RESUMO
Constant domains in antibody molecules at the level of the Fab (CH1 and CL) have long been considered to be simple scaffolding elements that physically separate the paratope-defining variable (V) region from the effector function-mediating constant (C) regions. However, due to recent findings that C domains of different isotypes can modulate the fine specificity encoded in the V region, elucidating the role of C domains in shaping the paratope and influencing specificity is a critical area of interest. To dissect the relative contributions of each C domain to this phenomenon, we generated antibody fragments with different C regions omitted, using a set of antibodies targeting capsular polysaccharides from the fungal pathogen, Cryptococcus neoformans. Antigen specificity mapping and functional activity measurements revealed that V region-only antibody fragments exhibited poly-specificity to antigenic variants and extended to recognition of self-antigens, while measurable hydrolytic activity of the capsule was greatly attenuated. To better understand the mechanistic origins of the remarkable loss of specificity that accompanies the removal of C domains from identical paratopes, we performed molecular dynamics simulations which revealed increased paratope plasticity in the scFv relative to the corresponding Fab. Together, our results provide insight into how the remarkable specificity of immunoglobulins is governed and maintained at the level of the Fab through the enforcement of structural restrictions on the paratope by CH1 domains.
Assuntos
Cryptococcus neoformans , Epitopos , Cryptococcus neoformans/imunologia , Cryptococcus neoformans/química , Epitopos/química , Epitopos/imunologia , Regiões Constantes de Imunoglobulina/química , Regiões Constantes de Imunoglobulina/genética , Simulação de Dinâmica Molecular , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Humanos , Especificidade de Anticorpos , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/genética , Animais , Anticorpos Antifúngicos/imunologia , Anticorpos Antifúngicos/químicaRESUMO
AL amyloidosis is a life-threatening disease caused by deposition of immunoglobulin light chains. While the mechanisms underlying light chains amyloidogenesis in vivo remain unclear, several studies have highlighted the role that tissue environment and structural amyloidogenicity of individual light chains have in the disease pathogenesis. AL natural deposits contain both full-length light chains and fragments encompassing the variable domain (VL) as well as different length segments of the constant region (CL), thus highlighting the relevance that proteolysis may have in the fibrillogenesis pathway. Here, we investigate the role of major truncated species of the disease-associated AL55 light chain that were previously identified in natural deposits. Specifically, we study structure, molecular dynamics, thermal stability, and capacity to form fibrils of a fragment containing both the VL and part of the CL (133-AL55), in comparison with the full-length protein and its variable domain alone, under shear stress and physiological conditions. Whereas the full-length light chain forms exclusively amorphous aggregates, both fragments generate fibrils, although, with different kinetics, aggregate structure, and interplay with the unfragmented protein. More specifically, the VL-CL 133-AL55 fragment entirely converts into amyloid fibrils microscopically and spectroscopically similar to their ex vivo counterpart and increases the amorphous aggregation of full-length AL55. Overall, our data support the idea that light chain structure and proteolysis are both relevant for amyloidogenesis in vivo and provide a novel biocompatible model of light chain fibrillogenesis suitable for future mechanistic studies.
Assuntos
Amiloide , Cadeias Leves de Imunoglobulina , Amiloide/metabolismo , Amiloide/química , Humanos , Cadeias Leves de Imunoglobulina/metabolismo , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Simulação de Dinâmica Molecular , Regiões Constantes de Imunoglobulina/metabolismo , Regiões Constantes de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/química , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Amiloidose de Cadeia Leve de Imunoglobulina/patologia , Cinética , Domínios ProteicosRESUMO
Immunoglobulin G (IgG) is one of the five antibody classes produced in mammals as part of the humoral responses accountable for protecting the organisms from infection. Its antibody heavy chain constant region is encoded by the Ig heavy-chain gamma gene (IGHG). In humans, there are four IGHG genes which encode the four subclasses, each with a specialized effector function. Although four subclasses of IgG proteins have also been reported in macaques, this does not appear to be the rule for all primates. In Platyrrhini, IgG has been stated to be encoded by a single-copy gene. To date, it remains unknown how the IGHG has expanded or contracted in the primate order; consequently, we have analyzed data from 38 primate genome sequences to identify IGHG genes and describe the evolution of IGHG genes in primate order. IGHG belongs to a multigene family that evolves by the birth-death evolutionary model in primates. Whereas Strepsirrhini and Platyrrhini have a single-copy gene, in Catarrhini, it has expanded to several paralogs in their genomes; some deleted and others pseudogenized. Furthermore, episodic positive selection may have promoted a species-specific IgG effector function. We propose that IgG evolved to reach an optimal number of copies per genome to adapt their humoral immune responses to different environmental conditions. This study has implications for biomedical trials using non-human primates.
Assuntos
Regiões Constantes de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas , Animais , Regiões Constantes de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulina G/genética , Evolução Molecular , Platirrinos , Filogenia , MamíferosRESUMO
General consensus states that immunoglobulins are exclusively expressed by B lymphocytes to form the first line of defense against common pathogens. Here, we provide compelling evidence for the expression of two heavy chain immunoglobulin genes in subpopulations of neurons in the mouse brain and spinal cord. RNA isolated from excitatory and inhibitory neurons through ribosome affinity purification revealed Ighg3 and Ighm transcripts encoding for the constant (Fc), but not the variable regions of IgG3 and IgM. Because, in the absence of the variable immunoglobulin regions, these transcripts lack the canonical transcription initiation site used in lymphocytes, we screened for alternative 5' transcription start sites and identified a novel 5' exon adjacent to a proposed promoter element. Immunohistochemical, Western blot, and in silico analyses strongly support that these neuronal transcripts are translated into proteins containing four Immunoglobulin domains. Our data thus demonstrate the expression of two Fc-encoding genes Ighg3 and Ighm in spinal and supraspinal neurons of the murine CNS and suggest a hitherto unknown function of the encoded proteins.
Assuntos
Sistema Nervoso Central/metabolismo , Regiões Constantes de Imunoglobulina/genética , Neurônios/metabolismo , Animais , Linfócitos B/metabolismo , Sequência de Bases/genética , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Regiões Constantes de Imunoglobulina/imunologia , Domínios de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genética , Transcriptoma/genéticaRESUMO
Somatic hypermutation (SHM) and class-switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation-induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome, and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt's lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than on the constant (C) region, which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sµ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of polymerase II (Pol II) and its S2 phosphorylated form at the IgH locus. Knockout of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of the P-TEFb complex) on the V region, and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreases SHM and the abundance of Pol II S2P at the IgH locus. Since all these factors play a role in transcription elongation, our studies reinforce the idea that the chromatin context and dynamics of transcription are critical for SHM.
Assuntos
Histona-Lisina N-Metiltransferase/imunologia , Histonas/imunologia , Hipermutação Somática de Imunoglobulina , Animais , Linfócitos B/imunologia , Linfoma de Burkitt/enzimologia , Linfoma de Burkitt/genética , Linfoma de Burkitt/imunologia , Linhagem Celular Tumoral , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Switching de Imunoglobulina , Regiões Constantes de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Lisina/genética , Lisina/imunologia , Metilação , CamundongosRESUMO
Cytogenetic abnormalities are a recognized factor in the pathogenesis of multiple myeloma (MM). While chromosomal translocations involving the IGH gene have been investigated and reported, the implications of deletions or amplifications in the IGH gene have been less frequently examined. We conducted a retrospective analysis of 260 patients with MM from Northern Israel. Fluorescent in situ hybridization (FISH) analysis of separated CD-138 positive cells was done on bone marrow samples collected between 2016 and 2018. We used IGH break apart probes to identify IGH abnormalities and performed statistical analysis of clinical and prognostic features, comparing the different cytogenetic groups. Deletions in the variable region of the IGH (IGHv) were found in 17.3 % (n = 45) of patients and correlated with significantly worse progression free survival (PFS) after two years of follow up (p = 0.008), as well as with a worse response to 1st line treatment (p = 0.037). The median PFS was 7.1 and 17.7 months in patients with and without IGHv deletion, respectively. PFS differences remained significant (p = 0.017) in subgroup analysis of patients with high-risk cytogenetics (n = 108, 19 with IGHv deletion). Overall survival was not significantly different in the two groups. Constant region (IGHc) amplifications, were less frequently found (6.15 %, n = 16), yet significantly correlated with worse PFS after two years of follow up (p = 0.023). This difference remained valid in the high-risk subgroup (p = 0.001). In Conclusion, we identified that deletion of the IGH variable region and amplification in the IGH constant region, are both associated with poor prognosis and inferior outcome in MM.
Assuntos
Genes de Cadeia Pesada de Imunoglobulina , Mieloma Múltiplo/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ciclofosfamida/administração & dosagem , Dexametasona/administração & dosagem , Feminino , Seguimentos , Amplificação de Genes , Deleção de Genes , Humanos , Regiões Constantes de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Hibridização in Situ Fluorescente , Israel/epidemiologia , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/mortalidade , Plasmócitos/ultraestrutura , Prognóstico , Intervalo Livre de Progressão , Estudos Retrospectivos , Teniposídeo/administração & dosagem , Resultado do TratamentoRESUMO
Polymorphic diversity in antibody constant domains has long been defined by allotypic motifs that cross react with the sera of other individuals. Improvements in sequencing technologies have led to the discovery of a large number of new allelic sequences that underlie this diversity. Many of the point mutations lie outside traditional allotypic motifs suggesting they do not elicit immunogenic responses. As antibodies play an important role in immune defense and biotechnology, understanding how this newly resolved diversity influences the function of antibodies is important. This review investigates the current known diversity of antibody alleles at a protein level for each antibody isotype as well as the kappa and lambda light chains. We focus on evidence emerging for how these mutations perturb antibody interactions with antigens and Fc receptors that are critical for function, as well as the influence this might have on the use of antibodies as therapeutics and reagents.
Assuntos
Alótipos de Imunoglobulina/imunologia , Regiões Constantes de Imunoglobulina/genética , Alelos , Animais , Formação de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Reações Cruzadas , Variação Genética , Humanos , Receptores Fc/metabolismoRESUMO
The constant region of immunoglobulin (Ig) G antibodies is responsible for their effector immune mechanism and prolongs serum half-life, while the fragment variable (Fv) region is responsible for cellular or tissue targeting. Therefore, antibody engineering for cancer therapeutics focuses on both functional efficacy of the constant region and tissue- or cell-specificity of the Fv region. In the functional aspect of therapeutic purposes, antibody engineers in both academia and industry have capitalized on the constant region of different IgG subclasses and engineered the constant region to enhance therapeutic efficacy against cancer, leading to a number of successes for cancer patients in clinical settings. In this article, we review IgG subclasses for cancer therapeutics, including i) IgG1, ii) IgG2, 3, and 4, iii) recent findings on Fc receptor functions, and iv) future directions of reprogramming the constant region of IgG to maximize the efficacy of antibody drug molecules in cancer patients.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Imunoconjugados , Regiões Constantes de Imunoglobulina , Imunoglobulina G , Neoplasias/tratamento farmacológico , Engenharia de Proteínas , Imunoconjugados/genética , Imunoconjugados/uso terapêutico , Regiões Constantes de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/uso terapêutico , Imunoglobulina G/genética , Imunoglobulina G/uso terapêutico , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/uso terapêutico , Neoplasias/metabolismoRESUMO
Despite great values in many applications, heavy chain-only antibodies (HcAbs) are naturally only produced in camelids and sharks, which are not easy to access and handle. Production of the type of antibodies in small laboratory animals would remarkably facilitate their applications. We previously reported a mouse line in which the CH1 exon of mouse γ1 was deleted that could express heavy chain-only IgG1 antibodies. However, these mice showed an extremely weak IgG1 response to specific antigens when immunized, and we could only achieve single VH domains with low affinity to antigens using these mice. One possibility is that the mouse germline VH repertoire was not sufficient to support the expression of functional heavy chain-only antibodies. In this study, we report the generation of a rat line in which the CH1 exon of the γ2a gene was removed and the γ1 and γ2b genes were silenced. Although the genetically modified rats expressed heavy chain-only IgG2a, they also exhibited a very weak IgG2a response to antigen immunization. Panning of a phage library constructed using IgG2a VH segments amplified from immunized rats identified antigen-specific single VH antibodies, which also exhibited much lower affinity than that of commercial mAbs. Together with our previous report, this study suggests that the simple genetic removal of the CH1 exon does not guarantee the successful expression of functional heavy chain-only antibodies.
Assuntos
Anticorpos/imunologia , Antígenos/imunologia , Éxons , Regiões Constantes de Imunoglobulina/genética , Imunoglobulina G/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Animais , Animais Geneticamente Modificados , Imunização , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Biblioteca de Peptídeos , RatosRESUMO
Erythropoietin (EPO) is a potential therapeutic for Alzheimer's disease (AD); however, limited blood-brain barrier (BBB) penetration reduces its applicability as a CNS therapeutic. Antibodies against the BBB transferrin receptor (TfRMAbs) act as molecular Trojan horses for brain drug delivery, and a fusion protein of EPO and TfRMAb, designated TfRMAb-EPO, is protective in a mouse model of AD. TfRMAbs have Fc effector function side effects, and removal of the Fc N-linked glycosylation site by substituting Asn with Gly reduces the Fc effector function. However, the effect of such Fc mutations on the pharmacokinetics (PK) of plasma clearance of TfRMAb-based fusion proteins, such as TfRMAb-EPO, is unknown. To examine this, the plasma PK of TfRMAb-EPO (wild-type), which expresses the mouse IgG1 constant heavy chain region and includes the Asn residue at position 292, was compared to the mutant TfRMAb-N292G-EPO, in which the Asn residue at position 292 is mutated to Gly. Plasma PK was compared following IV, IP, and SQ administration for doses between 0.3 and 3 mg/kg in adult male C57 mice. The results show a profound increase in clearance (6- to 8-fold) of the TfRMAb-N292G-EPO compared with the wild-type TfRMAb-EPO following IV administration. The clearance of both the wild-type and mutant TfRMAb-EPO fusion proteins followed nonlinear PK, and a 10-fold increase in dose resulted in a 7- to 11-fold decrease in plasma clearance. Following IP and SQ administration, the Cmax values of the TfRMAb-N292G-EPO mutant were profoundly (37- to 114-fold) reduced compared with the wild-type TfRMAb-EPO, owing to comparable increases in plasma clearance of the mutant fusion protein. The wild-type TfRMAb fusion protein was associated with reticulocyte suppression, and the N292G mutation mitigated this suppression of reticulocytes. Overall, the beneficial suppression of effector function via the N292G mutation may be offset by the deleterious effect this mutation has on the plasma levels of the TfRMAb-EPO fusion protein, especially following SQ administration, which is the preferred route of administration in humans for chronic neurodegenerative diseases including AD.
Assuntos
Anticorpos Monoclonais/farmacocinética , Barreira Hematoencefálica/metabolismo , Eritropoetina/farmacocinética , Imunoconjugados/farmacocinética , Receptores da Transferrina/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacocinética , Doença de Alzheimer/tratamento farmacológico , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/efeitos dos fármacos , Linhagem Celular , Células Endoteliais , Eritropoetina/administração & dosagem , Eritropoetina/genética , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/genética , Imunoconjugados/imunologia , Regiões Constantes de Imunoglobulina/administração & dosagem , Regiões Constantes de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/imunologia , Imunoglobulina G/administração & dosagem , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Injeções Intravenosas , Injeções Subcutâneas , Masculino , Camundongos , Mutação , Receptores da Transferrina/imunologia , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Reticulócitos/efeitos dos fármacosRESUMO
We report the production and application of a recombinant IgCw molecule, which is composed of only the constant domains of the heavy (CH) and light (CL) chains, lacking a variable (V) domain. We produced IgCw, especially human IgCw-γκ (98â¯kDa), composed of two human Cγ chains (37â¯kDa each) and two Cκ chains (12â¯kDa each), using HEK293F cell culture. We found that the yield of IgCw-γκ protein was â¼20â¯mg/L, which was comparable to that of full-size IgG; it bound to Fcγ receptor-positive cells with a low background noise on Fcγ receptor-negative cells; and IgCw-γκ can be used as a reference for measurement of Ig concentration. Moreover, Cγ and Cκ chains were easily isolated from IgCw-γκ by a single step of affinity chromatography in the presence of a reducing agent. These results demonstrate that the IgCw molecule has the potential to be used for certain in vitro and in vivo applications as an alternative to an irrelevant isotype control IgG, and to be used a favorable antigen for acquiring isotype-specific antibodies by immunizing animals.
Assuntos
Regiões Constantes de Imunoglobulina/química , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Células HeLa , Humanos , Regiões Constantes de Imunoglobulina/genética , Imunoglobulina G , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Receptores de IgG/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
DNA cytosine methylation is involved in the regulation of gene expression during development and its deregulation is often associated with disease. Mammalian genomes are predominantly methylated at CpG dinucleotides. Unmethylated CpGs are often associated with active regulatory sequences while methylated CpGs are often linked to transcriptional silencing. Previous studies on CpG methylation led to the notion that transcription initiation is more sensitive to CpG methylation than transcriptional elongation. The immunoglobulin heavy chain (IgH) constant locus comprises multiple inducible constant genes and is expressed exclusively in B lymphocytes. The developmental B cell stage at which methylation patterns of the IgH constant genes are established, and the role of CpG methylation in their expression, are unknown. Here, we find that methylation patterns at most cis-acting elements of the IgH constant genes are established and maintained independently of B cell activation or promoter activity. Moreover, one of the promoters, but not the enhancers, is hypomethylated in sperm and early embryonic cells, and is targeted by different demethylation pathways, including AID, UNG, and ATM pathways. Combined, the data suggest that, rather than being prominently involved in the regulation of the IgH constant locus expression, DNA methylation may primarily contribute to its epigenetic pre-marking.
Assuntos
Metilação de DNA , Genes de Cadeia Pesada de Imunoglobulina , Regiões Constantes de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Ilhas de CpG/genética , Citosina/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Ativação Linfocitária/genética , Camundongos , Regiões Promotoras GenéticasRESUMO
T-cell receptors (TCR) have considerable potential as therapeutics and antibody-like reagents to monitor disease progression and vaccine efficacy. Whereas antibodies recognize only secreted and surface-bound proteins, TCRs recognize otherwise inaccessible disease-associated intracellular proteins when they are presented as processed peptides bound to major histocompatibility complexes (pMHC). TCRs have been primarily explored for cancer therapy applications but could also target infectious diseases such as cytomegalovirus (CMV). However, TCRs are more difficult to express and engineer than antibodies, and advanced methods are needed to enable their widespread use. Here, we engineered the human CMV-specific TCR RA14 for high-affinity and robust soluble expression. To achieve this, we adapted our previously reported mammalian display system to present TCR extracellular domains and used this to screen CDR3 libraries for clones with increased pMHC affinity. After three rounds of selection, characterized clones retained peptide specificity and activation when expressed on the surface of human Jurkat T cells. We obtained high yields of soluble, monomeric protein by fusing the TCR extracellular domains to antibody hinge and Fc constant regions, adding a stabilizing disulfide bond between the constant domains and disrupting predicted glycosylation sites. One variant exhibited 50 nm affinity for its cognate pMHC, as measured by surface plasmon resonance, and specifically stained cells presenting this pMHC. Our work has identified a human TCR with high affinity for the immunodominant CMV peptide and offers a new strategy to rapidly engineer soluble TCRs for biomedical applications.
Assuntos
Citomegalovirus/imunologia , Expressão Gênica , Biblioteca Gênica , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Células CHO , Cricetulus , Citomegalovirus/genética , Humanos , Regiões Constantes de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/imunologia , Células Jurkat , Camundongos , Domínios Proteicos , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , SolubilidadeRESUMO
F-652 is a recombinant fusion protein consisting of two human interleukin-22 (IL-22) molecules linked to an immunoglobulin constant region (IgG2-Fc). IL-22 plays critical roles in promoting tissue repair and suppressing bacterial infection. The safety, pharmacokinetics (PK), tolerability, and biomarkers of F-652 were evaluated following a single dose in healthy male volunteers in a randomized, double-blind, placebo-controlled study. Following single-dose subcutaneous (SC) injection of F-652 at 2.0 µg/kg into healthy subjects, six out of six subjects experienced delayed injection site reactions, which presented as erythematous and/or discoid eczematous lesions 10 to 17 days post-dosing. F-652 was then administered to the healthy subjects via an intravenous (IV) infusion at 2.0, 10, 30, and 45 µg/kg. No severe adverse event (SAE) was observed during the study. Among the IV-dosed cohorts, eye and skin treatment emergent adverse events (TEAEs) were observed in the 30 and 45 µg/kg cohorts. F-652 IV dosing resulted in linear increases in Cmax and AUC(0-t), and the T1/2 ranged from 39.4 to 206 h in the cohorts. An IV injection of F-652 induced dose-dependent increases in serum marker serum amyloid A, C-reactive protein, and FIB, and decreased serum triglycerides. The serum levels of 36 common pro-inflammatory cytokines/chemokines were not altered by the treatment of F-652 at 45 µg/kg. In conclusion, IV administration of F-652 to healthy male volunteers is safe and well-tolerated and demonstrates favorable PK and pharmacodynamic properties. These results warrant further clinical development of F-652 to treat inflammatory diseases.
Assuntos
Infecções Bacterianas/tratamento farmacológico , Proteínas Recombinantes de Fusão/uso terapêutico , Cicatrização/efeitos dos fármacos , Adulto , Biomarcadores/sangue , Citocinas/sangue , Dimerização , Relação Dose-Resposta a Droga , Método Duplo-Cego , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Voluntários Saudáveis , Humanos , Regiões Constantes de Imunoglobulina/genética , Mediadores da Inflamação/sangue , Infusões Intravenosas , Reação no Local da Injeção/etiologia , Interleucinas/genética , Masculino , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/farmacocinética , Proteína Amiloide A Sérica/metabolismo , Adulto Jovem , Interleucina 22RESUMO
Age-related changes can significantly alter the state of adaptive immune system and often lead to attenuated response to novel pathogens and vaccination. In present study we employed 5'RACE UMI-based full length and nearly error-free immunoglobulin profiling to compare plasma cell antibody repertoires in young (19-26 years) and middle-age (45-58 years) individuals vaccinated with a live yellow fever vaccine, modeling a newly encountered pathogen. Our analysis has revealed age-related differences in the responding antibody repertoire ranging from distinct IGH CDR3 repertoire properties to differences in somatic hypermutation intensity and efficiency and antibody lineage tree structure. Overall, our findings suggest that younger individuals respond with a more diverse antibody repertoire and employ a more efficient somatic hypermutation process than elder individuals in response to a newly encountered pathogen.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Imunidade Ativa , Receptores de Antígenos de Linfócitos B/metabolismo , Vacina contra Febre Amarela/imunologia , Adulto , Animais , Anticorpos Antivirais/imunologia , Feminino , Humanos , Imunidade Ativa/genética , Regiões Constantes de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos B/genética , Hipermutação Somática de Imunoglobulina , Vacinação , Febre Amarela/prevenção & controle , Adulto JovemRESUMO
Nano-antibodies possess great potential in many applications. However, they are naturally derived from heavy chain-only antibodies (HcAbs), which lack light chains and the CH1 domain, and are only found in camelids and sharks. In this study, we investigated whether the precise genetic removal of the CH1 exon of the γ1 gene enabled the production of a functional heavy chain-only IgG1 in mice. IgG1 heavy chain dimers lacking associated light chains were detected in the sera of the genetically modified mice. However, the genetic modification led to decreased expression of IgG1 but increased expression of other IgG subclasses. The genetically modified mice showed a weaker immune response to specific antigens compared with wild type mice. Using a phage-display approach, antigen-specific, single domain VH antibodies could be screened from the mice but exhibited much weaker antigen binding affinity than the conventional monoclonal antibodies. Although the strategy was only partially successful, this study confirms the feasibility of producing desirable nano-bodies with appropriate genetic modifications in mice.
Assuntos
Anticorpos Monoclonais/imunologia , Cadeias gama de Imunoglobulina/imunologia , Engenharia de Proteínas , Anticorpos de Domínio Único/imunologia , Animais , Anticorpos Monoclonais/genética , Éxons/genética , Éxons/imunologia , Estudos de Viabilidade , Regiões Constantes de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/imunologia , Cadeias gama de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Biblioteca de Peptídeos , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Anticorpos de Domínio Único/genéticaRESUMO
The advantages of site-specific over stochastic bioconjugation technologies include homogeneity of product, minimal perturbation of protein structure/function, and - increasingly - the ability to perform structure activity relationship studies at the conjugate level. When selecting the optimal location for site-specific payload placement, many researchers turn to in silico modeling of protein structure to identify regions predicted to offer solvent-exposed conjugatable sites while conserving protein function. Here, using the aldehyde tag as our site-specific technology platform and human IgG1 antibody as our target protein, we demonstrate the power of taking an unbiased scanning approach instead. Scanning insertion of the human formylglycine generating enzyme (FGE) recognition sequence, LCTPSR, at each of the 436 positions in the light and heavy chain antibody constant regions followed by co-expression with FGE yielded a library of antibodies bearing an aldehyde functional group ready for conjugation. Each of the variants was expressed, purified, and conjugated to a cytotoxic payload using the Hydrazinyl Iso-Pictet-Spengler ligation to generate an antibody-drug conjugate (ADC), which was analyzed in terms of conjugatability (assessed by drug-to-antibody ratio, DAR) and percent aggregate. We searched for insertion sites that could generate manufacturable ADCs, defined as those variants yielding reasonable antibody titers, DARs of ≥ 1.3, and ≥ 95% monomeric species. Through this process, we discovered 58 tag insertion sites that met these metrics, including 14 sites in the light chain, a location that had proved refractory to the placement of manufacturable tag sites using in silico modeling/rational approaches.
Assuntos
Aldeídos/imunologia , Imunoconjugados/imunologia , Regiões Constantes de Imunoglobulina/imunologia , Imunoglobulina G/imunologia , Aldeídos/química , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Composição de Medicamentos/métodos , Glicina/análogos & derivados , Glicina/química , Glicina/genética , Glicina/imunologia , Humanos , Imunoconjugados/química , Imunoconjugados/genética , Regiões Constantes de Imunoglobulina/química , Regiões Constantes de Imunoglobulina/genética , Imunoglobulina G/química , Imunoglobulina G/genética , Biblioteca de Peptídeos , Ligação ProteicaRESUMO
Immunoglobulins are important elements of the adaptive immune system that bind to an immense variety of microbial antigens to neutralize infectivity and specify effector functions. In the present study, the immunoglobulin heavy chain constant region (IGHC) genes from marine mammals were identified and compared with those of their terrestrial relatives to explore their genomic organization and evolutionary characteristics. The genomic organization of marine mammal IGHC genes was shown to be conservative with other eutherian mammals. Stronger signals of positive selection on IGHC were revealed in terrestrial mammals than that in marine mammals with the branch-site model, displaying different selective pressure, which might suggest their divergent adaptations to contrasted environments.
Assuntos
Adaptação Fisiológica/genética , Organismos Aquáticos/genética , Regiões Constantes de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Mamíferos/genética , Animais , Evolução Molecular , Genoma/genética , Genômica/métodos , Filogenia , Seleção Genética/genéticaRESUMO
We have designed a complete antibody-like construct where the CH1 and Cκ domains are exchanged for a pair of the CH3 domains and efficient pairing of the heavy and light variable domain is achieved using "Knobs-into-Holes" strategy. This construct, composed of only naturally occurring immunoglobulin sequences without artificial linkers, expressed at a high level in mammalian cells, however exhibited low solubility. Rational mutagenesis aimed at the amino acid residues located at the interface of the variable domains and the exchanged CH3 domains was applied to improve the biophysical properties of the molecule. The domain-exchanged construct, including variable domains of the HER2/neu specific antibody trastuzumab, was able to bind to the surface of the strongly HER2/neu positive cell line SK-BR3 4-fold weaker than trastuzumab, but could nevertheless incite a more potent response in an antibody-dependent cell cytotoxicity (ADCC) reporter assay with FcγRIIIa-overexpressing T-cells. This could be explained with a stronger binding to the FcγRIIIa. Importantly, the novel construct could mediate a specific ADCC effect with natural killer cells similar to the parental antibody.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Animais , Anticorpos Monoclonais/química , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular , Humanos , Regiões Constantes de Imunoglobulina/química , Regiões Constantes de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Células Matadoras Naturais/imunologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios Proteicos , Engenharia de Proteínas , Receptor ErbB-2/imunologia , Receptores de IgG/química , Receptores de IgG/genética , Receptores de IgG/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Trastuzumab/química , Trastuzumab/genética , Trastuzumab/imunologiaRESUMO
We report a method to generate bifunctional antibodies by grafting full-length proteins into constant region loops of a full-length antibody or an antigen-binding fragment (Fab). The fusion proteins retain the antigen binding activity of the parent antibody but have an additional activity associated with the protein insert. The engineered antibodies have excellent in vitro activity, physiochemical properties, and stability. Among these, a Her2 × CD3 bispecific antibody (BsAb) was constructed by inserting an anti-Her2 single-chain variable fragment (ScFv) into an anti-CD3 Fab. This bispecific antibody efficiently induces targeted cell lysis in the presence of effector cells at as low as sub-picomolar concentrations in vitro. Moreover, the Her2 × CD3 BsAb shows potent in vivo antitumor activity in mouse Her22+ and Her21+ xenograft models. These results demonstrate that insertion of a full-length protein into non-CDR loops of antibodies provides a feasible approach to generate multifunctional antibodies for therapeutic applications.