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1.
Life Sci ; 256: 117985, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32562692

RESUMO

AIMS: To assess the combination therapy of anti-CD20 mabs and adenovirus-mediated interleukin-10 (IL-10) gene delivery on the prevention of type 1 diabetes (T1D) in non-obese diabetes (NOD) mice. MAIN METHODS: In present study, we simultaneously blocked the B cell interactions and recovered the Th cell subset proportion by using through anti-CD20 Mab and adenovirus-mediated gene delivery of IL-10, respectively. After 9 consecutive days of combination therapy, various measurements, including hematoxylin-eosin staining (HE), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling assay (TUNEL), immunohistochemistry, ELISA, PCR and western blot were applied to further assess the efficacy. KEY FINDINGS: The results suggested that the combination intervention reduced the T1D-associated morbidity of NOD mice, promote insulin secretion, control blood glucose and ease pancreatitis. Moreover, the combination therapy might play a protective role in pancreatic ß cells by suppressing the expression of TNF-α and Fas, blocking the Caspase-8 and Caspase-3 apoptotic pathways and activating the Bcl-2 anti-apoptotic pathway. Finally, the combination intervention may up-regulate the gene expression of CK-19 and PDX-1 and further accelerate the differentiation and proliferation of pancreatic ß cells. SIGNIFICANCE: Therefore, the combination intervention with anti-CD20 mabs and the IL-10 gene plays a role in the prevention of T1D to some extent in NOD mice.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Antígenos CD20/administração & dosagem , Apoptose/efeitos dos fármacos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Interleucina-10/administração & dosagem , Pancreatite/tratamento farmacológico , Adenoviridae/genética , Animais , Anticorpos Monoclonais/genética , Antígenos CD20/genética , Apoptose/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Quimioterapia Combinada , Feminino , Interleucina-10/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pancreatite/genética , Pancreatite/patologia
2.
PLoS One ; 15(5): e0232713, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379792

RESUMO

For an antibody to be a successful therapeutic many competing factors require optimization, including binding affinity, biophysical characteristics, and immunogenicity risk. Additional constraints may arise from the need to formulate antibodies at high concentrations (>150 mg/ml) to enable subcutaneous dosing with reasonable volume (ideally <1.0 mL). Unfortunately, antibodies at high concentrations may exhibit high viscosities that place impractical constraints (such as multiple injections or large needle diameters) on delivery and impede efficient manufacturing. Here we describe the optimization of an anti-PDGF-BB antibody to reduce viscosity, enabling an increase in the formulated concentration from 80 mg/ml to greater than 160 mg/ml, while maintaining the binding affinity. We performed two rounds of structure guided rational design to optimize the surface electrostatic properties. Analysis of this set demonstrated that a net-positive charge change, and disruption of negative charge patches were associated with decreased viscosity, but the effect was greatly dependent on the local surface environment. Our work here provides a comprehensive study exploring a wide sampling of charge-changes in the Fv and CDR regions along with targeting multiple negative charge patches. In total, we generated viscosity measurements for 40 unique antibody variants with full sequence information which provides a significantly larger and more complete dataset than has previously been reported.


Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Becaplermina/imunologia , Desenho Assistido por Computador , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Modelos Moleculares , Mutação , Conformação Proteica , Propriedades de Superfície , Viscosidade
3.
Adv Exp Med Biol ; 1194: 359-371, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32468552

RESUMO

Monoclonal antibodies (mAbs) constitute a promising class of therapeutics, since ca. 25% of all biotech drugs in development are mAbs. Even though their therapeutic value is now well established, human- and murine-derived mAbs do have deficiencies, such as short in vivo lifespan and low stability. However, the most difficult obstacle to overcome, toward the exploitation of mAbs for disease treatment, is the prevention of the formation of protein aggregates. ANTISOMA is a pipeline for the reduction of the aggregation tendency of mAbs through the decrease in their intrinsic aggregation propensity, based on an automated amino acid substitution approach. The method takes into consideration the special features of mAbs and aims at proposing specific point mutations that could lead to the redesign of those promising therapeutics, without affecting their epitope-binding ability. The method is available online at http://bioinformatics.biol.uoa.gr/ANTISOMA .


Assuntos
Anticorpos Monoclonais , Biologia Computacional , Agregação Patológica de Proteínas , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Biologia Computacional/métodos , Epitopos/genética , Humanos , Camundongos , Agregação Patológica de Proteínas/tratamento farmacológico
4.
PLoS Comput Biol ; 16(3): e1007147, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32119655

RESUMO

Targeted cancer therapies are powerful alternatives to chemotherapies or can be used complementary to these. Yet, the response to targeted treatments depends on a variety of factors, including mutations and expression levels, and therefore their outcome is difficult to predict. Here, we develop a mechanistic model of gastric cancer to study response and resistance factors for cetuximab treatment. The model captures the EGFR, ERK and AKT signaling pathways in two gastric cancer cell lines with different mutation patterns. We train the model using a comprehensive selection of time and dose response measurements, and provide an assessment of parameter and prediction uncertainties. We demonstrate that the proposed model facilitates the identification of causal differences between the cell lines. Furthermore, our study shows that the model provides predictions for the responses to different perturbations, such as knockdown and knockout experiments. Among other results, the model predicted the effect of MET mutations on cetuximab sensitivity. These predictive capabilities render the model a basis for the assessment of gastric cancer signaling and possibly for the development and discovery of predictive biomarkers.


Assuntos
Cetuximab/farmacologia , Neoplasias Gástricas/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cetuximab/genética , Cetuximab/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/metabolismo , Humanos , Modelos Biológicos , Modelos Estatísticos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores R , Transdução de Sinais/fisiologia , Neoplasias Gástricas/tratamento farmacológico , Proteínas ras/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(14): 7981-7989, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32209664

RESUMO

Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement (ADE) in vitro and extend their half-lives. Here we report on prophylactic coadministration of the Fc-modified antibodies to pregnant rhesus macaques challenged three times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission, protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Transmissão Vertical de Doença Infecciosa/prevenção & controle , Complicações Infecciosas na Gravidez/prevenção & controle , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Feto/imunologia , Feto/virologia , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/administração & dosagem , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/virologia , Engenharia de Proteínas , RNA Viral/isolamento & purificação , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Zika virus/genética , Zika virus/patogenicidade , Infecção por Zika virus/imunologia , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologia
6.
J Biotechnol ; 312: 11-22, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32114154

RESUMO

An increasing number of engineered therapeutic recombinant proteins with unpredictable manufacturability are currently filling industrial cell line development pipelines. These proteins can be "difficult-to-express" (DTE) in that production of a sufficient quantity of correctly processed recombinant product by engineered mammalian cells is difficult to achieve. In these circumstances, identification of appropriate cell engineering strategies to increase yield is difficult as constraints are cell line and product-specific. Here we describe and validate the development of a high-throughput microscale platform for multiparallel testing of multiple functional genetic components at varying stoichiometry followed by assessment of their effect on cell functional performance. The platform was used to compare and identify optimal cell engineering solutions for both transient and stable production of a model DTE IgG1 monoclonal antibody. We simultaneously tested the functional effect of 32 genes encoding discrete ER or secretory pathway components, each at varying levels of expression and utilized in different combinations. We show that optimization of functional gene load and relative stoichiometry is critical and optimal cell engineering solutions for stable and transient production contexts are significantly different. Our analysis indicates that cell engineering workflows should be cell line, protein product and production-process specific; and that next-generation cell engineering technology that enables precise control of the relative expression of multiple functional genetic components is necessary to achieve this.


Assuntos
Células CHO , Engenharia Celular/métodos , Engenharia Genética/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Células CHO/metabolismo , Técnicas de Cultura de Células , Cricetinae , Cricetulus , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Via Secretória/genética , Via Secretória/fisiologia
7.
Mol Biotechnol ; 62(3): 151-167, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32036549

RESUMO

Single chain variable fragments (scFvs) are generated by joining together the variable heavy and light chain of a monoclonal antibody (mAb) via a peptide linker. They offer some advantages over the parental mAb such as low molecular weight, heterologous production, multimeric form, and multivalency. The scFvs were produced against more than 50 antigens till date using 10 different plant species as the expression system. There were considerable improvements in the expression and purification strategies of scFv in the last 24 years. With the growing demand of scFv in therapeutic and diagnostic fields, its biosynthesis needs to be increased. The easiness in development, maintenance, and multiplication of transgenic plants make them an attractive expression platform for scFv production. The review intends to provide comprehensive information about the use of plant expression system to produce scFv. The developments, advantages, pitfalls, and possible prospects of improvement for the exploitation of plants in the industrial level are discussed.


Assuntos
Anticorpos Monoclonais , Expressão Gênica , Anticorpos de Cadeia Única , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Humanos , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética
8.
PLoS One ; 15(1): e0228164, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31995598

RESUMO

Most of the approved monoclonal antibodies used in the clinic were initially discovered in mice. However, many targets of therapeutic interest are highly conserved proteins that do not elicit a robust immune response in mice. There is a need for non-mammalian antibody discovery platforms which would allow researchers to access epitopes that are not recognized in mammalian hosts. Recently, we introduced the OmniChicken®, a transgenic animal carrying human VH3-23 and VK3-15 at its immunoglobulin loci. Here, we describe a new version of the OmniChicken which carries VH3-23 and either VL1-44 or VL3-19 at its heavy and light chain loci, respectively. The Vλ-expressing birds showed normal B and T populations in the periphery. A panel of monoclonal antibodies demonstrated comparable epitope coverage of a model antigen compared to both wild-type and Vκ-expressing OmniChickens. Kinetic analysis identified binders in the picomolar range. The Vλ-expressing bird increases the antibody diversity available in the OmniChicken platform, further enabling discovery of therapeutic leads.


Assuntos
Animais Geneticamente Modificados/genética , Galinhas/genética , Cadeias lambda de Imunoglobulina/genética , Animais , Animais Geneticamente Modificados/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Galinhas/imunologia , Humanos , Imunidade Humoral , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/imunologia , Progranulinas/imunologia , Linfócitos T/imunologia , Transgenes/genética
9.
Sci Signal ; 13(616)2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992582

RESUMO

Monoclonal antibodies recognize epitopes so specifically that altering a single residue can disrupt binding. In this issue of Science Signaling, Schüchner et al and Frohner et al report that flanking amino acids and an underappreciated posttranslational modification perturb epitope affinity for two groups of widely used monoclonal antibodies.


Assuntos
Anticorpos Monoclonais , Afinidade de Anticorpos/genética , Especificidade de Anticorpos/genética , Epitopos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Humanos
10.
Neuron ; 105(4): 645-662.e11, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-31831332

RESUMO

The intronic C9orf72 G4C2 expansion, the most common genetic cause of ALS and FTD, produces sense- and antisense-expansion RNAs and six dipeptide repeat-associated, non-ATG (RAN) proteins, but their roles in disease are unclear. We generated high-affinity human antibodies targeting GA or GP RAN proteins. These antibodies cross the blood-brain barrier and co-localize with intracellular RAN aggregates in C9-ALS/FTD BAC mice. In cells, α-GA1 interacts with TRIM21, and α-GA1 treatment reduced GA levels, increased GA turnover, and decreased RAN toxicity and co-aggregation of proteasome and autophagy proteins to GA aggregates. In C9-BAC mice, α-GA1 reduced GA as well as GP and GR proteins, improved behavioral deficits, decreased neuroinflammation and neurodegeneration, and increased survival. Glycosylation of the Fc region of α-GA1 is important for cell entry and efficacy. These data demonstrate that RAN proteins drive C9-ALS/FTD in C9-BAC transgenic mice and establish a novel therapeutic approach for C9orf72 ALS/FTD and other RAN-protein diseases.


Assuntos
Esclerose Amiotrófica Lateral/genética , Anticorpos Monoclonais/genética , Proteína C9orf72/genética , Demência Frontotemporal/genética , Terapia Genética/métodos , Proteína ran de Ligação ao GTP/metabolismo , Idoso , Esclerose Amiotrófica Lateral/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/metabolismo , Encéfalo/metabolismo , Proteína C9orf72/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Demência Frontotemporal/metabolismo , Marcação de Genes/métodos , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Distribuição Aleatória , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína ran de Ligação ao GTP/antagonistas & inibidores
11.
Bull Cancer ; 107(1S): S85-S93, 2020 Jan.
Artigo em Francês | MEDLINE | ID: mdl-31547937

RESUMO

The extraordinary and unexpected success of cellular immunotherapy using genetically engineered T-cells to express a chimeric antigen receptor (CAR) targeting CD19, in the treatment of refractory or relapsing B-hematological malignancies, has provided a real therapeutic hope. Indeed, remission rates reach more than 80 % in patients at a stage, without any other possibilities of treatment, notably in the child's acute lymphoblastic leukemia. These results, initially resulting from academic research, led to Food and Drug accreditation for market access of two innovative autologous therapy drugs, Kimryah® and Yescarta®. Based on the impressive clinical results, mainly so far in hematological malignancies (LAL, MM, LBDGC, etc.), the development of several types of cells expressing a CAR receptor suggests a wide range of future applications, particularly in the field of solid tumors. However, while the development of CAR-T cells now appears to be in the hands of private pharmaceuticals companies, the logistical constraints, the cryopreservation and the very high cost of these personalized medicines may ultimately limit their use. The development of academic productions by CAR-T cells could bypass some of these disadvantages. The strong innovation capacity of healthcare institutions associated with research units allows them to identify the ideal tumor target and efficient performing cells. Thus, authorized production platforms could allow for shorter administration times and reasonable production costs for national health systems. The aim of this workshop is to identify the requirements for the academic production of CAR-T cells, while respecting the research standards useful to establish proof of concept, but also at the preclinical development stage, leading in fine to the manufacture, through an authorized pharmaceutical establishment, of the innovative therapy drug, and in accordance with Good Manufacturing Practice (GMP). The ultimate goal is to make these innovative and high-performance medicines available to as many patients as possible.


Assuntos
Imunoterapia Adotiva/normas , Centros Médicos Acadêmicos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/imunologia , Técnicas de Cultura de Células , Linhagem Celular , Indústria Farmacêutica , Europa (Continente) , França , Vetores Genéticos/genética , Humanos , Imunoterapia Adotiva/economia , Imunoterapia Adotiva/métodos , Lentivirus/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Transgenes
12.
Sci Adv ; 5(8): eaaw1822, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31489367

RESUMO

Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas, which secrete antibodies in the preferred format, species, and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab' fragments, isotype-switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemoenzymatic modification. We believe that this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research.


Assuntos
Anticorpos Monoclonais/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Hibridomas/fisiologia , Animais , Especificidade de Anticorpos/genética , Linhagem Celular Tumoral , Genômica/métodos , Fragmentos Fab das Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/genética
13.
J Immunol Methods ; 474: 112654, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31421081

RESUMO

The guinea pig serves as a useful animal model for a number of human diseases and has played an important role during development and testing of experimental vaccines and disease therapies. However, the availability of reagents to examine the immunological response in this species is very limited. Monoclonal antibodies (mAb) specific for cell surface proteins or products of immune cells have been useful tools for characterizing and quantifying immune responses in humans and in murine models of human disease, but very few similar reagents are available for characterizing and manipulating the immune response of guinea pigs. A rat IgG2a mAb specific for guinea pig CD4 has previously been described and was shown to inhibit T cell proliferation, but was inefficient at depleting CD4+ T cells in vivo. We hypothesized that the in vivo CD4+ T cell depletion function of this mAb could be improved by expression of the rat IgG2b heavy chain. We show that the purified mAb from an IgG2b class-switch variant, but not the parental IgG2a mAb, significantly depleted CD4+ T cells from secondary lymphoid tissue of guinea pigs. Further, treatment of guinea pigs with the IgG2b mAb at 2.0 mg/kg resulted in depletion of CD4+ T cells from peripheral blood and spleen. The use of this modified antibody to specifically alter the immune response of guinea pigs should prove useful in a number of guinea pig infectious disease models.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Linfócitos T CD4-Positivos/imunologia , Imunoglobulina G/imunologia , Depleção Linfocítica/métodos , Baço/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Feminino , Cobaias , Hibridomas , Switching de Imunoglobulina , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Ratos
14.
J Immunol Methods ; 474: 112647, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31421082

RESUMO

Cytokeratin 18 (CK18), the main scaffold protein of keratinocyte, is distributed in epithelial cells. This structural protein maintains the integrity and continuity of epithelial tissue. Cytokeratin is also frequently used as an immunohistochemical marker of tumor growth. In recent years, immune repertoire (IR) evaluation using next-generation sequencing (NGS) have become increasingly efficient. Here we deep sequenced the mouse IR of the immunoglobulin heavy chain (IGH) after CK18 immunization. We comprehensively analyzed the IR based on complementarity determining region 3 (CDR3) abundance, germline gene usage polarization, clone diversity, and lineage. We found many convergence characteristics after CK18 immunization. Convergence represents a phenomenon that antigen stimulation or pathogen exposure induces the antigen specific clone expansion and enrichment. The convergence could be used for the immune evaluation and antibody screen. After immunization, the IGHV5 gene clusters became preponderant. The abundance and length of the most frequent CDR3 both increased, nevertheless the IR diversity level decreased. From the convergent IGH repertoires, we selected and expressed six antibodies with the most frequent CDR3s and IGH V-J combinations. The ELISA results suggested all screened six antibodies bound CK18 specifically. The most potential antibody had 9.424E-10M M affinity for the interaction with the CK18. Therefore, this is the NGS platform has been first used for anti-CK18 monoclonal antibodies (MAbs) discovery. These analyses methods could also be used for vaccine evaluation.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Regiões Determinantes de Complementaridade/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Queratina-18/imunologia , Animais , Anticorpos Monoclonais/genética , Afinidade de Anticorpos , Diversidade de Anticorpos , Regiões Determinantes de Complementaridade/genética , Ensaio de Imunoadsorção Enzimática , Sequenciamento de Nucleotídeos em Larga Escala , Imunização , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Queratina-18/administração & dosagem , Masculino , Camundongos Endogâmicos BALB C
15.
Vet Immunol Immunopathol ; 215: 109913, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31420069

RESUMO

The development of a rapid and efficient system to generate porcine monoclonal antibodies (mAbs) is an important step toward the discovery of critical neutralizing targets for designing rational vaccines against porcine viruses. In this study, we established a platform for producing porcine mAbs based on single cell technologies. First, we singled out an optimal donor from 507 pigs based on serum antibody neutralizing activity against porcine reproductive and respiratory syndrome virus (PRRSV). After identifying the contribution of IgG to the neutralizing activity, single CD45R+IgG+Ag+ B cells were sorted from peripheral blood mononuclear cells (PBMCs). Single B cell RT-PCR was performed using primers designed to cover the germline repertoire of the porcine VH/VL gene segments. Paired VH/VLs were cloned into a eukaryotic expression vector and transfected into 293T cells. We demonstrate that full-length porcine mAbs were produced, and antigen-specific mAbs were obtained after further validation. The approach reported in this study can be applied to generate porcine mAbs against any given antigen and may help with the screening of neutralizing antibodies against porcine pathogens.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Suínos/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Especificidade de Anticorpos , Linfócitos B/imunologia , Células HEK293 , Humanos , Região Variável de Imunoglobulina/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Transfecção , Recombinação V(D)J
16.
Monoclon Antib Immunodiagn Immunother ; 38(4): 175-178, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31355694

RESUMO

The diacylglycerol kinases (DGKs) catalyze the phosphorylation of the cell membrane lipid diacylglycerol (DG), which is important in lipid biochemistry and signal transduction into phosphatidic acid. DG-mediated signal transduction downstream of the T cell receptor has been reported to be terminated by DGKζ, 1 of 10 DGK isoforms in most cases. We previously established an anti-DGKζ monoclonal antibody (mAb) DzMab-1 (rat IgG1, kappa), which reacts with both mouse DGKζ and human DGKζ (hDGKζ). In this study, we characterized the binding epitope of DzMab-1 using Western blotting, and found that Met1 and Pro3 residues of hDGKζ are important for facilitating DzMab-1 binding to hDGKζ. Furthermore, DzMab-1 was shown to be useful for immunohistochemical analyses for formalin-fixed paraffin-embedded HeLa cells. These findings could be applied for the production of more functional anti-hDGKζ mAbs.


Assuntos
Anticorpos Monoclonais/imunologia , Diacilglicerol Quinase/imunologia , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Imuno-Histoquímica/métodos , Anticorpos Monoclonais/genética , Diacilglicerol Quinase/antagonistas & inibidores , Diacilglicerol Quinase/genética , Células HeLa , Humanos
17.
Methods Mol Biol ; 2033: 81-93, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31332749

RESUMO

Covalent conjugation of chemical moieties to antibodies has numerous applications, including antibody-drug conjugates, antibody conjugation for diagnostics, and more. Most nonspecific chemical conjugation methods ligate onto any of a number of sites on the antibody, leading to multiple conjugated species, many of which perturb antibody function. To solve these problems, we used CRISPR/Cas9-edited hybridomas to introduce a Sortase tag (LPXTG) and a Flag tag at the 3' end of the CH3 heavy chain region of a mouse monoclonal antibody. The Flag tag allows easy purification of the antibody, while the LPXTG is then acted on by the bacterial transpeptidase Sortase to site-specifically add on any of a number of chemical moieties that possess a triglycine repeat. This technique thus allows rapid production of an antibody onto which a wide array of chemical moieties can be site-specifically conjugated.


Assuntos
Anticorpos Monoclonais/genética , Edição de Genes/métodos , Engenharia Genética/métodos , Imunoconjugados/genética , Animais , Anticorpos Monoclonais/imunologia , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/imunologia , Humanos , Hibridomas/imunologia , Imunoconjugados/imunologia , Camundongos , Oligopeptídeos/genética , Oligopeptídeos/imunologia
18.
Appl Microbiol Biotechnol ; 103(18): 7505-7518, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31350616

RESUMO

The production potential of recombinant monoclonal antibody (mAb) expressing cell lines depends, among other factors, on the intrinsic antibody structure determined by the amino acid sequence. In this study, we investigated the influence of somatic mutations in the V(D)J sequence of four individual, mature model mAbs on the expression potential. Therefore, we defined four couples, each consisting of one naturally occurring mAb (2G12, Ustekinumab, 4B3, and 2F5) and the corresponding germline-derived cognate mAb (353/11, 554/12, 136/63, and 236/14). For all eight mAb variants, recombinant Chinese hamster ovary (CHO) cell lines were developed with mAbs expressed from a defined chromosomal locus. The presented workflow investigates critical parameters including productivity, intra- and extracellular product profile, XBP1 splicing, thermal stability, and in silico hydrophobicity. Significant differences in productivity were even observed between the germline-derived mAbs which did not undergo somatic mutagenesis. Accordingly, back-to-germline mutations of mature mAbs are not necessarily reflecting improved expression and stability but indicate opportunities and limits of mAb engineering. From our studies, we conclude that germinalization represents a potential to improve mAb properties depending on the antibody's germline family, highlighting the fact that mAbs should be treated individually.


Assuntos
Anticorpos Monoclonais/genética , Mutação em Linhagem Germinativa , Proteínas Recombinantes/genética , Temperatura , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Células CHO , Cricetinae , Cricetulus , Mutação , Estabilidade Proteica , Proteínas Recombinantes/imunologia
19.
Med Sci (Paris) ; 35(6-7): 497-500, 2019.
Artigo em Francês | MEDLINE | ID: mdl-31274074

Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias Hematológicas/terapia , Imunoterapia Adotiva/métodos , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Animais , Anticorpos Monoclonais/genética , Antígenos de Neoplasias/imunologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/metabolismo , Caspase 9/genética , Proteínas de Fusão bcr-abl/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Transgênicos Suicidas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunoterapia Adotiva/efeitos adversos , Proteína Acessória do Receptor de Interleucina-1/genética , Proteína Acessória do Receptor de Interleucina-1/imunologia , Células Matadoras Naturais/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/transplante , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos Quiméricos/imunologia
20.
Biotechnol J ; 14(11): e1800573, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31329373

RESUMO

The monoclonal antibody (mAb) industry is witnessing unprecedented growth, with an increasing range of new molecules and biosimilars as well as disease targets approved than ever before. Competition necessitates pharmaceutical companies to reduce development/production costs and time-to-market. To this aim, mathematical modeling can aid traditional experiment-only-based process development by reducing the design space, integrating scales, and assisting in identifying optimal operating conditions in less time and with lower expense. Mathematical models have been employed by other industries for control and optimization purposes and are important decisional tools for testing scenarios, process configurations, operating conditions, etc. Herein, a predictive, experimentally validated mathematical model that captures cellular metabolism and growth with cell cycle, cell death (apoptosis), and mAb production in GS-NS0 cells is presented. The model utilizes cellular, metabolic, and gene expression data, highlighting how multiple data sources can be integrated in one tool with the aim of optimizing mammalian cell bioprocessing.


Assuntos
Anticorpos Monoclonais/biossíntese , Apoptose , Ciclo Celular , Modelos Teóricos , Mieloma Múltiplo/metabolismo , Proteínas do Mieloma/metabolismo , Animais , Anticorpos Monoclonais/genética , Medicamentos Biossimilares , Linhagem Celular Tumoral , Técnicas de Cultura/métodos , Regulação Neoplásica da Expressão Gênica , Camundongos , Mieloma Múltiplo/genética
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