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2.
Biotechnol Prog ; 38(6): e3290, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36537257

RESUMO

Antigen binding fragments (Fab) are a promising class of therapeutics as they maintain high potency while having significantly smaller size relative to full-length antibodies. Because Fab molecules are aglycosylated, many expression platforms, including prokaryotic, yeast, and mammalian cells, have been developed for their expression, with Escherichia coli being the most commonly used Fab expression system. In this study, we have examined production of a difficult to express Fab molecule in a targeted integration (TI) Chinese Hamster Ovary (CHO) host. Without a need for extensive host or process optimization, as is usually required for E. coli, by simply using different vector configurations, clones with very high Fab expression titers were obtained. In this case, by increasing heavy chain (HC) gene copy numbers, clones with titers of up to 7.4 g/L in the standard fed-batch production culture were obtained. Our findings suggest that having a predetermined transgene integration site, as well as the option to optimize gene copy number/dosage, makes CHO TI hosts an effective system for expression of Fab molecules, allowing Fab expression using platform process and without significant process development efforts.


Assuntos
Fragmentos Fab das Imunoglobulinas , Proteínas Recombinantes , Animais , Cricetinae , Células CHO , Cricetulus , Dosagem de Genes , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transgenes
3.
Biochem Biophys Res Commun ; 636(Pt 1): 184-189, 2022 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-36334443

RESUMO

Matrix metalloproteinase 9 (MMP9) contributes to several aspects of inflammation and cancer pathology, including invasion, metastasis, and angiogenesis. In this study, we expressed a recombinant fragment antigen-binding (Fab)-type anti-MMP9 antibody in Escherichia coli with high purity within five days and confirmed the nanomolar order of antigen-binding efficiency of the recombinant Fab. Moreover, we optimized the experimental time for performing enzyme-linked immunosorbent assay (ELISA), and decreased the reaction time from the conventional 20.5 h to 3.5 h. The rapid and sensitive MMP9 detection system developed in this study can be applied to a range of applications, including the diagnosis of diseases with MMP9 overexpression including inflammatory and cancer-related diseases.


Assuntos
Escherichia coli , Fragmentos Fab das Imunoglobulinas , Fragmentos Fab das Imunoglobulinas/genética , Proteínas Recombinantes , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Metaloproteases
4.
Theranostics ; 12(16): 7067-7079, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36276653

RESUMO

The accelerated approval of the monoclonal antibody (mAb) aducanumab as a treatment option for Alzheimer's Disease and the continued discussions about its efficacy have shown that a better understanding of immunotherapy for the treatment of neurodegenerative diseases is needed. 89Zr-immuno-PET could be a suitable tool to open new avenues for the diagnosis of CNS disorders, monitoring disease progression, and assessment of novel therapeutics. Herein, three different 89Zr-labeling strategies and direct radioiodination with 125I of a bispecific anti-amyloid-beta aducanumab derivate, consisting of aducanumab with a C-terminal fused anti-transferrin receptor binding single chain Fab fragment derived from 8D3 (Adu-8D3), were compared ex vivo and in vivo with regard to brain uptake and target engagement in an APP/PS1 Alzheimer's disease mouse model and wild type animals. Methods: Adu-8D3 and a negative control antibody, based on the HIV specific B12 antibody also carrying C-terminal fused 8D3 scFab (B12-8D3), were each conjugated with NCS-DFO, NCS-DFO*, or TFP-N-suc-DFO-Fe-ester, followed by radiolabeling with 89Zr. 125I was used as a substitute for 124I for labeling of both antibodies. 30 µg of radiolabeled mAb, corresponding to approximately 6 MBq 89Zr or 2.5 MBq 125I, were injected per mouse. PET imaging was performed 1, 3 and 7 days post injection (p.i.). All mice were sacrificed on day 7 p.i. and subjected to ex vivo biodistribution and brain autoradiography. Immunostaining on brain tissue was performed after autoradiography for further validation. Results: Ex vivo biodistribution revealed that the brain uptake of [89Zr]Zr-DFO*-NCS-Adu-8D3 (2.19 ±0.12 %ID/g) was as high as for its 125I-analog (2.21 ±0.15 %ID/g). [89Zr]Zr-DFO-NCS-Adu-8D3 and [89Zr]Zr-DFO-N-suc-Adu-8D3 showed significantly lower uptake (< 0.65 %ID/g), being in the same range as for the 89Zr-labeled controls (B12-8D3). Autoradiography of [89Zr]Zr-DFO*-NCS-Adu-8D3 and [125I]I-Adu-8D3 showed an amyloid-beta related granular uptake pattern of radioactivity. In contrast, the [89Zr]Zr-DFO-conjugates and the control antibody groups did not show any amyloid-beta related uptake pattern, indicating that DFO is inferior for 89Zr-immuno-PET imaging of the brain in comparison to DFO* for Adu-8D3. This was confirmed by day 7 PET images showing only amyloid-beta related brain uptake for [89Zr]Zr-DFO*-NCS-Adu-8D3. In wild type animals, such an uptake was not observed. Immunostaining showed a co-localization of all administered Adu-8D3 conjugates with amyloid-beta plaques. Conclusion: We successfully demonstrated that 89Zr-immuno-PET is suitable for imaging and quantifying amyloid-beta specific brain uptake using a bispecific aducanumab brain shuttling antibody, Adu-8D3, but only when using the novel chelator DFO*, and not DFO, for labeling with 89Zr.


Assuntos
Doença de Alzheimer , Anticorpos Biespecíficos , Animais , Camundongos , Radioisótopos do Iodo , Quelantes , Desferroxamina , Zircônio , Distribuição Tecidual , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/tratamento farmacológico , Linhagem Celular Tumoral , Tomografia por Emissão de Pósitrons/métodos , Anticorpos Monoclonais/uso terapêutico , Peptídeos beta-Amiloides , Fragmentos Fab das Imunoglobulinas , Ésteres
5.
Nat Commun ; 13(1): 6314, 2022 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-36274064

RESUMO

Immunoglobulin M (IgM) is the most ancient of the five isotypes of immunoglobulin (Ig) molecules and serves as the first line of defence against pathogens. Here, we use cryo-EM to image the structure of the human full-length IgM pentamer, revealing antigen binding domains flexibly attached to the asymmetric and rigid core formed by the Cµ4 and Cµ3 constant regions and the J-chain. A hinge is located at the Cµ3/Cµ2 domain interface, allowing Fabs and Cµ2 to pivot as a unit both in-plane and out-of-plane. This motion is different from that observed in IgG and IgA, where the two Fab arms are able to swing independently. A biased orientation of one pair of Fab arms results from asymmetry in the constant domain (Cµ3) at the IgM subunit interacting most extensively with the J-chain. This may influence the multi-valent binding to surface-associated antigens and complement pathway activation. By comparison, the structure of the Fc fragment in the IgM monomer is similar to that of the pentamer, but is more dynamic in the Cµ4 domain.


Assuntos
Imunoglobulina A , Fragmentos Fc das Imunoglobulinas , Humanos , Imunoglobulina M , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G , Fragmentos Fab das Imunoglobulinas/química
6.
Artigo em Inglês | MEDLINE | ID: mdl-36219924

RESUMO

Although mammals are still the main source of polyclonal antibodies production, the use of egg yolk immunoglobulins (IgY) is increasing, and thus improvement of its safety to reduce undesired reactions is needed. Removal of IgY constant domains by pepsin enzymatic treatment is expected to reduce potential adverse effects derived from the parenteral administration of these antibodies, while also increasing its distribution volume. Current Fab' production requires the previous purification of IgY to be used as starting material. In this context, relevant economic benefits may be gained if initial IgY purification could be avoided, by using rawer starting materials. In this work, a three-step process for Fab' production from crude egg yolk water-soluble fractions is described using scalable and simple low-cost technologies such as ultrafiltration and anion exchange chromatography. The overall process yield of 33% highly pure Fab' from water-soluble fractions favorably compares to the manufacture of related medicinal products from mammalian antibodies, such as antivenoms.


Assuntos
Gema de Ovo , Fragmentos Fab das Imunoglobulinas , Animais , Água , Galinhas , Imunoglobulinas , Anticorpos , Mamíferos
7.
Org Biomol Chem ; 20(40): 7972-7980, 2022 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-36193721

RESUMO

The ability of a series of electron-deficient aromatic compounds to form charge-transfer complexes with tryptophan in water has been evaluated by X-ray diffraction studies, UV-vis spectra and NMR. As dinitrophenyl (DNP) ligands are well-known to generate antibody-mediated responses and the π-π stacking interactions with tryptophan residues of the antibody Fab fragment have been reported, most of the aromatic receptors studied here are nitro derivatives. Charge-transfer interactions between the rich indole ring of tryptophan and the electron-deficient aromatic receptors have been observed in the solid state, as four crystal structures of the complexes were obtained. The aromatic donor-acceptor interactions in solution were also verified by UV-vis and NMR spectroscopy. The association of the tripeptide Trp-Gly-Trp, a motif found in antigen Ag43, with the electron-deficient aromatic diimide was also studied by UV-vis and NMR spectroscopy. Our results show that these simple electron-deficient molecules could potentially behave as novel haptens and be incorporated in more elaborated drugs targeting protein-protein interactions, due to the synergistic effect of multiple non-covalent interactions.


Assuntos
Elétrons , Triptofano , Triptofano/química , Água/química , Haptenos , Indóis/química , Fragmentos Fab das Imunoglobulinas
8.
Stroke ; 53(11): 3506-3513, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36128904

RESUMO

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.


Assuntos
AVC Isquêmico , Acidente Vascular Cerebral , Humanos , Animais , Camundongos , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Ativador de Plasminogênio Tecidual/uso terapêutico , Glicoproteínas da Membrana de Plaquetas/uso terapêutico , Ligantes , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Fragmentos Fab das Imunoglobulinas/farmacologia , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Colágeno
9.
Am J Cardiol ; 183: 129-136, 2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36089419

RESUMO

The role of digoxin in clinical practice has narrowed over time. Data on digoxin toxicity trends and outcomes are variable and lack granularity for treatment outcomes. This study aimed to address data gaps in digoxin toxicity trends and outcomes in patients treated with or without digoxin immune fab (DIF). This single-center analysis examined patients with signs/symptoms concerning digoxin toxicity, defined as hospital admission or emergency department visit with elevated digoxin serum concentrations (>2 ng/ml) and/or a primary diagnosis code of digoxin toxicity and/or DIF order. Between 2000 and 2020, 727 patients were identified with signs concerning for digoxin toxicity with a mortality rate of 12.7% during admission and 42.7% at 1 year. DIF was ordered in 9% of cases. Incidence of digoxin toxicity per 1,000 patients with a digoxin prescription and frequency of DIF treatment fluctuated over time without a clear trend toward increase or reduction. DIF-treated patients demonstrated a heavier co-morbidity burden and lower presenting heart rates (median 53 [39.5 to 69.5] vs 77 [64.0 to 91.5] beats/min, p <0.001), worse renal function (median estimated glomerular filtration rate, 30.3 [14.8 to 48.6] vs 40.0 [24.2 to 61.2] ml/min/1.73 m2, p = 0.013), and higher potassium (median 4.5 [4.0 to 5.3] vs 4.3 [3.9 to 4.8] mEq/L, p = 0.022). Compared with a matched cohort, DIF-treated patients experienced a nonsignificant, numerically lower in-hospital mortality (8.2% vs 15.8%, p = 0.199) and 30-day all-cause hospitalization (14.3% vs 24.7%, p = 0.112) and similar 6-month and 1-year hospitalization and mortality. In conclusion, digoxin toxicity remains a pertinent public health issue despite reduction in digoxin utilization. DIF therapy is used in a medically complex population with a high-acuity illness at presentation and is associated with nonsignificant trends toward reduced in-hospital mortality and early readmission that are attenuated over time.


Assuntos
Fármacos Cardiovasculares , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Fármacos Cardiovasculares/uso terapêutico , Digoxina , Progressão da Doença , Frequência Cardíaca , Hospitalização , Potássio , Fragmentos Fab das Imunoglobulinas
10.
Nat Commun ; 13(1): 5222, 2022 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-36064790

RESUMO

The trimeric serine protease HTRA1 is a genetic risk factor associated with geographic atrophy (GA), a currently untreatable form of age-related macular degeneration. Here, we describe the allosteric inhibition mechanism of HTRA1 by a clinical Fab fragment, currently being evaluated for GA treatment. Using cryo-EM, X-ray crystallography and biochemical assays we identify the exposed LoopA of HTRA1 as the sole Fab epitope, which is approximately 30 Å away from the active site. The cryo-EM structure of the HTRA1:Fab complex in combination with molecular dynamics simulations revealed that Fab binding to LoopA locks HTRA1 in a non-competent conformational state, incapable of supporting catalysis. Moreover, grafting the HTRA1-LoopA epitope onto HTRA2 and HTRA3 transferred the allosteric inhibition mechanism. This suggests a conserved conformational lock mechanism across the HTRA family and a critical role of LoopA for catalysis, which was supported by the reduced activity of HTRA1-3 upon LoopA deletion or perturbation. This study reveals the long-range inhibition mechanism of the clinical Fab and identifies an essential function of the exposed LoopA for activity of HTRA family proteases.


Assuntos
Serina Peptidase 1 de Requerimento de Alta Temperatura A , Degeneração Macular , Serina Endopeptidases , Cristalografia por Raios X , Epitopos , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Degeneração Macular/tratamento farmacológico , Degeneração Macular/genética , Degeneração Macular/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
11.
J Chromatogr A ; 1681: 463464, 2022 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-36099694

RESUMO

Fragment antigen-binding (Fab) has several advantages in the treatment and diagnosis of some diseases. The lack of highly efficient affinity chromatography platform creates a purification bottleneck for the downstream processing of Fab-based products, which raises the urgent need for a novel immunoglobin G (IgG)-binding domain (IgBD) with both high affinity and broad specificity for Fab. SpGC3FabRR (designated CFab) was previously identified as a Fab-selective IgBD, which triggered our interest in evaluating the potential of CFab for Fab purification. However, we found that monomeric CFab showed weak Fab-binding. To increase its affinity, a self-trimerizing domain (tri) was fused to CFab to produce CFab-tri. It was found that CFab-tri existed as a trimer and showed promising binding to Fab derived from IgG of humans, rhesus monkeys, mice, rats, and rabbits. Affinity chromatography demonstrated that the recovery rates of Fab derived from IgG of humans, rats, mice, and rabbits by CFab-tri-HP column were 2- to 5-fold of those by protein G-HP column. Human Fab was effectively purified by both protein L- and CFab-tri-HP column. However, unlike CFab-tri-HP column, protein L-HP column was inefficient for purification of Fab derived from IgG of rats, mice, and rabbits. Notably, rat Fab spiked into the extract of Escherichia coli (E. coli) was effectively recovered by CFab-tri-HP column. These results indicate that CFab-tri outperforms protein G and protein L as a ligand for Fab purification, and CFab-tri-based affinity chromatography might be developed as a novel platform for Fab purification.


Assuntos
Escherichia coli , Fragmentos Fab das Imunoglobulinas , Animais , Escherichia coli/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/química , Ligantes , Camundongos , Coelhos , Ratos , Proteínas Recombinantes/genética
12.
Anal Chem ; 94(40): 14054-14060, 2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36174111

RESUMO

In this work, we utilized polycyclic aromatic hydrocarbon (PAH) derivatives as ligands to develop a zinc-based metal-organic framework (Zn-MOF) as an effective detection probe to construct an electrochemiluminescence (ECL) sensor for trenbolone detection. As traditional ECL emitters, PAHs and their derivatives have limited luminescence efficiency because of the aggregation-induced quenching (ACQ) effect. Therefore, Zn-PTC was designed by the coordination of 3,4,9,10-perylenetetracarboxylic (PTC) in the MOF to eliminate the ACQ effect. Meanwhile, Zn-PTC formed based on an aromatic ligand possessed the metal-to-ligand charge-transfer (MLCT) effect, which could transfer the energy of Zn2+ to the aromatic ligand for strong luminescence. The ECL efficiency of Zn-PTC was calculated to be approximately 2.2 times that of the ligand (K4PTC). Second, the Ag@Fe core-shell bimetallic nanocrystal was prepared for efficient activation of persulfate (S2O82-), thereby generating more sulfate radicals (SO4•-) to further promote ECL emission. According to ECL characterizations, UV-vis and fluorescence spectra, and density functional theory calculations, the luminescence and signal amplification mechanisms were investigated. In addition, NKFRGKYKC (NKF) was introduced as an affinity ligand to directionally immobilize the target antibodies, thus releasing specific sites in their Fab fragment to enhance binding activity. Based on the above strategies, the constructed biosensor exhibited high sensitivity, realizing trace detection of TBE with a wide detection range (10 fg/mL-100 ng/mL) and a low detection limit (3.28 fg/mL). This study provided an important reference for sensitive monitoring of steroid pollutants in the environment.


Assuntos
Técnicas Biossensoriais , Poluentes Ambientais , Estruturas Metalorgânicas , Hidrocarbonetos Policíclicos Aromáticos , Técnicas Eletroquímicas , Fragmentos Fab das Imunoglobulinas , Ligantes , Limite de Detecção , Medições Luminescentes , Estruturas Metalorgânicas/química , Sulfatos , Acetato de Trembolona , Zinco
13.
MAbs ; 14(1): 2124902, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36166705

RESUMO

ALPHABETICAL LIST OF ABBREVIATIONS: Fab Fragment antigen-binding; Fc Fragment crystallizable; HMW High molecular weight; ∆HMW Difference between HMW species at stress temperature and 5°C controls; IgG Immunoglobulin G; mAbs Monoclonal antibodies; MV-VHH Multivalent VHH molecule with the format aC-L1-aC-L1-aD; NMR Nuclear magnetic resonance; scFv Single-chain fragment variable; SEC Size-exclusion chromatography; VHH Variable domain of Heavy chain of Heavy chain-only antibody.


Assuntos
Excipientes , Cadeias Pesadas de Imunoglobulinas , Anticorpos Monoclonais , Fragmentos Fab das Imunoglobulinas , Fragmentos Fc das Imunoglobulinas , Imunoglobulina G , Cadeias Pesadas de Imunoglobulinas/química , Espectroscopia de Ressonância Magnética
14.
Infect Immun ; 90(9): e0030622, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-36000876

RESUMO

Lyme disease vaccines based on recombinant Outer surface protein A (OspA) elicit protective antibodies that interfere with tick-to-host transmission of the disease-causing spirochete Borreliella burgdorferi. Another hallmark of OspA antisera and certain OspA monoclonal antibodies (MAbs) is their capacity to induce B. burgdorferi agglutination in vitro, a phenomenon first reported more than 30 years ago but never studied in molecular detail. In this report, we demonstrate that transmission-blocking OspA MAbs, individually and in combination, promote dose-dependent and epitope-specific agglutination of B. burgdorferi. Agglutination occurred within minutes and persisted for hours. Spirochetes in the core of the aggregates exhibited evidence of outer membrane (OM) stress, revealed by propidium iodide uptake. The most potent agglutinator was the mouse MAb LA-2, which targets the OspA C terminus (ß-strands 18 to 20). Human MAb 319-44, which also targets the OspA C terminus (ß-strand 20), and 857-2, which targets the OspA central ß-sheet (strands 8 to 10), were less potent agglutinators, while MAb 221-7, which targets ß-strands 10 to 11, had little to no measurable agglutinating activity, even though its affinity for OspA exceeded that of LA-2. Remarkably, monovalent Fab fragments derived from LA-2, and to a lesser degree 319-44, retained the capacity to induce B. burgdorferi aggregation and OM stress, a particularly intriguing observation considering that "LA-2-like" Fabs have been shown to experimentally entrap B. burgdorferi within infected ticks and prevent transmission during feeding to a mammalian host. It is therefore tempting to speculate that B. burgdorferi aggregation triggered by OspA-specific antibodies in vitro may in fact reflect an important biological activity in vivo.


Assuntos
Grupo Borrelia Burgdorferi , Borrelia burgdorferi , Doença de Lyme , Carrapatos , Aglutinação , Animais , Anticorpos Antibacterianos , Anticorpos Monoclonais , Antígenos de Superfície , Proteínas da Membrana Bacteriana Externa , Vacinas Bacterianas , Epitopos , Humanos , Soros Imunes , Fragmentos Fab das Imunoglobulinas , Lipoproteínas , Vacinas contra Doença de Lyme , Mamíferos , Camundongos , Propídio
15.
Cells ; 11(16)2022 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-36010551

RESUMO

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.


Assuntos
Neoplasias do Colo , Vesículas Extracelulares , Comunicação Celular , Neoplasias do Colo/patologia , Vesículas Extracelulares/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Tetraspanina 29/metabolismo , Microambiente Tumoral
16.
J Control Release ; 350: 146-157, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35973471

RESUMO

Chronic ulcerative oral mucosal inflammatory diseases, including oral lichen planus and recurrent aphthous stomatitis, are painful and highly prevalent, yet lack effective clinical management. In recent years, systemic biologic therapies, including monoclonal antibodies that block the activity of cytokines, have been increasingly used to treat a range of immune-mediated inflammatory conditions such as rheumatoid arthritis and psoriasis. The ability to deliver similar therapeutic agents locally to the oral epithelium could radically alter treatment options for oral mucosal inflammatory diseases, where pro-inflammatory cytokines, in particular tumour-necrosis factor-α (TNFα), are major drivers of pathogenesis. To address this, an electrospun dual-layer mucoadhesive patch comprising medical-grade polymers was investigated for the delivery of F(ab) biologics to the oral mucosa. A fluorescent-labelled F(ab) was incorporated into mucoadhesive membranes using electrospinning with 97% v/v ethanol as a solvent. The F(ab) was detected within the fibres in aggregates when visualised by confocal microscopy. Biotinylated F(ab) was rapidly eluted from the patch (97 ± 5% released within 3 h) without loss of antigen-binding activity. Patches applied to oral epithelium models successfully delivered the F(ab), with fluorescent F(ab) observed within the tissue and 5.1 ± 1.5% cumulative transepithelial permeation reached after 9 h. Neutralising anti-TNFα F(ab) fragments were generated from whole IgG by papain cleavage, as confirmed by SDS-PAGE, then incorporated into patches. F(ab)-containing patches had TNFα neutralising activity, as shown by the suppression of TNFα-mediated CXCL8 release from oral keratinocytes cultured as monolayers. Patches were applied to lipopolysaccharide-stimulated immune-competent oral mucosal ulcer equivalents that contained primary macrophages. Anti-TNFα patch treatment led to reduced levels of active TNFα along with a reduction in the levels of disease-implicated T-cell chemokines (CCL3, CCL5, and CXCL10) to baseline concentrations. This is the first report of an effective device for the delivery of antibody-based biologics to the oral mucosa, enabling the future development of new therapeutic strategies to treat painful conditions.


Assuntos
Mucosite , Humanos , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Fragmentos Fab das Imunoglobulinas/imunologia , Mucosite/tratamento farmacológico , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/imunologia
18.
J Virol ; 96(16): e0062722, 2022 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-35924923

RESUMO

Rotavirus live-attenuated vaccines, both mono- and pentavalent, generate broadly heterotypic protection. B-cells isolated from adults encode neutralizing antibodies, some with affinity for VP5*, that afford broad protection in mice. We have mapped the epitope of one such antibody by determining the high-resolution cryo-EM structure of its antigen-binding fragment (Fab) bound to the virion of a candidate vaccine strain, CDC-9. The Fab contacts both the distal end of a VP5* ß-barrel domain and the two VP8* lectin-like domains at the tip of a projecting spike. Its interactions with VP8* do not impinge on the likely receptor-binding site, suggesting that the mechanism of neutralization is at a step subsequent to initial attachment. We also examined structures of CDC-9 virions from two different stages of serial passaging. Nearly all the VP4 (cleaved to VP8*/VP5*) spikes on particles from the earlier passage (wild-type isolate) had transitioned from the "upright" conformation present on fully infectious virions to the "reversed" conformation that is probably the end state of membrane insertion, unable to mediate penetration, consistent with the very low in vitro infectivity of the wild-type isolate. About half the VP4 spikes were upright on particles from the later passage, which had recovered substantial in vitro infectivity but had acquired an attenuated phenotype in neonatal rats. A mutation in VP4 that occurred during passaging appears to stabilize the interface at the apex of the spike and could account for the greater stability of the upright spikes on the late-passage, attenuated isolate. IMPORTANCE Rotavirus live-attenuated vaccines generate broadly heterotypic protection, and B-cells isolated from adults encode antibodies that are broadly protective in mice. Determining the structural and mechanistic basis of broad protection can contribute to understanding the current limitations of vaccine efficacy in developing countries. The structure of an attenuated human rotavirus isolate (CDC-9) bound with the Fab fragment of a broadly heterotypic protective antibody shows that protection is probably due to inhibition of the conformational transition in the viral spike protein (VP4) critical for viral penetration, rather than to inhibition of receptor binding. A comparison of structures of CDC-9 virus particles at two stages of serial passaging supports a proposed mechanism for initial steps in rotavirus membrane penetration.


Assuntos
Anticorpos Amplamente Neutralizantes , Proteínas do Capsídeo , Epitopos de Linfócito B , Rotavirus , Vacinas Atenuadas , Vírion , Animais , Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Amplamente Neutralizantes/ultraestrutura , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/ultraestrutura , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Camundongos , Conformação Proteica , Ratos , Rotavirus/química , Rotavirus/classificação , Rotavirus/imunologia , Rotavirus/fisiologia , Inoculações Seriadas , Vacinas Atenuadas/química , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/metabolismo , Vírion/imunologia , Vírion/metabolismo , Vírion/ultraestrutura
19.
J Control Release ; 350: 584-599, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36037975

RESUMO

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.


Assuntos
Antígenos CD20 , Apoptose , Animais , Caspases/farmacologia , Humanos , Fragmentos Fab das Imunoglobulinas , Substâncias Macromoleculares , Camundongos , Morfolinos , Rituximab/farmacologia , Albumina Sérica Humana
20.
Int J Pharm ; 624: 122031, 2022 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-35863594

RESUMO

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.


Assuntos
Peptídeos , Vitamina D , Animais , Biologia , Meia-Vida , Humanos , Fragmentos Fab das Imunoglobulinas , Peptídeos/química , Ratos , Suínos , Porco Miniatura , Vitaminas
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