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1.
Appl Microbiol Biotechnol ; 108(1): 179, 2024 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-38280035

RESUMO

Several COVID-19 vaccines use adenovirus vectors to deliver the SARS-CoV-2 spike (S) protein. Immunization with these vaccines promotes immunity against the S protein, but against also the adenovirus itself. This could interfere with the entry of the vaccine into the cell, reducing its efficacy. Herein, we evaluate the efficiency of an adenovirus-vectored vaccine (chimpanzee ChAdOx1 adenovirus, AZD1222) in boosting the specific immunity compared to that induced by a recombinant receptor-binding domain (RBD)-based vaccine without viral vector. Mice immunized with the AZD1222 human vaccine were given a booster 6 months later, with either the homologous vaccine or a recombinant vaccine based on RBD of the delta variant, which was prevalent at the start of this study. A significant increase in anti-RBD antibody levels was observed in rRBD-boosted mice (31-61%) compared to those receiving two doses of AZD1222 (0%). Significantly higher rates of PepMix™- or RBD-elicited proliferation were also observed in IFNγ-producing CD4 and CD8 cells from mice boosted with one or two doses of RBD, respectively. The lower efficiency of the ChAdOx1-S vaccine in boosting specific immunity could be the result of a pre-existing anti-vector immunity, induced by increased levels of anti-adenovirus antibodies found both in mice and humans. Taken together, these results point to the importance of avoiding the recurrent use of the same adenovirus vector in individuals with immunity and memory against them. It also illustrates the disadvantages of ChAdOx1 adenovirus-vectored vaccine with respect to recombinant protein vaccines, which can be used without restriction in vaccine-booster programs. KEY POINTS: • ChAdOx1 adenovirus vaccine (AZD1222) may not be effective in boosting anti-SARS-CoV-2 immunity • A recombinant RBD protein vaccine is effective in boosting anti-SARS-CoV-2 immunity in mice • Antibodies elicited by the rRBD-delta vaccine persisted for up to 3 months in mice.


Assuntos
Vacinas contra Adenovirus , COVID-19 , Vacinas , Humanos , Animais , Camundongos , Pan troglodytes , ChAdOx1 nCoV-19 , Vacinas contra COVID-19/genética , SARS-CoV-2 , COVID-19/prevenção & controle , Adenoviridae/genética , Vacinação , Anticorpos Antivirais , Anticorpos Neutralizantes
2.
Int J Mol Sci ; 24(5)2023 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-36902040

RESUMO

We recently reported the isolation and characterization of anti-SARS-CoV-2 antibodies from a phage display library built with the VH repertoire of a convalescent COVID-19 patient, paired with four naïve synthetic VL libraries. One of the antibodies, called IgG-A7, neutralized the Wuhan, Delta (B.1.617.2) and Omicron (B.1.1.529) strains in authentic neutralization tests (PRNT). It also protected 100% transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE-2) from SARS-CoV-2 infection. In this study, the four synthetic VL libraries were combined with the semi-synthetic VH repertoire of ALTHEA Gold Libraries™ to generate a set of fully naïve, general-purpose, libraries called ALTHEA Gold Plus Libraries™. Three out of 24 specific clones for the RBD isolated from the libraries, with affinity in the low nanomolar range and sub-optimal in vitro neutralization in PRNT, were affinity optimized via a method called "Rapid Affinity Maturation" (RAM). The final molecules reached sub-nanomolar neutralization potency, slightly superior to IgG-A7, while the developability profile over the parental molecules was improved. These results demonstrate that general-purpose libraries are a valuable source of potent neutralizing antibodies. Importantly, since general-purpose libraries are "ready-to-use", it could expedite isolation of antibodies for rapidly evolving viruses such as SARS-CoV-2.


Assuntos
COVID-19 , Animais , Humanos , Camundongos , Anticorpos Neutralizantes , Anticorpos Antivirais , Imunoglobulina G , Camundongos Transgênicos , SARS-CoV-2
3.
Int J Mol Sci ; 23(17)2022 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-36077159

RESUMO

Since the first COVID-19 reports back in December of 2019, this viral infection caused by SARS-CoV-2 has claimed millions of lives. To control the COVID-19 pandemic, the Food and Drug Administration (FDA) and/or European Agency of Medicines (EMA) have granted Emergency Use Authorization (EUA) to nine therapeutic antibodies. Nonetheless, the natural evolution of SARS-CoV-2 has generated numerous variants of concern (VOCs) that have challenged the efficacy of the EUA antibodies. Here, we review the most relevant characteristics of these therapeutic antibodies, including timeline of approval, neutralization profile against the VOCs, selection methods of their variable regions, somatic mutations, HCDR3 and LCDR3 features, isotype, Fc modifications used in the therapeutic format, and epitope recognized on the receptor-binding domain (RBD) of SARS-CoV-2. One of the conclusions of the review is that the EUA therapeutic antibodies that still retain efficacy against new VOCs bind an epitope formed by conserved residues that seem to be evolutionarily conserved as thus, critical for the RBD:hACE-2 interaction. The information reviewed here should help to design new and more efficacious antibodies to prevent and/or treat COVID-19, as well as other infectious diseases.


Assuntos
Tratamento Farmacológico da COVID-19 , Glicoproteína da Espícula de Coronavírus , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Epitopos , Humanos , Glicoproteínas de Membrana/metabolismo , Testes de Neutralização , Pandemias , SARS-CoV-2 , Estados Unidos , Proteínas do Envelope Viral/genética
4.
Proteins ; 82(8): 1656-67, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24638881

RESUMO

The functional role of human antihinge (HAH) autoantibodies in normal health and disease remains elusive, but recent evidence supports their role in the host response to IgG cleavage by proteases that are prevalent in certain disorders. Characterization and potential exploitation of these HAH antibodies has been hindered by the absence of monoclonal reagents. 2095-2 is a rabbit monoclonal antibody targeting the IdeS-cleaved hinge of human IgG1. We have determined the crystal structure of the Fab of 2095-2 and its complex with a hinge analog peptide. The antibody is selective for the C-terminally cleaved hinge ending in G236 and this interaction involves an uncommon disulfide in VL CDR3. We probed the importance of the disulfide in VL CDR3 through engineering variants. We identified one variant, QAA, which does not require the disulfide for biological activity or peptide binding. The structure of this variant offers a starting point for further engineering of 2095-2 with the same specificity, but lacking the potential manufacturing liability of an additional disulfide. Proteins 2014; 82:1656-1667. © 2014 Wiley Periodicals, Inc.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Conformação Proteica , Proteólise , Coelhos
5.
Proteins ; 82(8): 1553-62, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24668560

RESUMO

To assess the state of the art in antibody 3D modeling, 11 unpublished high-resolution x-ray Fab crystal structures from diverse species and covering a wide range of antigen-binding site conformations were used as a benchmark to compare Fv models generated by seven structure prediction methodologies. The participants included: Accerlys Inc, Chemical Computer Group (CCG), Schrodinger, Jeff Gray's lab at John Hopkins University, Macromoltek, Astellas Pharma/Osaka University and Prediction of ImmunoGlobulin Structure (PIGS). The sequences of benchmark structures were submitted to the modelers and PIGS, and a set of models were generated for each structure. We provide here an overview of the organization, participants and main results of this second antibody modeling assessment (AMA-II). Also, we compare the results with the first antibody assessment published in this journal (Almagro et al., 2011;79:3050).


Assuntos
Fragmentos Fab das Imunoglobulinas/química , Sequência de Aminoácidos , Animais , Sítios de Ligação de Anticorpos , Cristalografia por Raios X , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Coelhos
6.
Pharmaceuticals (Basel) ; 17(2)2024 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-38399400

RESUMO

Monomeric ubiquitin (Ub) is a 76-amino-acid highly conserved protein found in eukaryotes. The biological activity of Ub first described in the 1970s was extracellular, but it quickly gained relevance due to its intracellular role, i.e., post-translational modification of intracellular proteins (ubiquitination) that regulate numerous eukaryotic cellular processes. In the following years, the extracellular role of Ub was relegated to the background, until a correlation between higher survival rate and increased serum Ub concentrations in patients with sepsis and burns was observed. Although the mechanism of action (MoA) of extracellular ubiquitin (eUb) is not yet well understood, further studies have shown that it may ameliorate the inflammatory response in tissue injury and multiple sclerosis diseases. These observations, compounded with the high stability and low immunogenicity of eUb due to its high conservation in eukaryotes, have made this small protein a relevant candidate for biotherapeutic development. Here, we review the in vitro and in vivo effects of eUb on immunologic, cardiovascular, and nervous systems, and discuss the potential MoAs of eUb as an anti-inflammatory, antimicrobial, and cardio- and brain-protective agent.

7.
Cancers (Basel) ; 15(6)2023 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-36980702

RESUMO

Transferrin receptor 1 (TfR1), also known as CD71, is a transmembrane protein involved in the cellular uptake of iron and the regulation of cell growth. This receptor is expressed at low levels on a variety of normal cells, but is upregulated on cells with a high rate of proliferation, including malignant cells and activated immune cells. Infection with the human immunodeficiency virus (HIV) leads to the chronic activation of B cells, resulting in high expression of TfR1, B-cell dysfunction, and ultimately the development of acquired immunodeficiency syndrome-related B-cell non-Hodgkin lymphoma (AIDS-NHL). Importantly, TfR1 expression is correlated with the stage and prognosis of NHL. Thus, it is a meaningful target for antibody-based NHL therapy. We previously developed a mouse/human chimeric IgG3 specific for TfR1 (ch128.1/IgG3) and showed that this antibody exhibits antitumor activity in an in vivo model of AIDS-NHL using NOD-SCID mice challenged intraperitoneally with 2F7 human Burkitt lymphoma (BL) cells that harbor the Epstein-Barr virus (EBV). We have also developed an IgG1 version of ch128.1 that shows significant antitumor activity in SCID-Beige mouse models of disseminated multiple myeloma, another B-cell malignancy. Here, we aim to explore the utility of ch128.1/IgG1 and its humanized version (hu128.1) in mouse models of AIDS-NHL. To accomplish this goal, we used the 2F7 cell line variant 2F7-BR44, which is more aggressive than the parental cell line and forms metastases in the brain of mice after systemic (intravenous) administration. We also used the human BL cell line JB, which in contrast to 2F7, is EBV-negative, allowing us to study both EBV-infected and non-infected NHL tumors. Treatment with ch128.1/IgG1 or hu128.1 of SCID-Beige mice challenged locally (subcutaneously) with 2F7-BR44 or JB cells results in significant antitumor activity against different stages of disease. Treatment of mice challenged systemically (intravenously) with either 2F7-BR44 or JB cells also showed significant antitumor activity, including long-term survival. Taken together, our results suggest that targeting TfR1 with antibodies, such as ch128.1/IgG1 or hu128.1, has potential as an effective therapy for AIDS-NHL.

8.
MAbs ; 15(1): 2254676, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37698877

RESUMO

Prolactin (PRL) has recently been demonstrated to elicit female-selective nociceptor sensitization and increase pain-like behaviors in female animals. Here we report the discovery and characterization of first-in-class, humanized PRL neutralizing monoclonal antibodies (PRL mAbs). We obtained two potent and selective PRL mAbs, PL 200,031 and PL 200,039. PL 200,031 was engineered as human IgG1 whereas PL 200,039 was reformatted as human IgG4. Both mAbs have sub-nanomolar affinity for human PRL (hPRL) and produce concentration-dependent and complete inhibition of hPRL signaling at the hPRL receptor (hPRLR). These two PRL mAbs are selective for hPRL as they do not inhibit other hPRLR agonists such as human growth hormone or placental lactogen. They also cross-react with non-human primate PRL but not with rodent PRL. Further, both mAbs show long clearance half-lives after intravenous administration in FcRn-humanized mice. Consistent with their isotypes, these mAbs only differ in binding affinities to Fcγ receptors, as expected by design. Finally, PL 200,019, the murine parental mAb of PL 200,031 and PL 200,039, fully blocked stress-induced and PRL-dependent pain behaviors in female PRL-humanized mice, thereby providing in vivo preclinical proof-of-efficacy for PRL mAbs in mechanisms relevant to pain in females.


Assuntos
Prolactina , Receptores da Prolactina , Feminino , Camundongos , Animais , Gravidez , Prolactina/metabolismo , Prolactina/farmacologia , Receptores da Prolactina/metabolismo , Anticorpos Monoclonais , Placenta/metabolismo , Ligação Proteica
9.
Viruses ; 15(8)2023 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-37632075

RESUMO

We recently reported the isolation and characterization of an anti-SARS-CoV-2 antibody, called IgG-A7, that protects transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE-2) from an infection with SARS-CoV-2 Wuhan. We show here that IgG-A7 protected 100% of the transgenic mice infected with Delta (B.1.617.2) and Omicron (B.1.1.529) at doses of 0.5 and 5 mg/kg, respectively. In addition, we studied the pharmacokinetic (PK) profile and toxicology (Tox) of IgG-A7 in CD-1 mice at single doses of 100 and 200 mg/kg. The PK parameters at these high doses were proportional to the doses, with serum half-life of ~10.5 days. IgG-A7 was well tolerated with no signs of toxicity in urine and blood samples, nor in histopathology analyses. Tissue cross-reactivity (TCR) with a panel of mouse and human tissues showed no evidence of IgG-A7 interaction with the tissues of these species, supporting the PK/Tox results and suggesting that, while IgG-A7 has a broad efficacy profile, it is not toxic in humans. Thus, the information generated in the CD-1 mice as a PK/Tox model complemented with the mouse and human TCR, could be of relevance as an alternative to Non-Human Primates (NHPs) in rapidly emerging viral diseases and/or quickly evolving viruses such as SARS-CoV-2.


Assuntos
COVID-19 , Animais , Camundongos , SARS-CoV-2 , Anticorpos Antivirais , Camundongos Transgênicos , Anticorpos Neutralizantes , Imunoglobulina G , Receptores de Antígenos de Linfócitos T
10.
Antibodies (Basel) ; 11(1)2022 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-35225871

RESUMO

This report describes the discovery and characterization of antibodies with potential broad SARS-CoV-2 neutralization profiles. The antibodies were obtained from a phage display library built with the VH repertoire of a convalescent COVID-19 patient who was infected with SARS-CoV-2 B.1.617.2 (Delta). The patient received a single dose of Ad5-nCoV vaccine (Convidecia™, CanSino Biologics Inc.) one month before developing COVID-19 symptoms. Four synthetic VL libraries were used as counterparts of the immune VH repertoire. After three rounds of panning with SARS-CoV-2 receptor-binding domain wildtype (RBD-WT) 34 unique scFvs, were identified, with 27 cross-reactive for the RBD-WT and RBD Delta (RBD-DT), and seven specifics for the RBD-WT. The cross-reactive scFvs were more diverse than the RBD-WT specific ones, being encoded by several IGHV genes from the IGHV1 and IGHV3 families combined with short HCDR3s. Six cross-reactive scFvs and one RBD-WT specific scFv were converted to human IgG1 (hIgG1). Out of the seven antibodies, six blocked the RBD-WT binding to angiotensin converting enzyme 2 (ACE2), suggesting these antibodies may neutralize the SARS-CoV-2 infection. Importantly, one of the antibodies also recognized the RBD from the B.1.1.529 (Omicron) isolate, implying that the VH repertoire of the convalescent patient would protect against SARS-CoV-2 Wildtype, Delta, and Omicron. From a practical viewpoint, the triple cross-reactive antibody provides the substrate for developing therapeutic antibodies with a broad SARS-CoV-2 neutralization profile.

11.
Antibodies (Basel) ; 11(3)2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36134953

RESUMO

Neutralizing antibodies targeting the receptor-binding domain (RBD) of SARS-CoV-2 are among the most promising strategies to prevent and/or treat COVID-19. However, as SARS-CoV-2 has evolved into new variants, most of the neutralizing antibodies authorized by the US FDA and/or EMA to treat COVID-19 have shown reduced efficacy or have failed to neutralize the variants of concern (VOCs), particularly B.1.1.529 (Omicron). Previously, we reported the discovery and characterization of antibodies with high affinity for SARS-CoV-2 RBD Wuhan (WT), B.1.617.2 (Delta), and B.1.1.529 (Omicron) strains. One of the antibodies, called IgG-A7, also blocked the interaction of human angiotensin-converting enzyme 2 (hACE2) with the RBDs of the three strains, suggesting it may be a broadly SARS-CoV-2 neutralizing antibody. Herein, we show that IgG-A7 efficiently neutralizes all the three SARS-CoV-2 strains in plaque reduction neutralization tests (PRNTs). In addition, we demonstrate that IgG-A7 fully protects K18-hACE2 transgenic mice infected with SARS-CoV-2 WT. Taken together, our findings indicate that IgG-A7 could be a suitable candidate for development of antibody-based drugs to treat and/or prevent SARS-CoV-2 VOCs infection.

12.
Proteins ; 79(11): 3050-66, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21935986

RESUMO

A blinded study to assess the state of the art in three-dimensional structure modeling of the variable region (Fv) of antibodies was conducted. Nine unpublished high-resolution x-ray Fab crystal structures covering a wide range of antigen-binding site conformations were used as benchmark to compare Fv models generated by four structure prediction methodologies. The methodologies included two homology modeling strategies independently developed by CCG (Chemical Computer Group) and Accerlys Inc, and two fully automated antibody modeling servers: PIGS (Prediction of ImmunoGlobulin Structure), based on the canonical structure model, and Rosetta Antibody Modeling, based on homology modeling and Rosetta structure prediction methodology. The benchmark structure sequences were submitted to Accelrys and CCG and a set of models for each of the nine antibody structures were generated. PIGS and Rosetta models were obtained using the default parameters of the servers. In most cases, we found good agreement between the models and x-ray structures. The average rmsd (root mean square deviation) values calculated over the backbone atoms between the models and structures were fairly consistent, around 1.2 Å. Average rmsd values of the framework and hypervariable loops with canonical structures (L1, L2, L3, H1, and H2) were close to 1.0 Å. H3 prediction yielded rmsd values around 3.0 Å for most of the models. Quality assessment of the models and the relative strengths and weaknesses of the methods are discussed. We hope this initiative will serve as a model of scientific partnership and look forward to future antibody modeling assessments.


Assuntos
Anticorpos/química , Sítios de Ligação de Anticorpos , Região Variável de Imunoglobulina/química , Modelos Moleculares , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Ratos , Alinhamento de Sequência , Software
13.
Antibodies (Basel) ; 8(3)2019 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-31544850

RESUMO

Phage display technology has played a key role in the remarkable progress of discovering and optimizing antibodies for diverse applications, particularly antibody-based drugs. This technology was initially developed by George Smith in the mid-1980s and applied by John McCafferty and Gregory Winter to antibody engineering at the beginning of 1990s. Here, we compare nine phage display antibody libraries published in the last decade, which represent the state of the art in the discovery and development of therapeutic antibodies using phage display. We first discuss the quality of the libraries and the diverse types of antibody repertoires used as substrates to build the libraries, i.e., naïve, synthetic, and semisynthetic. Second, we review the performance of the libraries in terms of the number of positive clones per panning, hit rate, affinity, and developability of the selected antibodies. Finally, we highlight current opportunities and challenges pertaining to phage display platforms and related display technologies.

14.
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
15.
Front Biosci ; 13: 1619-33, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17981654

RESUMO

Humanization has played a fundamental role in the remarkable progress of antibodies as therapeutic reagents. Here we have reviewed the publications on antibody humanization since the first report on CDR grafting in the second half of the 1980's up to June 2007. We describe the two main trends in the field: rational and empirical methods to humanize antibodies. Rational methods rely on the so-called design cycle. It consists of generating a small set of variants, which are designed based on the antibody structure and/or sequence information, and assessing their binding or any other characteristic of interest. Rational methods include CDR grafting, Resurfacing, Superhumanization and Human String Content Optimization. In contrast to rational methods, empirical methods are based on generating large combinatorial libraries and selecting the desired variants by enrichment technologies such as phage, ribosome or yeast display, or by high throughput screening techniques. The latter methods rest on selection rather than making assumptions on the impact of mutations on the antibody structure. These methods include Framework Libraries, Guided Selection, Framework Shuffling and Humaneering.


Assuntos
Anticorpos/química , Técnicas Imunológicas , Engenharia de Proteínas/métodos , Animais , Anticorpos/uso terapêutico , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Afinidade de Anticorpos , Bacteriófagos , Sítios de Ligação de Anticorpos , Proteínas do Sistema Complemento/química , Humanos , Fragmentos Fab das Imunoglobulinas , Região Variável de Imunoglobulina , Camundongos , Biblioteca de Peptídeos
16.
Mol Immunol ; 43(11): 1836-45, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16337682

RESUMO

We have constructed a chimeric antibody single-chain Fv (scFv) fragments phage-displayed library that combines an invariant human V(L) chain with the repertoire of V(H) domains amplified from a horse immunized against scorpion venom. To gain insight into the equine V(H) repertoire, the V(H) sequences of 46 unique clones randomly chosen from the library prior to antigenic selection were analyzed. Comparisons with previously reported equine V(H) sequences, as well as with the repertoire of human IGHV germline genes and known V(H) sequences of sheep, cattle and pig, suggest that the equine IGH locus harbors at least three IGHV gene families. Two families belong to clan II while the other was classified into clan I. The horse sequences were also found to encode a diverse repertoire of canonical structures. The most populated equine IGHV gene family, named IGHV1, and another family termed IGHV3, encode two out of the three canonical structures so far described for CDR1. The IGHV2 gene family has the third canonical structure at CDR1. In CDR2, nine loop lengths were found, with four of them matching the pattern of typical canonical structures. The remaining five CDR2 loop lengths are shorter or longer than those reported for human IGHV germline genes and known sequences of sheep, cattle and pig. The analysis of CDR3 loops indicates a length distribution broader than previous reports for horses; being similar to that of humans, sheep and pigs. Moreover, equine CDR3 loops were found to have a combination of lower content of cysteine and higher proportion of glycine not seen in the other species. This implies less constrained loops and therefore more apt for searching the conformational space of antigen-binding sites. Altogether, these findings reveal a more diverse perspective of the horse V(H) repertoire than previous estimations and lay foundations for future studies of the equine IGH locus.


Assuntos
Células Germinativas/metabolismo , Cavalos/genética , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/imunologia , Carneiro Doméstico/genética , Suínos/genética , Sequência de Aminoácidos , Animais , Bovinos , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Biblioteca Gênica , Cavalos/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Dados de Sequência Molecular , Nucleotídeos/genética , Biblioteca de Peptídeos , Filogenia , Alinhamento de Sequência , Análise de Sequência de Proteína , Carneiro Doméstico/imunologia , Suínos/imunologia
17.
Front Immunol ; 8: 1751, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29379493

RESUMO

The remarkable progress in engineering and clinical development of therapeutic antibodies in the last 40 years, after the seminal work by Köhler and Milstein, has led to the approval by the United States Food and Drug Administration (FDA) of 21 antibodies for cancer immunotherapy. We review here these approved antibodies, with emphasis on the methods used for their discovery, engineering, and optimization for therapeutic settings. These methods include antibody engineering via chimerization and humanization of non-human antibodies, as well as selection and further optimization of fully human antibodies isolated from human antibody phage-displayed libraries and immunization of transgenic mice capable of generating human antibodies. These technology platforms have progressively led to the development of therapeutic antibodies with higher human content and, thus, less immunogenicity. We also discuss the genetic engineering approaches that have allowed isotype switching and Fc modifications to modulate effector functions and bioavailability (half-life), which together with the technologies for engineering the Fv fragment, have been pivotal in generating more efficacious and better tolerated therapeutic antibodies to treat cancer.

18.
Insect Biochem Mol Biol ; 34(4): 343-52, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15041018

RESUMO

The single intron of the heavy-chain fibroin gene in domesticated (Bombyx mori) and wild (B. mandarina) silk moths has a length of approximately 1000 nucleotides. It is located only 57 bp from the gene's core promoter and harbors multiple AT-rich regulatory elements that have been found to enhance the basal level of transcription in vitro. In this work, the intronic nucleotide variability among members of both Bombyx species is analyzed. The intron sequences of B. mori strains k120 and Nistari, as well as B. mandarina specimens from Japan and Korea, were obtained. This information was compared with the previously reported sequences of B. mori strains p50 and C-108, as well as an additional B. mandarina specimen collected in Japan. We found a total of 26 variant positions, including variants shared by members of both species and species-specific changes. The potential functional role of these variants was investigated by using the program MatInspector to search for putative binding sites of transcription factors within the intron. We detected a multitude of putative binding elements distributed along the entire intronic sequence. Among them, 22 correspond to protein binding domains that are known to regulate fibroin transcription. The mapping of multiple variant positions within these putative binding sequences as well as in known regulatory elements of the intron argue for functional significance on the regulation of transcription.


Assuntos
Bombyx/química , Bombyx/genética , Fibroínas/genética , Proteínas de Insetos/genética , Íntrons/genética , Animais , Animais Domésticos , Animais Selvagens , Sequência de Bases , Sítios de Ligação , Fibroínas/metabolismo , Fatores de Transcrição Forkhead , Variação Genética , Proteínas de Insetos/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Filogenia , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade da Espécie , Fatores de Transcrição/metabolismo
19.
MAbs ; 6(3): 628-36, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24481222

RESUMO

To gain insight into the functional antibody repertoire of rabbits, the VH and VL repertoires of bone marrow (BM) and spleen (SP) of a naïve New Zealand White rabbit (NZW; Oryctolagus cuniculus) and that of lymphocytes collected from a NZW rabbit immunized (IM) with a 16-mer peptide were deep-sequenced. Two closely related genes, IGHV1S40 (VH1a3) and IGHV1S45 (VH4), were found to dominate (~90%) the VH repertoire of BM and SP, whereas, IGHV1S69 (VH1a1) contributed significantly (~40%) to IM. BM and SP antibodies recombined predominantly with IGHJ4. A significant proportion (~30%) of IM sequences recombined with IGHJ2. The VK repertoire was encoded by nine IGKV genes recombined with one IGKJ gene, IGKJ1. No significant bias in the VK repertoire of the BM, SP and IM samples was observed. The complementarity-determining region (CDR)-H3 and -L3 length distributions were similar in the three samples following a Gaussian curve with average length of 12.2 ± 2.4 and 11.1 ± 1.1 amino acids, respectively. The amino acid composition of the predominant CDR-H3 and -L3 loop lengths was similar to that of humans and mice, rich in Tyr, Gly, Ser and, in some specific positions, Asp. The average number of mutations along the IGHV/KV genes was similar in BM, SP and IM; close to 12 and 15 mutations for VH and VL, respectively. A monoclonal antibody specific for the peptide used as immunogen was obtained from the IM rabbit. The CDR-H3 sequence was found in 1,559 of 61,728 (2.5%) sequences, at position 10, in the rank order of the CDR-H3 frequencies. The CDR-L3 was found in 24 of 11,215 (0.2%) sequences, ranking 102. No match was found in the BM and SP samples, indicating positive selection for the hybridoma sequence. Altogether, these findings lay foundations for engineering of rabbit V regions to enhance their potential as therapeutics, i.e., design of strategies for selection of specific rabbit V regions from NGS data mining, humanization and design of libraries for affinity maturation campaigns.


Assuntos
Anticorpos/genética , Anticorpos/imunologia , Coelhos/genética , Coelhos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Medula Óssea/imunologia , Regiões Determinantes de Complementaridade/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridomas/imunologia , Imunização , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Mutação , Peptídeos/imunologia , Engenharia de Proteínas , Homologia de Sequência de Aminoácidos , Baço/imunologia
20.
Front Immunol ; 3: 342, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23162556

RESUMO

Antibodies are the fastest-growing segment of the biologics market. The success of antibody-based drugs resides in their exquisite specificity, high potency, stability, solubility, safety, and relatively inexpensive manufacturing process in comparison with other biologics. We outline here the structural studies and fundamental principles that define how antibodies interact with diverse targets. We also describe the antibody repertoires and affinity maturation mechanisms of humans, mice, and chickens, plus the use of novel single-domain antibodies in camelids and sharks. These species all utilize diverse evolutionary solutions to generate specific and high affinity antibodies and illustrate the plasticity of natural antibody repertoires. In addition, we discuss the multiple variations of man-made antibody repertoires designed and validated in the last two decades, which have served as tools to explore how the size, diversity, and composition of a repertoire impact the antibody discovery process.

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