Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 641
Filtrar
1.
Anticancer Res ; 44(5): 1955-1962, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38677771

RESUMEN

BACKGROUND/AIM: The epidermal growth factor receptor (EGFR) is over-expressed in several types of cancer, and monoclonal antibody therapy has been the strategy that has shown the best results. This study focused on the construction of a humanized single chain antibody (huscFv) directed against EGFR (HER1). MATERIALS AND METHODS: The CDR grafting method was used to incorporate murine complementarity determining regions (CDRs) of cetuximab into human sequences. A dot blot assay was used to examine the affinity of the huscFv secreted by HEK293T for EGFR. The inhibitory effect on the viability of A549 cells was evaluated using the WST-1 assay. RESULTS: The incorporation of murine CDRs of cetuximab into human sequences increased the degree of humanness by 16.4%. The increase in the humanization of scFv did not affect the affinity for EGFR. Metformin had a dose-dependent effect, with an IC50 of 46 mM, and in combination with huscFv, the cell viability decreased by 45% compared to the 15% demonstrated by huscFv alone. CONCLUSION: The CDR grafting technique is efficient for the humanization of scFv, maintaining its affinity for EGFR and demonstrating its inhibitory effect when combined with metformin in A549 cells.


Asunto(s)
Receptores ErbB , Metformina , Anticuerpos de Cadena Única , Humanos , Metformina/farmacología , Receptores ErbB/inmunología , Receptores ErbB/antagonistas & inhibidores , Células A549 , Anticuerpos de Cadena Única/farmacología , Anticuerpos de Cadena Única/inmunología , Células HEK293 , Cetuximab/farmacología , Supervivencia Celular/efectos de los fármacos , Anticuerpos Monoclonales Humanizados/farmacología , Animales , Ratones , Regiones Determinantes de Complementariedad/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/inmunología
2.
J Virol ; 98(3): e0199523, 2024 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-38323813

RESUMEN

Historically, antibody reactivity to pathogens and vaccine antigens has been evaluated using serological measurements of antigen-specific antibodies. However, it is difficult to evaluate all antibodies that contribute to various functions in a single assay, such as the measurement of the neutralizing antibody titer. Bulk antibody repertoire analysis using next-generation sequencing is a comprehensive method for analyzing the overall antibody response; however, it is unreliable for estimating antigen-specific antibodies due to individual variation. To address this issue, we propose a method to subtract the background signal from the repertoire of data of interest. In this study, we analyzed changes in antibody diversity and inferred the heavy-chain complementarity-determining region 3 (CDRH3) sequences of antibody clones that were selected upon influenza virus infection in a mouse model using bulk repertoire analysis. A decrease in the diversity of the antibody repertoire was observed upon viral infection, along with an increase in neutralizing antibody titers. Using kernel density estimation of sequences in a high-dimensional sequence space with background signal subtraction, we identified several clusters of CDRH3 sequences induced upon influenza virus infection. Most of these repertoires were detected more frequently in infected mice than in uninfected control mice, suggesting that infection-specific antibody sequences can be extracted using this method. Such an accurate extraction of antigen- or infection-specific repertoire information will be a useful tool for vaccine evaluation in the future. IMPORTANCE: As specific interactions between antigens and cell-surface antibodies trigger the proliferation of B-cell clones, the frequency of each antibody sequence in the samples reflects the size of each clonal population. Nevertheless, it is extremely difficult to extract antigen-specific antibody sequences from the comprehensive bulk antibody sequences obtained from blood samples due to repertoire bias influenced by exposure to dietary antigens and other infectious agents. This issue can be addressed by subtracting the background noise from the post-immunization or post-infection repertoire data. In the present study, we propose a method to quantify repertoire data from comprehensive repertoire data. This method allowed subtraction of the background repertoire, resulting in more accurate extraction of expanded antibody repertoires upon influenza virus infection. This accurate extraction of antigen- or infection-specific repertoire information is a useful tool for vaccine evaluation.


Asunto(s)
Anticuerpos Antivirales , Infecciones por Orthomyxoviridae , Orthomyxoviridae , Animales , Ratones , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Células Clonales/citología , Células Clonales/inmunología , Regiones Determinantes de Complementariedad/inmunología , Vacunas contra la Influenza/inmunología , Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/sangre , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología
3.
Nature ; 611(7935): 352-357, 2022 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-36289331

RESUMEN

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens1. In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1-CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens2-22. Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes23,24. We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.


Asunto(s)
Anticuerpos , Selección Clonal Mediada por Antígenos , Cadenas Pesadas de Inmunoglobulina , Cadenas Ligeras de Inmunoglobulina , Animales , Secuencia de Aminoácidos , Anticuerpos/química , Anticuerpos/genética , Anticuerpos/inmunología , Antígenos/química , Antígenos/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/inmunología , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Mamíferos , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Memoria Inmunológica , Recombinación V(D)J , Selección Clonal Mediada por Antígenos/genética , Selección Clonal Mediada por Antígenos/inmunología
4.
Immunity ; 55(2): 341-354.e7, 2022 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-34990590

RESUMEN

The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates.


Asunto(s)
Anticuerpos ampliamente neutralizantes/genética , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/genética , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/inmunología , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Epítopos , Femenino , Genotipo , Hepacivirus/genética , Hepatitis C/inmunología , Anticuerpos contra la Hepatitis C/química , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Masculino , Persona de Mediana Edad , Mutación , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología
5.
Immunology ; 165(1): 74-87, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34428313

RESUMEN

Having a limited number of VH segments, cattle rely on uniquely long DH gene segments to generate CDRH3 length variation (3-70 aa) far greater than that in humans or mice. Bovine antibodies with ultralong CDRH3s (>50 aa) possess unusual structures and abilities to bind to special antigens. In this study, we replaced most murine endogenous DH segments with bovine DH genes, generating a mouse line termed B-DH. The use of bovine DH genes significantly increased the length variation of CDRH3 and consequently the Ig heavy chain repertoire in B-DH mice. However, no ultralong CDRH3 was observed in B-DH mice, suggesting that other factors, in addition to long DH genes, are also involved in the formation of ultralong CDRH3. The B-DH mice mounted a normal humoral immune response to various antigens, although the B-cell developmental paradigm was obviously altered compared with wild-type mice. Additionally, B-DH mice are not predisposed to the generation of autoantibodies despite the interspecies DH gene replacement. The B-DH mice reported in this study provide a unique model to answer basic questions regarding the synergistic evolution of DH and VH genes, VDJ recombination and BCR selection in B-cell development.


Asunto(s)
Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/genética , Animales , Formación de Anticuerpos/genética , Formación de Anticuerpos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Bovinos , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Marcación de Gen , Sitios Genéticos , Vectores Genéticos/genética , Inmunidad Humoral , Ratones , Ratones Transgénicos , Recombinación V(D)J
6.
Life Sci Alliance ; 5(1)2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34675071

RESUMEN

Single-domain antibody (sdAb) holds the promising strategies for diverse research and translational applications. Here, we describe a method for the adaptation of the in situ proximity ligation assay (isPLA) followed by sequencing (isPLA-seq) to facilitate screening of a high-sensitive, high-throughput sdAb library for a given protein at subcellular and single-cell resolution. Based on the sequence of complementarity-determining region 3 (CDR3), the recombinant sdAb can be produced for in vitro and in vivo utilities. This method provides a general means to identify the functional measure of sdAb and its complementary epitopes and its potential applications to investigate cellular processes.


Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Anticuerpos de Dominio Único/inmunología , Secuencia de Bases , Línea Celular , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/inmunología , Biblioteca de Genes , Humanos , Inmunofenotipificación , Imagen Molecular , Proteína Sequestosoma-1/inmunología , Anticuerpos de Dominio Único/química
7.
J Leukoc Biol ; 111(1): 283-289, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-33847407

RESUMEN

The potential protective or pathogenic role of the adaptive immune response to SARS-CoV-2 infection has been vigorously debated. While COVID-19 patients consistently generate a T lymphocyte response to SARS-CoV-2 antigens, evidence of significant immune dysregulation in these patients continues to accumulate. In this study, next generation sequencing of the T cell receptor beta chain (TRB) repertoire was conducted in hospitalized COVID-19 patients to determine if immunogenetic differences of the TRB repertoire contribute to disease course severity. Clustering of highly similar TRB CDR3 amino acid sequences across COVID-19 patients yielded 781 shared TRB sequences. The TRB sequences were then filtered for known associations with common diseases such as EBV and CMV. The remaining sequences were cross-referenced to a publicly accessible dataset that mapped COVID-19 specific TCRs to the SARS-CoV-2 genome. We identified 158 SARS-CoV-2 specific TRB sequences belonging to 134 clusters in our COVID-19 patients. Next, we investigated 113 SARS-CoV-2 specific clusters binding only one peptide target in relation to disease course. Distinct skewing of SARS-CoV-2 specific TRB sequences toward the nonstructural proteins (NSPs) encoded within ORF1a/b of the SARS-CoV-2 genome was observed in clusters associated with critical disease course when compared to COVID-19 clusters associated with a severe disease course. These data imply that T-lymphocyte reactivity towards peptides from NSPs of SARS-CoV-2 may not constitute an effective adaptive immune response and thus may negatively affect disease severity.


Asunto(s)
COVID-19/inmunología , COVID-19/patología , Hospitalización , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Índice de Severidad de la Enfermedad , Proteínas Virales/inmunología , Anciano , Secuencia de Aminoácidos , COVID-19/virología , Regiones Determinantes de Complementariedad/inmunología , Genoma Viral , Humanos , Poliproteínas/química , Poliproteínas/inmunología , Poliproteínas/metabolismo , SARS-CoV-2/genética , Factores de Tiempo , Proteínas Virales/química , Proteínas Virales/metabolismo
8.
Bioorg Chem ; 119: 105563, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34942468

RESUMEN

Monoclonal antibodies emerged as an important therapeutic drug class with remarkable specificity and binding affinity. Nonetheless, these heterotetrameric immunoglobulin proteins come with high manufacturing and therapeutic costs which can take extraordinary proportions, besides other limitations such as their limited in cellulo access imposed by their molecular size (ca. 150 kDa). These drawbacks stimulated the development of downsized functional antibody fragments (ca. 15-50 kDa), together with smaller synthetic peptides (ca. 1-3 kDa) derived from the antibodies' crucial complementarity-determining regions (CDR). Despite the general lack of success in the literal translation of CDR loops in peptide mimetics, rational structure-based and computational approaches have shown their potential for obtaining functional CDR-based peptide mimetics. In this review, we describe the efforts made in the development of antibody and nanobody paratope-derived peptide mimetics with particular focus on the used design strategies, in addition to highlighting the challenges associated with their development.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Regiones Determinantes de Complementariedad/inmunología , Péptidos/inmunología , Anticuerpos Monoclonales/química , Regiones Determinantes de Complementariedad/química , Humanos , Modelos Moleculares , Estructura Molecular , Péptidos/química
9.
Eur J Immunol ; 52(2): 247-260, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34708869

RESUMEN

In addition to conventional immunoglobulin, camelids and cartilaginous fish express a special class of antibody that consists only of heavy (H) chain (HCAbs). In the holocephalan elephantfish, there are two HCAb classes, one of which has evolved surprising features. The H-chain genes in cartilaginous fish are organized as 20-200 minigenes, or clusters, each consisting of VH, 1-3 DH, JH gene segments with one set of constant region exons. We report that HHC2 (holocephalan H-chain antibody 2) evolved from IgM H-chain clusters, but its DH gene segments have diverged considerably. The three DH in HHC2 clusters are A-rich, so that one to three potential reading frames for each DH encode lysine and arginine. All three are incorporated into the rearranged VDJ, ensuring that the ligand-binding site carries multiple basic residues, as cDNA sequences demonstrate. The electropositive character in HHC2 CDR3 is accompanied by a paucity of aromatic amino acids, the latter feature at variance to the established, interactive role of tyrosine not only in ligand-binding but generally at interfaces of protein complexes. The selection for these divergent HHC2 features challenges currently accepted ideas on what determines antibody reactivity and molecular recognition.


Asunto(s)
Regiones Determinantes de Complementariedad , Proteínas de Peces , Peces , Cadenas Pesadas de Inmunoglobulina , Animales , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Evolución Molecular , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Peces/genética , Peces/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología
10.
Elife ; 102021 11 30.
Artículo en Inglés | MEDLINE | ID: mdl-34845983

RESUMEN

T-cell receptors (TCRs) encode clinically valuable information that reflects prior antigen exposure and potential future response. However, despite advances in deep repertoire sequencing, enormous TCR diversity complicates the use of TCR clonotypes as clinical biomarkers. We propose a new framework that leverages experimentally inferred antigen-associated TCRs to form meta-clonotypes - groups of biochemically similar TCRs - that can be used to robustly quantify functionally similar TCRs in bulk repertoires across individuals. We apply the framework to TCR data from COVID-19 patients, generating 1831 public TCR meta-clonotypes from the SARS-CoV-2 antigen-associated TCRs that have strong evidence of restriction to patients with a specific human leukocyte antigen (HLA) genotype. Applied to independent cohorts, meta-clonotypes targeting these specific epitopes were more frequently detected in bulk repertoires compared to exact amino acid matches, and 59.7% (1093/1831) were more abundant among COVID-19 patients that expressed the putative restricting HLA allele (false discovery rate [FDR]<0.01), demonstrating the potential utility of meta-clonotypes as antigen-specific features for biomarker development. To enable further applications, we developed an open-source software package, tcrdist3, that implements this framework and facilitates flexible workflows for distance-based TCR repertoire analysis.


Asunto(s)
Antígenos Virales/genética , COVID-19/inmunología , Antígenos HLA/genética , Receptores de Antígenos de Linfocitos T/genética , SARS-CoV-2/inmunología , Antígenos Virales/inmunología , Biomarcadores , COVID-19/genética , Regiones Determinantes de Complementariedad/inmunología , Biología Computacional/métodos , Epítopos/genética , Epítopos/inmunología , Genotipo , Antígenos HLA/inmunología , Humanos , Receptores de Antígenos de Linfocitos T/inmunología
11.
Int J Mol Sci ; 22(21)2021 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-34769474

RESUMEN

The anti-La mab 312B, which was established by hybridoma technology from human-La transgenic mice after adoptive transfer of anti-human La T cells, immunoprecipitates both native eukaryotic human and murine La protein. Therefore, it represents a true anti-La autoantibody. During maturation, the anti-La mab 312B acquired somatic hypermutations (SHMs) which resulted in the replacement of four aa in the complementarity determining regions (CDR) and seven aa in the framework regions. The recombinant derivative of the anti-La mab 312B in which all the SHMs were corrected to the germline sequence failed to recognize the La antigen. We therefore wanted to learn which SHM(s) is (are) responsible for anti-La autoreactivity. Humanization of the 312B ab by grafting its CDR regions to a human Ig backbone confirms that the CDR sequences are mainly responsible for anti-La autoreactivity. Finally, we identified that a single amino acid replacement (D > Y) in the germline sequence of the CDR3 region of the heavy chain of the anti-La mab 312B is sufficient for anti-La autoreactivity.


Asunto(s)
Anticuerpos Antinucleares/genética , Autoanticuerpos/genética , Hipermutación Somática de Inmunoglobulina/genética , Secuencia de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Anticuerpos Antinucleares/inmunología , Anticuerpos Antinucleares/metabolismo , Autoanticuerpos/química , Autoanticuerpos/inmunología , Autoanticuerpos/metabolismo , Autoinmunidad/genética , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Regiones Determinantes de Complementariedad/metabolismo , Epítopos/genética , Epítopos/inmunología , Células HeLa , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de Proteína
12.
Sci Rep ; 11(1): 21362, 2021 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-34725391

RESUMEN

The design of superior biologic therapeutics, including antibodies and engineered proteins, involves optimizing their specific ability to bind to disease-related molecular targets. Previously, we developed and applied the Assisted Design of Antibody and Protein Therapeutics (ADAPT) platform for virtual affinity maturation of antibodies (Vivcharuk et al. in PLoS One 12(7):e0181490, https://doi.org/10.1371/journal.pone.0181490 , 2017). However, ADAPT is limited to point mutations of hot-spot residues in existing CDR loops. In this study, we explore the possibility of wholesale replacement of the entire H3 loop with no restriction to maintain the parental loop length. This complements other currently published studies that sample replacements for the CDR loops L1, L2, L3, H1 and H2. Given the immense sequence space theoretically available to H3, we focused on the virtual grafting of over 5000 human germline-derived H3 sequences from the IGMT/LIGM database increasing the diversity of the sequence space when compared to using crystalized H3 loop sequences. H3 loop conformations are generated and scored to identify optimized H3 sequences. Experimental testing of high-ranking H3 sequences grafted into the framework of the bH1 antibody against human VEGF-A led to the discovery of multiple hits, some of which had similar or better affinities relative to the parental antibody. In over 75% of the tested designs, the re-designed H3 loop contributed favorably to overall binding affinity. The hits also demonstrated good developability attributes such as high thermal stability and no aggregation. Crystal structures of select re-designed H3 variants were solved and indicated that although some deviations from predicted structures were seen in the more solvent accessible regions of the H3 loop, they did not significantly affect predicted affinity scores.


Asunto(s)
Anticuerpos/química , Secuencia de Aminoácidos , Anticuerpos/inmunología , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/inmunología , Humanos , Modelos Moleculares , Agregado de Proteínas , Conformación Proteica , Estabilidad Proteica , Factor A de Crecimiento Endotelial Vascular/inmunología
13.
Immunol Lett ; 240: 71-76, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34666136

RESUMEN

A diverse immune repertoire is capable of recognizing the enormous universe of foreign antigens encountered over life. Aging has a profound impact on the immune repertoires. However, whether continuous age-related changes in the immune repertoires differ between sexes is unclear. In this study, the characteristics of immune repertoires stratified by sex during aging are deciphered by analyzing T-cell receptor ß-chain (TRB) and immunoglobulin heavy chain (IGH) sequences in 361 healthy adults. A similar change was observed between males and females across their lifespan, whereas age-subgroup analysis revealed sex-specific signatures in TRB and IGH repertoires. In regard to TRB, in males, repertoire richness and evenness increases slightly before the age of 32 years and 45 years respectively, and decreases sharply thereafter. Intriguingly, in females, they decrease significantly until around the age 57 years old, and subsequently undergo a stable stage up to the age of 83 years. Although IGH repertoire evenness increases significantly with age in both sexes, richness decreases significantly with age in males but remains stable in females. Moreover, average length of IGH CDR3 increases with age. In conclusion, these findings provide fundamental insights into the mechanisms underlying sex differences in adaptive immunity.


Asunto(s)
Envejecimiento , Cadenas Pesadas de Inmunoglobulina , Receptores de Antígenos de Linfocitos T alfa-beta , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Envejecimiento/inmunología , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Femenino , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología
14.
J Immunol ; 207(10): 2445-2455, 2021 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-34654689

RESUMEN

Preterm labor (PTL) is the leading cause of neonatal morbidity and mortality worldwide. Whereas many studies have investigated the maternal immune responses that cause PTL, fetal immune cell activation has recently been raised as an important contributor to the pathogenesis of PTL. In this study, we analyzed lymphocyte receptor repertoires in maternal and cord blood from 14 term and 10 preterm deliveries, hypothesizing that the high prevalence of infection in patients with PTL may result in specific changes in the T cell and B cell repertoires. We analyzed TCR ß-chain (TCR-ß) and IgH diversity, CDR3 lengths, clonal sharing, and preferential usage of variable and joining gene segments. Both TCR-ß and IgH repertoires had shorter CDR3s compared with those in maternal blood. In cord blood samples, we found that CDR3 lengths correlated with gestational age, with shorter CDR3s in preterm neonates suggesting a less developed repertoire. Preterm cord blood displayed preferential usage of a number of genes. In preterm pregnancies, we observed significantly higher prevalence of convergent clones between mother/baby pairs than in term pregnancies. Together, our results suggest the repertoire of preterm infants displays a combination of immature features and convergence with maternal TCR-ß clones compared with that of term infants. The higher clonal convergence in PTL could represent mother and fetus both responding to a shared stimulus like an infection. These data provide a detailed analysis of the maternal-fetal immune repertoire in term and preterm patients and contribute to a better understanding of neonate immune repertoire development and potential changes associated with PTL.


Asunto(s)
Cadenas Pesadas de Inmunoglobulina/inmunología , Recién Nacido/inmunología , Trabajo de Parto Prematuro/inmunología , Nacimiento Prematuro/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Regiones Determinantes de Complementariedad/inmunología , Femenino , Humanos , Recien Nacido Prematuro/inmunología , Embarazo
15.
Biomolecules ; 11(9)2021 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-34572576

RESUMEN

The isolation of nanobodies from pre-immune libraries by means of biopanning is a straightforward process. Nevertheless, the recovered candidates often require optimization to improve some of their biophysical characteristics. In principle, CDRs are not mutated because they are likely to be part of the antibody paratope, but in this work, we describe a mutagenesis strategy that specifically addresses CDR1. Its sequence was identified as an instability hot spot by the PROSS program, and the available structural information indicated that four CDR1 residues bound directly to the antigen. We therefore modified the loop flexibility with the addition of an extra glycine rather than by mutating single amino acids. This approach significantly increased the nanobody yields but traded-off with moderate affinity loss. Accurate modeling coupled with atomistic molecular dynamics simulations enabled the modifications induced by the glycine insertion and the rationale behind the engineering design to be described in detail.


Asunto(s)
Regiones Determinantes de Complementariedad/inmunología , Proteínas Recombinantes/biosíntesis , Anticuerpos de Dominio Único/inmunología , Secuencia de Aminoácidos , Regiones Determinantes de Complementariedad/química , Simulación de Dinámica Molecular , Anticuerpos de Dominio Único/química
16.
Immunohorizons ; 5(8): 675-686, 2021 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-34433623

RESUMEN

Ab repertoire diversity plays a critical role in the host's ability to fight pathogens. CDR3 is partially responsible for Ab-Ag binding and is a significant source of diversity in the repertoire. CDR3 diversity is generated during VDJ rearrangement because of gene segment selection, gene segment trimming and splicing, and the addition of nucleotides. We analyzed the Ab repertoire diversity across multiple experiments examining the effects of spaceflight on the Ab repertoire after vaccination. Five datasets from four experiments were analyzed using rank-abundance curves and Shannon indices as measures of diversity. We discovered a trend toward lower diversity as a result of spaceflight but did not find the same decrease in our physiological model of microgravity in either the spleen or bone marrow. However, the bone marrow repertoire showed a reduction in diversity after vaccination. We also detected differences in Shannon indices between experiments and tissues. We did not detect a pattern of CDR3 usage across the experiments. Overall, we were able to find differences in the Ab repertoire diversity across experimental groups and tissues.


Asunto(s)
Médula Ósea/inmunología , Regiones Determinantes de Complementariedad/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Vuelo Espacial/métodos , Bazo/inmunología , Vacunación/métodos , Secuencia de Aminoácidos , Animales , Diversidad de Anticuerpos/genética , Diversidad de Anticuerpos/inmunología , Médula Ósea/metabolismo , Regiones Determinantes de Complementariedad/genética , Femenino , Cadenas Pesadas de Inmunoglobulina/genética , Ratones Endogámicos C57BL , RNA-Seq/métodos , Bazo/metabolismo
17.
Nat Commun ; 12(1): 4699, 2021 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-34349111

RESUMEN

Similarity in T-cell receptor (TCR) sequences implies shared antigen specificity between receptors, and could be used to discover novel therapeutic targets. However, existing methods that cluster T-cell receptor sequences by similarity are computationally inefficient, making them impractical to use on the ever-expanding datasets of the immune repertoire. Here, we developed GIANA (Geometric Isometry-based TCR AligNment Algorithm) a computationally efficient tool for this task that provides the same level of clustering specificity as TCRdist at 600 times its speed, and without sacrificing accuracy. GIANA also allows the rapid query of large reference cohorts within minutes. Using GIANA to cluster large-scale TCR datasets provides candidate disease-specific receptors, and provides a new solution to repertoire classification. Querying unseen TCR-seq samples against an existing reference differentiates samples from patients across various cohorts associated with cancer, infectious and autoimmune disease. Our results demonstrate how GIANA could be used as the basis for a TCR-based non-invasive multi-disease diagnostic platform.


Asunto(s)
Algoritmos , Receptores de Antígenos de Linfocitos T/clasificación , COVID-19/diagnóstico , COVID-19/inmunología , Análisis por Conglomerados , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/inmunología , Diagnóstico Diferencial , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Humanos , Esclerosis Múltiple/diagnóstico , Esclerosis Múltiple/inmunología , Neoplasias/diagnóstico , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , SARS-CoV-2 , Alineación de Secuencia
18.
JCI Insight ; 6(13)2021 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-34236054

RESUMEN

TCR repertoire diversification constitutes a foundation for successful immune reconstitution after allogeneic hematopoietic cell transplantation (allo-HCT). Deep TCR Vß sequencing of 135 serial specimens from a cohort of 35 allo-HCT recipients/donors was performed to dissect posttransplant TCR architecture and dynamics. Paired analysis of clonotypic repertoires showed a minimal overlap with donor expansions. Rarefied and hyperexpanded clonotypic patterns were hallmarks of T cell reconstitution and influenced clinical outcomes. Donor and pretransplant TCR diversity as well as divergence of class I human leukocyte antigen genotypes were major predictors of recipient TCR repertoire recovery. Complementary determining region 3-based specificity spectrum analysis indicated a predominant expansion of pathogen- and tumor-associated clonotypes in the late post-allo-HCT phase, while autoreactive clones were more expanded in the case of graft-versus-host disease occurrence. These findings shed light on post-allo-HCT adaptive immune reconstitution processes and possibly help in tracking alloreactive responses.


Asunto(s)
Inmunidad Adaptativa , Regiones Determinantes de Complementariedad/inmunología , Enfermedad Injerto contra Huésped/inmunología , Antígenos HLA/inmunología , Trasplante de Células Madre Hematopoyéticas , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Células Clonales/inmunología , Epítopos , Perfil Genético , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Reconstitución Inmune , Receptores de Antígenos de Linfocitos T/inmunología , Análisis de Secuencia de Proteína , Trasplante Homólogo/efectos adversos , Trasplante Homólogo/métodos
19.
Sci Rep ; 11(1): 15430, 2021 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-34326410

RESUMEN

Mesothelin (MSLN) is an attractive candidate of targeted therapy for several cancers, and hence there are increasing needs to develop MSLN-targeting strategies for cancer therapeutics. Antibody-drug conjugates (ADCs) targeting MSLN have been demonstrated to be a viable strategy in treating MSLN-positive cancers. However, developing antibodies as targeting modules in ADCs for toxic payload delivery to the tumor site but not to normal tissues is not a straightforward task with many potential hurdles. In this work, we established a high throughput engineering platform to develop and optimize anti-MSLN ADCs by characterizing more than 300 scFv CDR-variants and more than 50 IgG CDR-variants of a parent anti-MSLN antibody as candidates for ADCs. The results indicate that only a small portion of the complementarity determining region (CDR) residues are indispensable in the MSLN-specific targeting. Also, the enhancement of the hydrophilicity of the rest of the CDR residues could drastically increase the overall solubility of the optimized anti-MSLN antibodies, and thus substantially improve the efficacies of the ADCs in treating human gastric and pancreatic tumor xenograft models in mice. We demonstrated that the in vivo treatments with the optimized ADCs resulted in almost complete eradication of the xenograft tumors at the treatment endpoints, without detectable off-target toxicity because of the ADCs' high specificity targeting the cell surface tumor-associated MSLN. The technological platform can be applied to optimize the antibody sequences for more effective targeting modules of ADCs, even when the candidate antibodies are not necessarily feasible for the ADC development due to the antibodies' inferior solubility or affinity/specificity to the target antigen.


Asunto(s)
Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Inmunoconjugados/administración & dosificación , Terapia Molecular Dirigida/métodos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Línea Celular Tumoral , Regiones Determinantes de Complementariedad/inmunología , Modelos Animales de Enfermedad , Proteínas Ligadas a GPI/inmunología , Xenoinjertos , Humanos , Inmunoconjugados/inmunología , Inmunoglobulina G/inmunología , Inyecciones Intravenosas , Masculino , Mesotelina , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Pancreáticas/patología , Ingeniería de Proteínas/métodos , Neoplasias Gástricas/patología , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos
20.
Nat Commun ; 12(1): 3815, 2021 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-34155209

RESUMEN

Since the COVID-19 pandemic onset, the antibody response to SARS-CoV-2 has been extensively characterized. Antibodies to the receptor binding domain (RBD) on the spike protein are frequently encoded by IGHV3-53/3-66 with a short complementarity-determining region (CDR) H3. Germline-encoded sequence motifs in heavy chain CDRs H1 and H2 have a major function, but whether any common motifs are present in CDR H3, which is often critical for binding specificity, is not clear. Here, we identify two public clonotypes of IGHV3-53/3-66 RBD antibodies with a 9-residue CDR H3 that pair with different light chains. Distinct sequence motifs on CDR H3 are present in the two public clonotypes that seem to be related to differential light chain pairing. Additionally, we show that Y58F is a common somatic hypermutation that results in increased binding affinity of IGHV3-53/3-66 RBD antibodies with a short CDR H3. These results advance understanding of the antibody response to SARS-CoV-2.


Asunto(s)
Enzima Convertidora de Angiotensina 2/inmunología , Anticuerpos Neutralizantes/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Secuencia de Aminoácidos , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/metabolismo , Formación de Anticuerpos , COVID-19/metabolismo , COVID-19/virología , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/inmunología , Regiones Determinantes de Complementariedad/metabolismo , Cristalografía por Rayos X , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...