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Toxoplasma gondii holds significant therapeutic potential; however, its nonspecific invasiveness results in off-target effects. The purpose of this study is to evaluate whether T. gondii specificity can be improved by surface display of scFv directed against dendritic cells' endocytic receptor, DEC205, and immune checkpoint PD-L1. Anti-DEC205 scFv was anchored to the T. gondii surface either directly via glycosylphosphatidylinositol (GPI) or by fusion with the SAG1 protein. Both constructs were successfully expressed, but the binding results suggested that the anti-DEC-SAG1 scFv had more reliable functionality towards recombinant DEC protein and DEC205-expressing MutuDC cells. Two anti-PD-L1 scFv constructs were developed that differed in the localization of the HA tag. Both constructs were adequately expressed, but the localization of the HA tag determined the functionality by binding to PD-L1 protein. Co-incubation of T. gondii displaying anti-PD-L1 scFv with tumor cells expressing/displaying different levels of PD-L1 showed strong binding depending on the level of available biomarker. Neutralization assays confirmed that binding was due to the specific interaction between anti-PD-L1 scFv and its ligand. A mixed-cell assay showed that T. gondii expressing anti-PD-L1 scFv predominately targets the PD-L1-positive cells, with negligible off-target binding. The recombinant RH-PD-L1-C strain showed increased killing ability on PD-L1+ tumor cell lines compared to the parental strain. Moreover, a co-culture assay of target tumor cells and effector CD8+ T cells showed that our model could inhibit PD1/PD-L1 interaction and potentiate T-cell immune response. These findings highlight surface display of antibody fragments as a promising strategy of targeting replicative T. gondii strains while minimizing nonspecific binding.
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Antígeno B7-H1 , Anticorpos de Cadeia Única , Toxoplasma , Toxoplasma/metabolismo , Toxoplasma/imunologia , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Humanos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/imunologia , Linhagem Celular Tumoral , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismoRESUMO
There is an urgent need for alternative therapies targeting human dendritic cells (DCs) that could reverse inflammatory syndromes in many autoimmune and inflammatory diseases and organ transplantations. Here, we describe a bispecific antibody (bsAb) strategy tethering two pathogen-recognition receptors at the surface of human DCs. This cross-linking switches DCs into a tolerant profile able to induce regulatory T-cell differentiation. The bsAbs, not parental Abs, induced interleukin 10 and transforming growth factor ß1 secretion in monocyte-derived DCs and human peripheral blood mononuclear cells. In addition, they induced interleukin 10 secretion by synovial fluid cells in rheumatoid arthritis and gout patients. This concept of bsAb-induced tethering of surface pathogen-recognition receptors switching cell properties opens a new therapeutic avenue for controlling inflammation and restoring immune tolerance.
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Anticorpos Biespecíficos , Linfócitos T Reguladores , Humanos , Interleucina-10/metabolismo , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/metabolismo , Leucócitos Mononucleares , Células DendríticasRESUMO
Trastuzumab emtansine (Kadcyla®) was the first antibody-drug conjugate (ADC) approved by the Food and Drug Administration in 2013 against a solid tumor, and the first ADC to treat human epidermal growth factor receptor 2 positive (HER2+) breast cancer. However, this second generation ADC is burden by several limitations included heterogeneity, limited activity against heterogeneous tumor (regarding antigen expression) and suboptimal tumor penetration. To address this, different development strategies are oriented towards homogeneous conjugation, new drugs, optimized linkers and/or smaller antibody formats. To reach better developed next generation ADCs, a key parameter to consider is the management of the hydrophobicity associated with the linker-drug, increasing with and limiting the drug-to-antibody ratio (DAR) of the ADC. Here, an innovative branched pegylated linker was developed, to control the hydrophobicity of the monomethyl auristatin E (MMAE) and its cathepsin B-sensitive trigger. This branched pegylated linker-MMAE was then used for the efficient generation of internalizing homogeneous ADC of DAR 8 and minibody-drug conjugate of DAR 4, targeting HER2. Both immunoconjugates were then evaluated in vitro and in vivo on breast cancer models. Interestingly, this study highlighted that the minibody-MMAE conjugate of DAR 4 was the best immunoconjugate regarding in vitro cellular internalization and cytotoxicity, gamma imaging, ex vivo biodistribution profile in mice and efficient reduction of tumor size in vivo. These results are very promising and encourage us to explore further fragment-drug conjugate development.
Assuntos
Aminobenzoatos , Neoplasias da Mama , Imunoconjugados , Oligopeptídeos , Estados Unidos , Camundongos , Humanos , Animais , Feminino , Neoplasias da Mama/tratamento farmacológico , Preparações Farmacêuticas , Distribuição Tecidual , Linhagem Celular Tumoral , Imunoconjugados/uso terapêutico , Ado-Trastuzumab Emtansina , Interações Hidrofóbicas e Hidrofílicas , PolietilenoglicóisRESUMO
Like pneumonia, coronavirus disease 2019 (COVID-19) is characterized by a massive infiltration of innate immune cells (such as polymorphonuclear leukocytes) into the airways and alveolar spaces. These cells release proteases that may degrade therapeutic antibodies and thus limit their effectiveness. Here, we investigated the in vitro and ex vivo impact on anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) IgG1s and other IgG subclasses (IgG2 and IgG4) of the neutrophil elastase, proteinase 3 and cathepsin G (the three main neutrophil serine proteases) found in endotracheal aspirates from patients with severe COVID-19. Although the IgGs were sensitive to neutrophil serine proteases, IgG2 was most resistant to proteolytic degradation. The two anti-SARS CoV2 antibodies (casirivimab and imdevimab) were sensitive to the lung's proteolytic environment, although neutrophil serine protease inhibitors only partly limited the degradation. Overall, our results show that the pneumonia-associated imbalance between proteases and their inhibitors in the airways contributes to degradation of antiviral antibodies.
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COVID-19 , Pneumonia , Humanos , RNA Viral , Serina Proteases/metabolismo , Neutrófilos/metabolismo , Pneumonia/metabolismo , COVID-19/metabolismo , Imunoglobulina G/metabolismoRESUMO
In the Americas and specially in Brazil, the Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta are the three most medically relevant brown spider species, and whose bites can lead to the condition known as loxoscelism. Here, we report the development of a tool capable of identifying a common epitope amongst Loxosceles sp. venom's toxins. A murine monoclonal antibody (LmAb12) and its recombinant fragments (scFv12P and diabody12P) have been produced and characterized. This antibody and its recombinant constructs were able to recognize proteins of Loxosceles spider venoms with specificity. The scFv12P variant was also able to detect low concentrations of Loxosceles venom in a competitive ELISA assay, displaying potential as a venom identification tool. The primary antigenic target of LmAb12 is a knottin, a venom neurotoxin, that has a shared identity of 100 % between the L. intermedia and L. gaucho species and high similarity to L. laeta. Furthermore, we observed LmAb12 was able to partially inhibit in vitro hemolysis, a cellular event typically induced by the Loxosceles sp. venoms. Such behavior might be due to LmAb12 cross-reactivity between the antigenic target of LmAb12 and the venom's dermonecrotic toxins, the PLDs, or even the existence of synergism between these two toxins.
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Venenos de Aranha , Aranhas , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antígenos/química , Antivenenos/química , Reações Cruzadas , Miniproteínas Nó de Cistina/química , Fosfolipase D/química , Venenos de Aranha/química , Aranhas/química , Epitopos/químicaRESUMO
Following our previous study on the development of EGFR-targeted nanomedicine (NM-scFv) for the active delivery of siRNA in EGFR-positive cancers, this study focuses on the development and the quality control of a radiolabeling method to track it in in vivo conditions with nuclear imaging. Our NM-scFv is based on the electrostatic complexation of targeted nanovector (NV-scFv), siRNA and two cationic polymers. NV-scFv comprises an inorganic core, a fluorescent dye, a polymer layer and anti-EGFR ligands. To track NM-scFv in vivo with nuclear imaging, the DTPA chemistry was used to radiolabel NM-scFv with 111In. DTPA was thiolated and introduced onto NV-scFv via the maleimide chemistry. To obtain suitable radiolabeling efficiency, different DTPA/NV-scFv ratios were tested, including 0.03, 0.3 and 0.6. At the optimized ratio (where the DTPA/NV-scFv ratio was 0.3), a high radiolabeling yield was achieved (98%) and neither DTPA-derivatization nor indium-radiolabeling showed any impact on NM-scFv's physicochemical characteristics (DH ~100 nm, PDi < 0.24). The selected NM-scFv-DTPA demonstrated good siRNA protection capacity and comparable in vitro transfection efficiency into EGFR-overexpressing cells in comparison to that of non-derivatized NM-scFv (around 67%). Eventually, it was able to track both qualitatively and quantitatively NM-scFv in in vivo environments with nuclear imaging. Both the radiolabeling and the NM-scFv showed a high in vivo stability level. Altogether, a radiolabeling method using DTPA chemistry was developed with success in this study to track our NM-scFv in in vivo conditions without any impact on its active targeting and physicochemical properties, highlighting the potential of our NM-scFv for future theranostic applications in EGFR-overexpressing cancers.
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According to Globocan 2020, breast cancer is considered one of the most common cancers affecting women and is one of the leading causes of death in over 100 countries. The available classical treatment options do not always give satisfactory outcomes, and some patients develop resistance to these treatments. This study aims to investigate the combination of nanovectorized siRNA directed against anti-apoptotic protein Survivin (siSurvivin) by targeted stealth magnetic siRNA nanovectors (TS-MSN), designed in our lab, with Doxorubicin (DOX), as an option for HER2+ breast cancer treatment. The hypothesis is that the pretreatment of the HER2+ breast cancer cell line SK-BR-3 with siSurvivin will induce apoptosis in the cancer cells and enhance the therapeutic efficacy of DOX, allowing a dose reduction of DOX and hence a reduction of potential side effects. TS-MSN are based on superparamagnetic iron oxide nanoparticles (SPIONs) covalently coupled with a fluorophore sulfocyanine-5 and polyethylene glycol 5000 (PEG5000) and functionalized with single-chain variable fragments (scFv) of an antibody targeting the HER2 membrane receptor. These covalently functionalized SPIONs are then complexed via electrostatic interactions with therapeutic siRNA and the cationic polymers, chitosan, and poly-L-arginine. TS-MSNsiSurvivin had an average size of 144 ± 30 nm, a PDI of 0.3, and a slightly positive zeta potential value of 10.56 ± 05.70 mV. The agarose gel electrophoresis assay confirmed that the siRNA is well-complexed into TS-MSN without leakage, as no free siRNA was detected. Moreover, siRNA in TS-MSN was protected from RNAse A degradation for up to 6 h at 37 °C. Formulations of TS-MSN with siSurvivin demonstrated in vitro gene knockdown up to 89% in the HER2+ breast cancer cell line SK-BR-3. Furthermore, qRT-PCR confirmed a significant Survivin mRNA relative expression inhibition (about 50%) compared to control siRNA or untreated cells. A combination protocol was evaluated between TS-MSN and Doxorubicin (DOX) for the first time. Therefore, SK-BR-3 cells were pretreated with TS-MSN formulated with siSurvivin at 50 nM for 24 h alone, before a DOX treatment at a concentration of 0.5 µM (corresponding to the IC50) was added for 48 h. The MTT cytotoxicity tests, performed after 72 h of treatment, revealed that the combination had a significant synergistic cytotoxic effect on SK-BR-3 cells compared to monotherapies or untreated cells. We confirmed that pretreatment of cells with siSurvivin potentializes the cytotoxic effect of DOX as an alternative approach for treating HER2+ breast cancer. In conclusion, a combination of anti-Survivin siRNA and DOX would be a good alternative in HER2+ breast cancer therapy.
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Bispecific antibodies (BsAbs) represent an important advance in innovative therapeutic strategies. Among the countless formats of BsAbs, fusion with molecules such as anticalins linked to a monoclonal antibody (mAb), represents an easy and low-cost way to obtain innovative molecules. We fused an anticalin against human fibronectin to a molecule biosimilar to trastuzumab (H0) or rituximab (R0), in four different positions, two on the N terminal region of heavy or light chains and two on the C terminal region. The eight BsAbs (H family (HF) 1 to 4 and R family (RF) 1 to 4) were produced and their affinity parameters and functional properties evaluated. The presence of anticalin did not change the glycosylation of the BsAb, shape or yield. The antigenic recognition of each BsAb family, Her2 for HF1 to 4 and CD20 for RF1 to 4, was slightly decreased (HF) or absent (RF) for the anticalin N-terminal in the light chain position. The anticalin recognition of FN was slightly decreased for the HF family, but a dramatic decrease was observed for RF members with lowest affinity for RF1. Moreover, functional properties of Abs, such as CD16 activation of NK, CD32-dependent phagocytosis and FcRn transcytosis, confirmed that this anticalin position leads to less efficient BsAbs, more so for RF than HF molecules. Nevertheless, all BsAbs demonstrated affinities for CD16, CD32 and FcRn, which suggests that more than affinity for FcRs is needed for a functioning antibody. Our strategy using anticalin and Abs allows for rapid generation of BsAbs, but as suggested by our results, some positions of anticalins on Abs result in less functionality.
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Antibody-drug conjugates (ADCs) derived from a full immunoglobulin-G (IgG) are associated with suboptimal solid-tumor penetration and Fc-mediated toxicities. Antibody fragment-drug conjugates (FDCs) could be an alternative. Nevertheless, innovative solutions are needed to implant cysteines as conjugation sites in the single-chain fragment variable (scFv) format, which is the backbone from which many other antibody formats are built. In addition, the bioconjugation site has the utmost importance to optimize the safety and efficacy of bioconjugates. Our previous intra-tag cysteine (ITC) strategy consisted of introducing a bioconjugation motif at the C-terminal position of the 4D5.2 scFv, but this motif was subjected to proteolysis when the scFv was produced in CHO cells. Considering these data, using three intra-domain cysteine (IDC) strategies, several parameters were studied to assess the impact of different locations of a site-specific bioconjugation motif in the variable domains of an anti-HER2 scFv. In comparison to the ITC strategy, our new IDC strategy allowed us to identify new fragment-drug conjugates (FDCs) devoid of proteolysis and exhibiting enhanced stability profiles, better affinity, and better ability to kill selectively HER2-positive SK-BR-3 cells in vitro at picomolar concentrations. Thus, this work represents an important optimization step in the design of more complex and effective conjugates.
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The use of monoclonal antibodies (mAbs) in therapy is gradually advancing and discussions entail its safety, rentability and effectiveness. To this date, around a hundred mAbs have been approved by the FDA for the treatment of various diseases. Aiming for their large-scale production, recombinant DNA technology is mainly employed, and antibodies can be expressed in various eukaryotic and prokaryotic systems. Moreover, considering their heterologous origin and potential immunogenicity, various strategies have been developed for mAb humanization, considering that around 50 % of commercial mAbs are humanized. Hence, we introduce LimAb7, a mouse mAb capable of binding and neutralizing brown spider's Loxosceles intermedia dermonecrotic toxins in vivo/in vitro. This antibody has been produced in mouse and humanized scFv and diabody formats, however results indicated losses in antigen-binding affinity, stability, and neutralizing ability. Intending to develop evolved, stable, and neutralizing antibody fragments, we report for the first time the design of humanized antibody V-domains produced as Fab fragments, against spider venom toxins. Improvements in constructs were observed regarding their physicochemical stability, target binding and binding pattern maintenance. As their neutralizing features remain to be characterized, we believe this data sheds new light on antibody humanization by producing a parental molecule in different recombinant formats.
Assuntos
Anticorpos Monoclonais , Fragmentos Fab das Imunoglobulinas , Animais , Anticorpos Monoclonais/química , Anticorpos Neutralizantes , CamundongosRESUMO
Antibody-drug conjugates (ADCs) are targeted therapies, mainly used in oncology, consisting in a three-component molecular architecture combining a highly potent drug conjugated via a linker onto a monoclonal antibody (mAb), designed for the selective delivery of the drug to the tumor site. The linker is a key component, defining the ADC stability and mechanism of action, and particularly the drug release strategy. In this study, we have developed and synthesized a cleavable linker, which possesses an Asn-Pro-Val (NPV) sequence sensitive to the human neutrophil elastase (HNE), overexpressed in the tumor microenvironment. This linker permitted the site-specific conjugation of the cell-permeable drug, monomethyl auristatin E (MMAE), onto trastuzumab, using a disulfide re-bridging technology. The resulting ADC was then evaluated in vitro. This conjugate demonstrated retained antigen (Ag) binding affinity and exhibited high subnanomolar potency against Ag-positive tumor cells after internalization, suggesting an intracellular mechanism of linker cleavage. While no internalization and cytotoxic activity of this ADC was observed on Ag-negative cells in classical conditions, the supplementation of exogenous HNE permitted to restore a nanomolar activity on these cells, suggesting an extracellular mechanism of drug release in these conditions. This in vitro proof of concept tends to prove that the NPV sequence could allow a dual intra- and extracellular mechanism of drug release. This work represents a first step in the design of original ADCs with a new dual intra- and extracellular drug delivery system and opens the way to further experimentations to evaluate their full potential in vivo.
Assuntos
Antineoplásicos/química , Imunoconjugados/química , Elastase de Leucócito/metabolismo , Oligopeptídeos/química , Trastuzumab/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Dipeptídeos/química , Dissulfetos/química , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Humanos , Imunoconjugados/farmacologia , Ligação Proteica , Conformação Proteica , Trastuzumab/farmacologiaRESUMO
Maternal-fetal transmission of Toxoplasma gondii tachyzoites acquired during pregnancy has potentially dramatic consequences for the fetus. Current reference-standard treatments are not specific to the parasite and can induce severe side effects. In order to provide treatments with a higher specificity against toxoplasmosis, we developed antibody fragments-single-chain fragment variable (scFv) and scFv fused with mouse immunoglobulin G2a crystallizable fragment (scFv-Fc)-directed against the major surface protein SAG1. After validating their capacity to inhibit T. gondii proliferation in vitro, the antibody fragments' biological activity was assessed in vivo using a congenital toxoplasmosis mouse model. Dams were treated by systemic administration of antibody fragments and with prevention of maternal-fetal transmission being used as the parameter of efficacy. We observed that both antibody fragments prevented T. gondii dissemination and protected neonates, with the scFv-Fc format having better efficacy. These data provide a proof of concept for the use of antibody fragments as effective and specific treatment against congenital toxoplasmosis and provide promising leads.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antiprotozoários/imunologia , Engenharia de Proteínas , Anticorpos de Cadeia Única , Toxoplasmose Congênita , Animais , Feminino , Camundongos , Gravidez , Anticorpos de Cadeia Única/imunologia , Toxoplasma/imunologia , Toxoplasmose Congênita/tratamento farmacológico , Toxoplasmose Congênita/prevenção & controleRESUMO
A targeted nanomedicine with humanized anti-EGFR scFv (NM-scFv) was developed for siRNA delivery into triple negative breast cancer (TNBC) cells. NM-scFv consisted of i) targeted nanovector (NV-scFv): nano-cargo with targeting properties; ii) siRNA: pharmacological agent and iii) cationic polymers (chitosan, poly-L-arginine): for siRNA complexation and endosomal escape. NV-scFv was based on superparamagnetic nanoparticle (SPION) labeled with Dylight™680, a PEG layer and a humanized anti-EGFR scFv. The PEG density was optimized from 236 ± 3 to 873 ± 4 PEGs/NV-scFv and the number of targeting ligands per NV-scFv was increased from 9 to 13. This increase presented a double benefit: i) enhanced cellular internalization by a factor of 2.0 for a 24 h incubation time and ii) few complement protein consumption reflecting a greater stealthiness (26.9 vs 45.3% of protein consumption at 150 µg of iron/mL of NHS). A design of experiments was performed to optimize the charge ratios of chitosan/siRNA (CS) and PLR/siRNA (CR) that influenced significantly: i) siRNA protection and ii) gene silencing effect. With optimal ratios (CS = 10 and CR = 10), anti-GFP siRNA was completely complexed and the transfection efficiency of NM-scFv was 69.4% vs 25.3% for non-targeted NM. These results demonstrated the promising application of our NM-scFv for the targeted siRNA delivery into TNBC cells.
Assuntos
Marcação de Genes , Nanomedicina , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Anticorpos de Cadeia Única/metabolismo , Transfecção , Neoplasias de Mama Triplo Negativas/terapia , Linhagem Celular Tumoral , Quitosana/química , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Nanopartículas , Peptídeos/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Anticorpos de Cadeia Única/química , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Single-domain antibodies (sdAbs) offer great features such as increased stability but are hampered by a limited serum half-life. Many strategies have been developed to improve the sdAb half-life, such as protein engineering and controlled release systems (CRS). In our study, we designed a new product that combined a hydrogel with a 3D-printed implant. The results demonstrate the implant's ability to sustain sdAb release up to 13 days through a reduced initial burst release followed by a continuous release. Furthermore, formulation screening helped to identify the best sdAb formulation conditions and improved our understanding of our CRS. Through the screening step, we gained knowledge about the influence of the choice of polymer and about potential interactions between the sdAb and the polymer. To conclude, this feasibility study confirmed the ability of our CRS to extend sdAb release and established the fundamental role of formulation screening for maximizing knowledge about our CRS.
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Envenoming due to Loxosceles spider bites still remains a neglected disease of particular medical concern in the Americas. To date, there is no consensus for the treatment of envenomed patients, yet horse polyclonal antivenoms are usually infused to patients with identified severe medical conditions. It is widely known that venom proteins in the 30-35 kDa range with sphingomyelinase D (SMasesD) activity, reproduce most of the toxic effects observed in loxoscelism. Hence, we believe that monoclonal antibody fragments targeting such toxins might pose an alternative safe and effective treatment. In the present study, starting from the monoclonal antibody LimAb7, previously shown to target SMasesD from the venom of L. intermedia and neutralize its dermonecrotic activity, we designed humanized antibody V-domains, then produced and purified as recombinant single-chain antibody fragments (scFvs). These molecules were characterized in terms of humanness, structural stability, antigen-binding activity, and venom-neutralizing potential. Throughout this process, we identified some blocking points that can impact the Abs antigen-binding activity and neutralizing capacity. In silico analysis of the antigen/antibody amino acid interactions also contributed to a better understanding of the antibody's neutralization mechanism and led to reformatting the humanized antibody fragment which, ultimately, recovered the functional characteristics for efficient in vitro venom neutralization.
Assuntos
Anticorpos Monoclonais , Antivenenos , Anticorpos de Cadeia Única , Venenos de Aranha/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Antivenenos/administração & dosagem , Antivenenos/imunologia , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Modelos Moleculares , Testes de Neutralização , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/imunologia , Picada de Aranha/terapia , Venenos de Aranha/efeitos adversos , Aranhas/imunologiaRESUMO
In order to increase the successful development of recombinant antibodies and fragments, it seems fundamental to enhance their expression and/or biophysical properties, such as the thermal, chemical, and pH stabilities. In this study, we employed a method bases on replacing the antibody framework region sequences, in order to promote more particularly single-chain Fragment variable (scFv) product quality. We provide evidence that mutations of the VH- C-C' loop might significantly improve the prokaryote production of well-folded and functional fragments with a production yield multiplied by 27 times. Additional mutations are accountable for an increase in the thermal (+19.6 °C) and chemical (+1.9 M) stabilities have also been identified. Furthermore, the hereby-produced fragments have shown to remain stable at a pH of 2.0, which avoids molecule functional and structural impairments during the purification process. Lastly, this study provides relevant information to the understanding of the relationship between the antibodies amino acid sequences and their respective biophysical properties.
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The Rift Valley fever virus (RVFV) is responsible for a serious mosquito-borne viral disease in humans and ruminants. The development of a new and safer vaccine is urgently needed due to the risk of introduction of this arbovirus into RVFV-free continents. We recently showed that a DNA vaccine encoding eGn, the ectodomain of the RVFV Gn glycoprotein, conferred a substantial protection in the sheep natural host and that the anti-eGn IgG levels correlated to protection. Addressing eGn to DEC205 reduced the protective efficacy while decreasing the antibody and increasing the IFNγ T cell responses in sheep. In order to get further insight into the involved mechanisms, we evaluated our eGn-encoding DNA vaccine strategy in the reference mouse species. A DNA vaccine encoding eGn induced full clinical protection in mice and the passive transfer of immune serum was protective. This further supports that antibodies, although non-neutralizing in vitro, are instrumental in the protection against RVFV. Addressing eGn to DEC205 was also detrimental to protection in mice, and in this species, both the antibody and the IFNγ T cell responses were strongly decreased. Conversely when using a plasmid encoding a different antigen, i.e., mCherry, DEC205 targeting promoted the antibody response. Altogether our results show that the outcome of targeting antigens to DEC205 depends on the species and on the fused antigen and is not favorable in the case of eGn. In addition, we bring evidences that eGn in itself is a pertinent antigen to be included in a DNA vaccine and that next developments should aim at promoting the anti-eGn antibody response.
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Glicoproteínas/imunologia , Imunidade Humoral/imunologia , Vírus da Febre do Vale do Rift/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Células CHO , Linhagem Celular , Cricetulus , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Envelope Viral/imunologiaRESUMO
Molecular engineering has made possible to reformat monoclonal antibodies into smaller antigen-binding structures like scFvs, diabodies, Fabs with new potential in vivo applications because they do not induce Fc-mediated functions. However, most of these molecules are from rodent origin. As a consequence, they are immunogenic and approval for administration to humans requires prior humanization. Today, there is no well-identified strategy to create recombinant humanized antibody V-domains that preserve the antigen-binding characteristics of the parental antibody associated with high stability and solubility. Here, we propose a strategy that consists in grafting CDRs onto properly chosen human antibody frameworks in order to reduce immunogenicity. A flowchart indicates the way to proceed in order to introduce an internal affinity purification tag while structural refinements are proposed to maintain antigen-binding characteristics. The best humanized candidates are identified through selection steps including in silico analysis, research scale production followed by early functional evaluation, purification assays, aggregation, and stability assessment.
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Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/farmacologia , Sítios de Ligação , Linhagem Celular , Regiões Determinantes de Complementaridade/genética , Biologia Computacional/métodos , Cricetinae , Bases de Dados Genéticas , Expressão Gênica , Humanos , Imunoensaio , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/farmacologia , Região Variável de Imunoglobulina , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Engenharia de Proteínas/métodos , Estabilidade Proteica , Proteínas Recombinantes , Reprodutibilidade dos Testes , Anticorpos de Cadeia Única , Relação Estrutura-Atividade , NavegadorRESUMO
Antibody-drug conjugates (ADC) are spearheading vectorized chemotherapy against cancer, with 4 FDA-approved ADCs and 79 in clinical trials. However, most ADCs are produced using a stochastic bioconjugation method, target hematological cancers, and are derived from a full immunoglobulin-G (IgG). These factors limit their efficacy, especially against solid tumors which remain difficult to treat. Here we report the site-specific conjugation of a single auristatin derivative onto an engineered anti-HER2 single chain fragment variable (scFv) of the trastuzumab antibody, generating new scFv-drug conjugates (SDC). Two cysteines were judiciously incorporated at the beginning of the scFv hexahistidine tag, in order to allow controlled bioconjugation of a heterobifunctional linker including a second generation maleimide (SGM), either cleavable (for monomethyl auristatin E) or noncleavable (for monomethyl auristatin F). Our data indicated that both SDCs conserved their affinity to HER2 in comparison to the native scFv, and were efficiently able to kill in vitro HER2-positive SK-BR-3 cells at subnanomolar concentrations (EC50 of 0.68 nM and 0.32 nM). No effect was observed on HER2-negative MCF-7 cells. Ours results showed efficient targeting of site-specific SDCs against HER2-positive breast cancer cells. This work represents a first important step in the design of more effective small conjugates, paving the way for future in vivo translation to evaluate their full potential.
Assuntos
Aminobenzoatos/química , Neoplasias da Mama/tratamento farmacológico , Imunoconjugados/química , Imunoconjugados/farmacologia , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Maleimidas/química , Oligopeptídeos/química , Receptor ErbB-2/efeitos dos fármacos , Anticorpos de Cadeia Única/química , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imunoconjugados/uso terapêutico , Fatores Imunológicos/uso terapêutico , Engenharia de Proteínas , Trastuzumab/química , Trastuzumab/imunologiaRESUMO
Toxoplasmosis is a major public health problem and the development of a human vaccine is of high priority. Efficient vaccination against Toxoplasma gondii requires both a mucosal and systemic Th1 immune response. Moreover, dendritic cells play a critical role in orchestrating the innate immune functions and driving specific adaptive immunity to T. gondii. In this study, we explore an original vaccination strategy that combines administration via mucosal and systemic routes of fusion proteins able to target the major T. gondii surface antigen SAG1 to DCs using an antibody fragment single-chain fragment variable (scFv) directed against DEC205 endocytic receptor. Our results show that SAG1 targeting to DCs by scFv via intranasal and subcutaneous administration improved protection against chronic T. gondii infection. A marked reduction in brain parasite burden is observed when compared with the intranasal or the subcutaneous route alone. DC targeting improved both local and systemic humoral and cellular immune responses and potentiated more specifically the Th1 response profile by more efficient production of IFN-γ, interleukin-2, IgG2a, and nasal IgA. This study provides evidence of the potential of DC targeting for the development of new vaccines against a range of Apicomplexa parasites.