60 mg/animal, once daily for 5 d) was delivered via the inhaled route. The investigational eFlow nebulizer used in this study generated a respirable aerosol of CDP7766 with no evidence of degradation, loss of potency, aggregation, or formation of particulates. Inhaled CDP7766 was well tolerated in the model (no adverse effects related to local irritation) and significantly inhibited BAL allergen-induced cytokine and chemokine upregulation (60 mg vs. vehicle: eotaxin-3, P < 0.0008; MIP [macrophage inflammatory protein]-1β, IL-8, IFN-γ, P ≤ 0.01). CDP7766 significantly inhibited the increase in pulmonary resistance stimulated by inhaled allergen, measured 15 minutes and 24 hours after allergen challenge. Inhaled CDP7766 potently inhibited the function of IL-13 generated during the airway response to inhaled allergen in cynomolgus macaques, demonstrating the potential of inhaled anti-IL-13 therapeutics for the treatment of allergic asthma.

CD28 Costimulation of T Helper 1 Cells Enhances Cytokine Release In Vivo.

Compared to naive T cells, differentiated T cells are thought to be less dependent on CD28 costimulation for full activation. To revisit the role of CD28 costimulation in mouse T cell recall responses, we adoptively transferred in vitro generated OT-II T helper (Th) 1 cells into C57BL/6 mice (Thy1.2+) and then either blocked CD28–ligand interactions with Fab fragments of the anti-CD28 monoclonal antibody (mAb) E18 or deleted CD28 expression using inducible CD28 knock-out OT-II mice as T cell donors. After injection of ovalbumin protein in adjuvant into the recipient mice we observed that systemic interferon (IFN)γ release strongly depended on CD28 costimulation of the Th1 cells, while secondary clonal expansion was not reduced in the absence of CD28 costimulation. For human memory CD4+ T cell responses we also noted that cytokine release was reduced upon inhibition of CD28 costimulation. Together, our data highlight the so far underestimated role of CD28 costimulation for the reactivation of fully differentiated CD4+ T cells.

A novel Python program for implementation of quality control in the ELISA

The use of semi-quantitative assays such as the enzyme-linked immunosorbent assay (ELISA) requires stringent quality control of the data. However, such quality control is often lacking in academic settings due to unavailability of software and knowledge. Therefore, our aim was to develop methods to easily implement Levey-Jennings quality control methods. For this purpose, we created a program written in Python (a programming language with an open-source license) and tested it using a training set of ELISA standard curves quantifying the Fab fragment of an anti-cocaine monoclonal antibody in mouse blood. A colorimetric ELISA was developed using a goat anti-human anti-Fab capture method. Mouse blood samples spiked with the Fab fragment were tested against a standard curve of known concentrations of Fab fragment in buffer over a period of 133days stored at 4°C to assess stability of the Fab fragment and to generate a test dataset to assess the program. All standard curves were analyzed using our program to batch process the data and to generate Levey-Jennings control charts and statistics regarding the datasets. The program was able to identify values outside of two standard deviations, and this identification of outliers was consistent with the results of a two-way ANOVA. This program is freely available, which will help laboratories implement quality control methods, thus improving reproducibility within and between labs. We report here successful testing of the program with our training set and development of a method for quantification of the Fab fragment in mouse blood.