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1.
Int J Mol Sci ; 20(8)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991723

RESUMO

Antibodies leverage on their unique architecture to bind with an array of antigens. The strength of interaction has a direct relation to the affinity of the antibodies towards the antigen. In vivo affinity maturation is performed through multiple rounds of somatic hypermutation and selection in the germinal centre. This unique process involves intricate sequence rearrangements at the gene level via molecular mechanisms. The emergence of in vitro display technologies, mainly phage display and recombinant DNA technology, has helped revolutionize the way antibody improvements are being carried out in the laboratory. The adaptation of molecular approaches in vitro to replicate the in vivo processes has allowed for improvements in the way recombinant antibodies are designed and tuned. Combinatorial libraries, consisting of a myriad of possible antibodies, are capable of replicating the diversity of the natural human antibody repertoire. The isolation of target-specific antibodies with specific affinity characteristics can also be accomplished through modification of stringent protocols. Despite the ability to screen and select for high-affinity binders, some 'fine tuning' may be required to enhance antibody binding in terms of its affinity. This review will provide a brief account of phage display technology used for antibody generation followed by a summary of different combinatorial library characteristics. The review will focus on available strategies, which include molecular approaches, next generation sequencing, and in silico approaches used for antibody affinity maturation in both therapeutic and diagnostic applications.


Assuntos
Anticorpos Monoclonais/genética , Técnicas de Visualização da Superfície Celular/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Mutagênese , Biblioteca de Peptídeos
2.
Nat Commun ; 10(1): 1953, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028254

RESUMO

Malaria vaccine design and prioritization has been hindered by the lack of a mechanistic correlate of protection. We previously demonstrated a strong association between protection and merozoite-neutralizing antibody responses following vaccination of non-human primates against Plasmodium falciparum reticulocyte binding protein homolog 5 (PfRH5). Here, we test the mechanism of protection. Using mutant human IgG1 Fc regions engineered not to engage complement or FcR-dependent effector mechanisms, we produce merozoite-neutralizing and non-neutralizing anti-PfRH5 chimeric monoclonal antibodies (mAbs) and perform a passive transfer-P. falciparum challenge study in Aotus nancymaae monkeys. At the highest dose tested, 6/6 animals given the neutralizing PfRH5-binding mAb c2AC7 survive the challenge without treatment, compared to 0/6 animals given non-neutralizing PfRH5-binding mAb c4BA7 and 0/6 animals given an isotype control mAb. Our results address the controversy regarding whether merozoite-neutralizing antibody can cause protection against P. falciparum blood-stage infections, and highlight the quantitative challenge of achieving such protection.


Assuntos
Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antiprotozoários/imunologia , Humanos , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Vacinas Antimaláricas/uso terapêutico , Malária Falciparum/metabolismo , Plasmodium falciparum/imunologia , Plasmodium falciparum/patogenicidade , Primatas
3.
Proc Natl Acad Sci U S A ; 116(15): 7425-7430, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30910977

RESUMO

Serum IgG, which is mainly generated from IgG-secreting plasma cells in the bone marrow (BM), protects our body against various pathogens. We show here that the protein SiiE of Salmonella is both required and sufficient to prevent an efficient humoral immune memory against the pathogen by selectively reducing the number of IgG-secreting plasma cells in the BM. Attenuated SiiE-deficient Salmonella induces high and lasting titers of specific and protective Salmonella-specific IgG and qualifies as an efficient vaccine against Salmonella A SiiE-derived peptide with homology to laminin ß1 is sufficient to ablate IgG-secreting plasma cells from the BM, identifying laminin ß1 as a component of niches for IgG-secreting plasma cells in the BM, and furthermore, qualifies it as a unique therapeutic option to selectively ablate IgG-secreting plasma cells in autoimmune diseases and multiple myeloma.


Assuntos
Células da Medula Óssea/imunologia , Imunidade Humoral , Imunoglobulina G/imunologia , Memória Imunológica , Plasmócitos/imunologia , Salmonella/imunologia , Animais , Células da Medula Óssea/citologia , Imunoglobulina G/genética , Laminina/genética , Laminina/imunologia , Camundongos , Camundongos Knockout , Plasmócitos/citologia , Salmonella/genética
4.
Methods Mol Biol ; 1953: 121-136, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912019

RESUMO

Single-domain antibodies represent an emerging class of antibody fragments with promising therapeutic and diagnostic potential. As a result, multiple strategies have been developed in order to improve their biophysical and/or biological properties. In particular, the fusion of single-domain antibodies to the Fc part of an IgG molecule has become a common protein engineering approach toward this aim. Here, we describe a detailed protocol for a streamlined laboratory-scale production of VH single-domain antibodies as Fc fusions in mammalian cells. Firstly, DNA sequence encoding VH domain of interest fused to an IgG Fc is synthesized as a double-stranded gene fragment. Secondly, the DNA fragment is directly assembled into a restriction enzyme-digested vector in an assembly reaction. Finally, vector carrying the VH-Fc-fusion construct is introduced into suspension-adapted mammalian cells for transient expression of the Fc chimeric fusion. One-week post-transfection, the expressed Fc-fusion protein is purified using protein A/G affinity chromatography. Using this protocol, we were able to clone, express, and purify milligrams of isolated anti-HER2 VH domain as a mouse IgG2c Fc fusion in less than 2 weeks. This protocol can be readily modified to express proteins of interest other than VH domains as Fc fusions.


Assuntos
Fragmentos Fc das Imunoglobulinas/genética , Proteínas Recombinantes de Fusão/genética , Anticorpos de Domínio Único/genética , Animais , Biotinilação , Linhagem Celular , Cromatografia de Afinidade/métodos , Clonagem Molecular/métodos , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Camundongos , Plasmídeos/genética , Receptor ErbB-2/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/isolamento & purificação , Transfecção/métodos
5.
J Immunol Res ; 2019: 4516041, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30886871

RESUMO

Bispecific antibodies (BsAbs) are a sort of dual functional proteins with specific binding to two distinct targets, which have become a focus of interest in antibody engineering and drug development research and have a promising future for wide applications in cancer immunotherapy and autoimmune disease. The key of clinical application and commercial-scale manufacturing of BsAbs is the amenability to assembly and purification of desired heterodimers. Advances in genetic engineering technology had resulted in the development of diverse BsAbs. Multiple recombinant strategies have been used to solve the mispairing problem between light and heavy chains, as well as to enforce accurate dimerization of heterologous heavy chains. There are 23 platforms available to generate 62 BsAbs which can be further divided into IgG-like ones and fragment-based ones, and more than 50 molecules are undergoing clinical trials currently. BsAbs with IgG-like architecture exhibit superior advantages in structure (similar to natural antibodies), pharmacokinetics, half-life, FcR-mediated function, and biological activity. This review considers various IgG-like BsAb generation approaches, summarizes the clinical applications of promising new BsAbs, and describes the mechanism of BsAbs in tumor therapy.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Imunoglobulina G/metabolismo , Imunoterapia/métodos , Neoplasias/terapia , Animais , Anticorpos Biespecíficos/genética , Dimerização , Engenharia Genética , Humanos , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Neoplasias/imunologia
6.
Fish Shellfish Immunol ; 88: 464-471, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30858097

RESUMO

This study reports the development of a monoclonal antibody (designated 3B10) against the muskellunge (Esox masquinongy) IgM. The 3B10 monoclonal antibody (mAb) belongs to the IgG3 kappa isotype. Western blotting demonstrated that 3B10 mAb reacted primarily to muskellunge IgM heavy chain. 3B10 also reacted strongly with the IgM heavy chain of other esocids, including the northern pike (Esox lucius), tiger muskellunge (E. masquinongy x E. lucius), and, to a much lesser extent, the chain pickerel (E. niger). The 3B10 mAb did not bind to IgM from 10 other fish species resident in the Great Lakes basin. Using the 3B10 mAb, it was possible to determine the muskellunge Ig ability to bind to antigens. Using trinitrophenyl hapten conjugated to keyhole limpet hemocyanin (TNP-KLH) as the eliciting antigen, muskellunge Ig subclasses exhibited a range of affinities with log aK values 5.56-6.25 that is considered intermediate compared to other fish species. 3B10 mAb was used to develop and evaluate an indirect ELISA for the detection and quantitation of circulating antibodies against the viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb). Using the newly optimized assay, anti-VHSV-IVb antibodies were detected in sera of VHSV-IVb vaccinated muskellunge as well as from those of wild muskellunge sampled from an endemic waterbody. In addition to its use in immunoassays, the developed 3B10 mAb will enable future investigation aiming at deciphering immune mechanism of this important fish species to pathogens.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/sangue , Esocidae/imunologia , Septicemia Hemorrágica Viral/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Animais , Anticorpos Monoclonais/genética , Ensaio de Imunoadsorção Enzimática , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Peixes/imunologia , Peixes/virologia , Genótipo , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Novirhabdovirus
7.
Iran J Immunol ; 16(1): 26-42, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30864553

RESUMO

BACKGROUND: We have recently produced an inhibitory mouse anti-human HER2 mAb (2A8) which displayed potent anti-tumor activity in combination with trastuzumab. OBJECTIVE: To describe chimerization and functional characterization of 2A8 mAb. METHODS: The VH and VL genes of 2A8 mAb were amplified from cDNA of the mouse hybridoma, ligated to constant regions of human immunoglobulin, and expressed in CHO cell line. Reactivity with four members of human HER family, the inhibitory effects and antibody-dependent cell cytotoxicity (ADCC) of purified chimeric mAb (c2A8) were assessed by ELISA, XTT, H3-tymidine incorporation and lactate dehydrogenase assays. Inhibition of ERK and AKT downstream signaling pathways by the chimeric antibody were analyzed by Western blotting. RESULTS: Chimeric 2A8 mAb bound to recombinant human HER2 and did not cross-react with the other members of HER family. Moreover, c2A8 was able to recognize HER2-overexpressing cancer cell line and inhibited growth and proliferation of these cells. The binding affinity of c2A8 was comparable to the mouse parental mAb. ADCC and Western blotting results showed that the mouse 2A8 mAb was successfully chimerized and could significantly inhibit phosphorylation of AKT in combination with trastuzumab. CONCLUSION: The c2A8 mAb is potentially a valuable tool for targeted immunotherapy of HER2 positive cancers.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Especificidade de Anticorpos/genética , Especificidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Reações Cruzadas/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Macaca fascicularis , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Transfecção , Trastuzumab/farmacologia
8.
Proc Natl Acad Sci U S A ; 116(9): 3758-3763, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808762

RESUMO

Echoviruses are amongst the most common causative agents of aseptic meningitis worldwide and are particularly devastating in the neonatal population, where they are associated with severe hepatitis, neurological disease, including meningitis and encephalitis, and even death. Here, we identify the neonatal Fc receptor (FcRn) as a pan-echovirus receptor. We show that loss of expression of FcRn or its binding partner beta 2 microglobulin (ß2M) renders cells resistant to infection by a panel of echoviruses at the stage of virus attachment, and that a blocking antibody to ß2M inhibits echovirus infection in cell lines and in primary human intestinal epithelial cells. We also show that expression of human, but not mouse, FcRn renders nonpermissive human and mouse cells sensitive to echovirus infection and that the extracellular domain of human FcRn directly binds echovirus particles and neutralizes infection. Lastly, we show that neonatal mice expressing human FcRn are more susceptible to echovirus infection by the enteral route. Our findings thus identify FcRn as a pan-echovirus receptor, which may explain the enhanced susceptibility of neonates to echovirus infections.


Assuntos
Enterovirus Humano B/genética , Antígenos de Histocompatibilidade Classe I/genética , Receptores Fc/genética , Receptores Virais/genética , Microglobulina beta-2/genética , Animais , Infecções por Echovirus/genética , Infecções por Echovirus/imunologia , Infecções por Echovirus/virologia , Enterovirus Humano B/patogenicidade , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Ligação Proteica , Microglobulina beta-2/imunologia
9.
Nat Commun ; 10(1): 893, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30792391

RESUMO

Our understanding of the conformational and electrostatic determinants that underlie targeting of human leukocyte antigens (HLA) by anti-HLA alloantibodies is principally based upon in silico modelling. Here we provide a biochemical/biophysical and functional characterization of a human monoclonal alloantibody specific for a common HLA type, HLA-A*11:01. We present a 2.4 Å resolution map of the binding interface of this antibody on HLA-A*11:01 and compare the structural determinants with those utilized by T-cell receptor (TCR), killer-cell immunoglobulin-like receptor (KIR) and CD8 on the same molecule. These data provide a mechanistic insight into the paratope-epitope relationship between an alloantibody and its target HLA molecule in a biological context where other immune receptors are concomitantly engaged. This has important implications for our interpretation of serologic binding patterns of anti-HLA antibodies in sensitized individuals and thus, for the biology of human alloresponses.


Assuntos
Antígeno HLA-A11/química , Antígeno HLA-A11/metabolismo , Isoanticorpos/química , Isoanticorpos/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/metabolismo , Sítios de Ligação de Anticorpos/genética , Cristalografia por Raios X , Epitopos/química , Epitopos/genética , Epitopos/metabolismo , Antígeno HLA-A11/genética , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Isoanticorpos/genética , Modelos Moleculares , Biblioteca de Peptídeos , Conformação Proteica
10.
Microb Pathog ; 129: 68-73, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30711546

RESUMO

Introducing an effective vaccine for tuberculosis (TB) is an urgent need. Mycobacterium tuberculosis (Mtb) Ag85 complex is suggested for making protective immunodominant antigens for design and development of novel TB vaccine. In the present study, a pDR2EF1-Fcγ1 vector has been used to make Ag85B:hFcγ1 recombinant fusion protein. Briefly, specific XbaI and NotI site incorporated primers were used to amplify Mtb-fbpB gene by PCR, TA-cloned and amplified in E.coli DH5α. The resulting vector then subcloned into the pDR2EF1.Fcγ1 vector and transferred to Chinese hamster ovary (CHO) cell line. DNA sequencing was performed to confirm that Ag85B:hFcγ1 construct is precise and in-frame. Then, Ag85B:hFcγ1 protein was produced by CHO expression system and recombinant protein was purified using HiTrap rProtein A Sepharose Fast Flow column. The presence of recombinant fusion protein confirmed by immunofluorescence (IFA) and Western blotting (WB). This fusion protein containing Fc fragment of human IgG1, apart from stability and adjuvanticity potential, could bind to FcRγI (CD64) on the surface of antigen-presenting cells (APCs) and induce cross-presentation in favour of host immune response and can be used as a potential candidate along with other subunit vaccines against Mtb.


Assuntos
Aciltransferases/metabolismo , Apresentação do Antígeno , Células Apresentadoras de Antígenos/metabolismo , Antígenos de Bactérias/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Aciltransferases/genética , Animais , Antígenos de Bactérias/genética , Células CHO , Cricetulus , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Ligação Proteica , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusão/genética
11.
MAbs ; 11(3): 516-531, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30663541

RESUMO

We describe here the design, construction and validation of ALTHEA Gold Libraries™. These single-chain variable fragment (scFv), semisynthetic libraries are built on synthetic human well-known IGHV and IGKV germline genes combined with natural human complementarity-determining region (CDR)-H3/JH (H3J) fragments. One IGHV gene provided a universal VH scaffold and was paired with two IGKV scaffolds to furnish different topographies for binding distinct epitopes. The scaffolds were diversified at positions identified as in contact with antigens in the known antigen-antibody complex structures. The diversification regime consisted of high-usage amino acids found at those positions in human antibody sequences. Functionality, stability and diversity of the libraries were improved throughout a three-step construction process. In a first step, fully synthetic primary libraries were generated by combining the diversified scaffolds with a set of synthetic neutral H3J germline gene fragments. The second step consisted of selecting the primary libraries for enhanced thermostability based on the natural capacity of Protein A to bind the universal VH scaffold. In the third and final step, the resultant stable synthetic antibody fragments were combined with natural H3J fragments obtained from peripheral blood mononuclear cells of a large pool of 200 donors. Validation of ALTHEA Gold Libraries™ with seven targets yielded specific antibodies in all the cases. Further characterization of the isolated antibodies indicated KD values as human IgG1 molecules in the single-digit and sub-nM range. The thermal stability (Tm) of all the antigen-binding fragments was 75°C-80°C, demonstrating that ALTHEA Gold Libraries™ are a valuable source of specific, high affinity and highly stable antibodies.


Assuntos
Regiões Determinantes de Complementaridade , Biblioteca Gênica , Imunoglobulina G , Anticorpos de Cadeia Única , Regiões Determinantes de Complementaridade/biossíntese , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/genética , Leucócitos Mononucleares/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética
12.
MAbs ; 11(3): 559-568, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30694096

RESUMO

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.


Assuntos
Anticorpos Monoclonais Murinos , Antígenos , Imunoglobulina A , Imunoglobulina G , Região Variável de Imunoglobulina , Proteínas Recombinantes de Fusão , Animais , Anticorpos Monoclonais Murinos/biossíntese , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/genética , Antígenos/biossíntese , Antígenos/química , Antígenos/genética , Células CHO , Cricetulus , Imunoglobulina A/biossíntese , Imunoglobulina A/química , Imunoglobulina A/genética , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/genética , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Camundongos , Peptídeos/química , Peptídeos/genética , Proteólise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Suínos
13.
Immunogenetics ; 71(4): 307-320, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30656359

RESUMO

Camelid ungulates produce homodimeric heavy chain-only antibodies (HCAbs) in addition to conventional antibodies consisting of paired heavy and light chains. In the llama, HCAbs are made up by at least two subclasses (long-hinge IgG2b and short-hinge IgG2c HCAbs vs. conventional heterotetrameric IgG1s). Here, we generated murine monoclonal antibodies (mAbs) specific for the hinge-CH2 boundary of llama IgG2b (mAb 1C10) and the Fc of llama IgG2c HCAbs (mAb 5E4). Flow cytometric analysis of llama peripheral blood lymphocytes revealed that IgG1+, IgG2b+ and IgG2c+ B cells could be distinguished using mAbs 1C10/5E4 but had equivalent expression of three other cell-surface markers. MiSeq sequencing of the peripheral B cell repertoires of three llamas showed that (i) IgG2b and IgG2c HCAbs were present in similar proportions in the repertoire, (ii) a subset of IgG2b and IgG2c HCAbs, but not IgG1s, entirely lacked a hinge exon and showed direct VHH-CH2 splicing; these "hingeless" HCAbs were clonally expanded, somatically mutated and derived from hinged HCAb precursors, (iii) substantial repertoire overlap existed between IgG subclasses, especially between IgG2b and IgG2c HCAbs, (iv) the complementarity-determining region (CDR)-H3 length distributions of IgG2b and IgG2c HCAbs were broader and biased towards longer lengths compared with IgG1s due to increased N-nucleotide addition, (v) IgG2b and IgG2c HCAbs used a more restricted set of IGHV genes compared with IgG1s, and (vi) IgG2b and IgG2c HCAbs had elevated somatic mutations rates of both CDRs and framework regions (FRs) compared with IgG1s, especially of CDR-H1 and FR3. The distinct molecular features of llama IgG1, IgG2b and IgG2c antibodies imply that these subclasses may have divergent immunological functions and suggest that specific mechanisms operate to diversify HCAb repertoires in the absence of a light chain.


Assuntos
Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Camelídeos Americanos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Animais , Linfócitos B/metabolismo , Camelídeos Americanos/genética , Regiões Determinantes de Complementaridade/genética , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Imunogenética/métodos , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Imunofenotipagem/métodos , Camundongos
14.
Methods Enzymol ; 614: 239-261, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30611426

RESUMO

The majority of proteins excreted by human cells and borne at the cell surface are modified with carbohydrates. Glycoproteins mediate a wide range of processes and adopt fundamental roles in many diseases. The carbohydrates covalently attached to proteins during maturation in the cell directly impact protein structure and function as integral and indispensable components. However, the ability to study the structure of glycoproteins to high resolution was historically limited by technical barriers including a limited availability of appropriate recombinant protein expression platforms, limited methods to generate compositional homogeneity, and difficulties analyzing glycoprotein composition. Furthermore, glycoproteins and in particular the glycan moieties themselves often exhibit a high degree of conformational heterogeneity. Solution NMR spectroscopy is a powerful tool to study biological macromolecules that is capable of characterizing mobile elements of molecules with atomic-level resolution. Methods to express glycoproteins, incorporate stable isotope labels, and analyze glycoproteins have recently opened new avenues to prepare and investigate glycoproteins. These methods are accessible to many laboratories with experience expressing and purifying proteins from prokaryotic expression hosts.


Assuntos
Glicoproteínas/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Marcação por Isótopo/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Sequência de Carboidratos , Linhagem Celular , Cromatografia de Afinidade , Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Serina/química , Serina/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Treonina/química , Treonina/metabolismo
15.
J Pharmacol Exp Ther ; 368(3): 435-445, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30563942

RESUMO

Follistatin is an endogenous glycoprotein that promotes growth and repair of skeletal muscle by sequestering inhibitory ligands of the transforming growth factor-ß superfamily and may therefore have therapeutic potential for neuromuscular diseases. Here, we sought to determine the suitability of a newly engineered follistatin fusion protein (FST288-Fc) to promote localized, rather than systemic, growth of skeletal muscle by capitalizing on the intrinsic heparin-binding ability of the follistatin-288 isoform. As determined by surface plasmon resonance and cell-based assays, FST288-Fc binds to activin A, activin B, myostatin (growth differentiation factor GDF8), and GDF11 with high affinity and neutralizes their activity in vitro. Intramuscular administration of FST288-Fc in mice induced robust, dose-dependent growth of the targeted muscle but not of surrounding or contralateral muscles, in contrast to the systemic effects of a locally administered fusion protein incorporating activin receptor type IIB (ActRIIB-Fc). Furthermore, systemic administration of FST288-Fc in mice did not alter muscle mass or body composition as determined by NMR, which again contrasts with the pronounced systemic activity of ActRIIB-Fc when administered by the same route. Subsequent analysis revealed that FST288-Fc in the circulation undergoes rapid proteolysis, thereby restricting its activity to individual muscles targeted by intramuscular administration. These results indicate that FST288-Fc can produce localized growth of skeletal muscle in a targeted manner with reduced potential for undesirable systemic effects. Thus, FST288-Fc and similar agents may be beneficial in the treatment of disorders with muscle atrophy that is focal, asymmetric, or otherwise heterogeneous.


Assuntos
Folistatina/administração & dosagem , Imunoglobulina G/administração & dosagem , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/crescimento & desenvolvimento , Proteínas Recombinantes de Fusão/administração & dosagem , Sequência de Aminoácidos , Animais , Relação Dose-Resposta a Droga , Folistatina/genética , Folistatina/metabolismo , Humanos , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Injeções Intramusculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
16.
MAbs ; 11(2): 401-410, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30569818

RESUMO

In vitro immunization can to used to produce monoclonal antibodies(mAbs), but this technology is limited by poor reproducibility and the requirement of pre-immunized lymphocytes. To improve this approach, we recently developed a method for rapid generation of antigen-specific B cells. Here, we report the application of this system to the production of human IgGs against tumor necrosis factor (TNF). We expressed mutant proteins with site-specific incorporated p-nitrophenylalanine (pNO2Phe), which stimulated an in vitro immune response in human immune cells. After constructing an antigen-specific antibody library from in vitro immunized B cells identified by fluorescence-activated cell sorting, we demonstrated that many point mutation events of the variable region occurred in our step-by-step co-cultivation system for affinity maturation in vitro. To mimic the class switching, we panned for high-affinity antigen-binding fragments by the phage display method, assembled them and identified hTNF-neutralizing human IgGs. This approach may provide a general method for raising high-affinity monoclonal antibodies against self-proteins. Furthermore, it supports mechanistic understanding in breaking human self-tolerance with pNO2Phe.


Assuntos
Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Imunoglobulina G/imunologia , Técnicas Imunológicas/métodos , Fenilalanina/análogos & derivados , Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/genética , Técnicas In Vitro , Biblioteca de Peptídeos , Fenilalanina/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores
17.
Methods Mol Biol ; 1904: 431-454, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30539485

RESUMO

Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigens, enabling therapeutic strategies not possible with conventional monoclonal antibodies (mAbs). Since bispecific antibodies are regarded as promising therapeutic agents, many different bispecific design modalities have been evaluated. Many of these are based on antibody fragments or on inclusion of non-antibody components. For some therapeutic applications, full-size, native IgG-like bsAbs may be the optimal format.To prepare bsAbs in IgG format, two challenges should be met. One is that each heavy chain will only pair with the heavy chain of the second specificity and that heavy chain homodimerization will be prevented. The second is that each heavy chain will only pair with the light chain of its own specificity and that pairing with the light chain of the second specificity will be prevented. The first solution to the first criterion (known as knobs into holes, KIH) was presented in 1996 by Genentech and additional solutions were presented more recently. However, until recently, out of >120 published formats, only a handful of solutions for the second criterion that make it possible to produce a bispecific IgG by a single expressing cell were suggested.Here, we present a protocol for preparing bsAbs in IgG format in transfected mammalian cells. For heavy chain dimerization we use KIH while as a solution for the second challenge-correct pairing of heavy and light chains of bispecific IgGs we present our "BIClonals" technology; an engineered (artificial) disulfide bond between the antibodies' variable domains that asymmetrically replaces the natural disulfide bond between CH1 and CL.During our studies of bsAbs we found that H-L chain pairing seems to be driven by VH-VL interfacial interactions that differ between different antibodies; hence, there is no single optimal solution for effective and precise assembly of bispecific IgGs that suits every antibody sequence, making it necessary to carefully evaluate the optimal solution for each new antibody.


Assuntos
Anticorpos Biespecíficos/biossíntese , Anticorpos Biespecíficos/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Linhagem Celular , Expressão Gênica , Vetores Genéticos/genética , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Engenharia de Proteínas , Transfecção
18.
J Pharm Biomed Anal ; 162: 91-100, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30227357

RESUMO

Metal ions can be enzyme cofactors and can directly influence the kinetics of biochemical reactions that also influence the biological production and quality attributes of therapeutic proteins, such as glycan formation and distribution. However, the concentrations of metals in commercially available chemically defined media can range from 1 to 25,000 ppb. Because such concentration changes can impact cell growth, manufacturing yield and product quality the alteration/fluctuation in media composition should be well controlled to maintain product quality. Here, we describe a platform of analytical methods to determine the composition of several metals in different sample matrices using an advanced automated Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). These methods, validated to ICH Q2R1 regulatory validation parameters, were successfully applied to- (a) screen cell culture media; (b) determine changes in the metal concentration during cell growth in spinner flasks, and, (c) determine effect on the glycosylation pattern and homogeneity of an IgG3:κ produced from a murine-hybridoma cell line in bench-top parallel bioreactors due to a spike in copper and iron concentration. Our results show that maintenance of metal content in the cell culture media is critical for product consistency of the IgG3:κ produced.


Assuntos
Anticorpos Monoclonais/biossíntese , Cobre/metabolismo , Meios de Cultura/metabolismo , Glucuronidase/biossíntese , Imunoglobulina G/biossíntese , Cadeias kappa de Imunoglobulina/biossíntese , Ferro/metabolismo , Espectrometria de Massas/métodos , Animais , Anticorpos Monoclonais/genética , Reatores Biológicos , Células CHO , Proliferação de Células , Cricetulus , Glucuronidase/genética , Glicosilação , Hibridomas , Imunoglobulina G/genética , Cadeias kappa de Imunoglobulina/genética , Espectrometria de Massas/normas , Camundongos , Controle de Qualidade , Reprodutibilidade dos Testes , Fatores de Tempo , Transfecção
19.
Immunology ; 156(1): 56-68, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30171602

RESUMO

B lymphocytes, known as antibody producers, mediate tumor cell destruction in the manner of antibody-dependent cell-mediated cytotoxicity; however, their anti-tumor function seems to be weakened during tumorigenesis, while the underlying mechanisms remain unclear. In this study, we found that IgG mediated anti-tumor effects, but IgG-producing B cells decreased in various tumors. Considering the underlying mechanism, glycometabolism was noteworthy. We found that tumor-infiltrating B cells were glucose-starved and accompanied by a deceleration of glycometabolism. Both inhibition of glycometabolism and deprivation of glucose through tumor cells, or glucose-free treatment, reduced the differentiation of B cells into IgG-producing cells. In this process, special AT-rich sequence-binding protein-1 (SATB1) was significantly silenced in B cells. Down-regulating SATB1 by inhibiting glycometabolism or RNA interference reduced the binding of signal transducer and activator of transcription 6 (STAT6) to the promoter of germline Cγ gene, subsequently resulting in fewer B cells producing IgG. Our findings provide the first evidence that glycometabolic inhibition by tumorigenesis suppresses differentiation of B cells into IgG-producing cells, and altering glycometabolism may be promising in improving the anti-tumor effect of B cells.


Assuntos
Adenocarcinoma/imunologia , Linfócitos B/metabolismo , Neoplasias Colorretais/imunologia , Glucose/metabolismo , Neoplasias Pulmonares/imunologia , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Neoplasias/imunologia , Idoso , Animais , Azoximetano , Linfócitos B/imunologia , Células Cultivadas , Neoplasias Colorretais/induzido quimicamente , Sulfato de Dextrana , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Masculino , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/genética , Fator de Transcrição STAT6/metabolismo
20.
Proc Natl Acad Sci U S A ; 115(34): 8615-8620, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30072430

RESUMO

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a classical nonhomologous end-joining (cNHEJ) factor. Loss of DNA-PKcs diminished mature B cell class switch recombination (CSR) to other isotypes, but not IgG1. Here, we show that expression of the kinase-dead DNA-PKcs (DNA-PKcsKD/KD ) severely compromises CSR to IgG1. High-throughput sequencing analyses of CSR junctions reveal frequent accumulation of nonproductive interchromosomal translocations, inversions, and extensive end resection in DNA-PKcsKD/KD , but not DNA-PKcs-/- , B cells. Meanwhile, the residual joints from DNA-PKcsKD/KD cells and the efficient Sµ-Sγ1 junctions from DNA-PKcs-/- B cells both display similar preferences for small (2-6 nt) microhomologies (MH). In DNA-PKcs-/- cells, Sµ-Sγ1 joints are more resistant to inversions and extensive resection than Sµ-Sε and Sµ-Sµ joints, providing a mechanism for the isotype-specific CSR defects. Together, our findings identify a kinase-dependent role of DNA-PKcs in suppressing MH-mediated end joining and a structural role of DNA-PKcs protein in the orientation of CSR.


Assuntos
Linfócitos B/enzimologia , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Switching de Imunoglobulina/fisiologia , Imunoglobulina G/biossíntese , Proteínas Nucleares/metabolismo , Recombinação Genética/fisiologia , Animais , Linfócitos B/citologia , Linhagem Celular , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Imunoglobulina G/genética , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética
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