RESUMO
Natural killer (NK) cells are key players in human innate immunity. Cell engager antibody formats that recruit and activate NK cells more effectively have emerged as a promising immunotherapy approach to target cancer cells through more effective antibody-dependent cell-mediated cytotoxicity (ADCC). Monoclonal antibody drugs with ADCC activity have shown clinical benefit and improved outcomes for patients with certain types of cancer. CD16a, a Fc gamma III receptor, is the major component that is responsible for the ADCC activity of NK cells. Screening AvantGen's yeast displayed human antibody libraries led to the isolation of 2 antibody clones, #1A2 and #2-2A2, that selectively recognize both isoforms (F and V) of CD16a on primary NK cells with high affinity, yet minimally (#1A2) or do not (#2-2A2) cross-react with both allelotypes of CD16b (NA1 and NA2) expressed by neutrophils. Epitope mapping studies revealed that they bind to an epitope dependent on residue Y158 of CD16a, since mutation of Y158 to the corresponding CD16b residue H158 completely abolishes binding to CD16a. When formatted as bispecific antibodies targeting CD16a and a tumor-associated antigen (TAA, e.g. CD19), they exhibit specific binding to NK cells and induce potent NK cell activation upon encountering tumor cells, resulting in effective tumor cell killing. Notably, these bispecific antibody engagers stimulate NK cell cytokine release during co-culture with target cells, resulting in target cell cytotoxicity. These anti-CD16a antibody clones are promising candidates for combination with any TAA of interest, offering the potential for novel NK cell engager-based cancer therapeutics that are minimally affected by the high concentrations of human IgG in the circulation.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Células Matadoras Naturais , Receptores de IgG , Humanos , Células Matadoras Naturais/imunologia , Receptores de IgG/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Mapeamento de Epitopos/métodos , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias/tratamento farmacológicoRESUMO
Direct identification and isolation of Ag-specific T cells became possible with the development of MHC tetramers, based on fluorescent avidins displaying biotinylated peptide-MHC complexes. This approach, extensively used for MHC class I-restricted T cells, has met very limited success with class II peptide-MHC complex tetramers (pMHCT-2) for the detection of CD4(+)-specific T cells. In addition, a very large number of these reagents, although capable of specifically activating T cells after being coated on solid support, is still unable to stain. To try to understand this puzzle and design usable tetramers, we examined each parameter critical for the production of pMHCT-2 using the I-A(d)-OVA system as a model. Through this process, the geometry of peptide-MHC display by avidin tetramers was examined, as well as the stability of rMHC molecules. However, we discovered that the most important factor limiting the reactivity of pMHCT-2 was the display of peptides. Indeed, long peptides, as presented by MHC class II molecules, can be bound to I-A/HLA-DQ molecules in more than one register, as suggested by structural studies. This mode of anchorless peptide binding allows the selection of a broader repertoire on single peptides and should favor anti-infectious immune responses. Thus, beyond the technical improvements that we propose, the redesign of pMHCT-2 will give us the tools to evaluate the real size of the CD4 T cell repertoire and help us in the production and testing of new vaccines.
Assuntos
Apresentação de Antígeno/imunologia , Animais , Apresentação de Antígeno/genética , Biotinilação/genética , Galinhas , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Engenharia de Proteínas , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The emergence of COVID-19 has led to a pandemic that has caused millions of cases of disease, variable morbidity and hundreds of thousands of deaths. Currently, only remdesivir and dexamethasone have demonstrated limited efficacy, only slightly reducing disease burden, thus novel approaches for clinical management of COVID-19 are needed. We identified a panel of human monoclonal antibody clones from a yeast display library with specificity to the SARS-CoV-2 spike protein receptor binding domain that neutralized the virus in vitro. Administration of the lead antibody clone to Syrian hamsters challenged with SARS-CoV-2 significantly reduced viral load and histopathology score in the lungs. Moreover, the antibody interrupted monocyte infiltration into the lungs, which may have contributed to the reduction of disease severity by limiting immunopathological exacerbation. The use of this antibody could provide an important therapy for treatment of COVID-19 patients.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Tratamento Farmacológico da COVID-19 , COVID-19 , Imunoglobulina G , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , COVID-19/sangue , COVID-19/imunologia , Chlorocebus aethiops , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Masculino , Mesocricetus , Índice de Gravidade de Doença , Células Vero , Carga Viral/efeitos dos fármacos , Carga Viral/imunologiaRESUMO
The emergence of COVID-19 has led to a pandemic that has caused millions of cases of disease, variable morbidity and hundreds of thousands of deaths. Currently, only remdesivir and dexamethasone have demonstrated limited efficacy, only slightly reducing disease burden, thus novel approaches for clinical management of COVID-19 are needed. We identified a panel of human monoclonal antibody clones from a yeast display library with specificity to the SARS-CoV-2 spike protein receptor binding domain that neutralized the virus in vitro . Administration of the lead antibody clone to Syrian hamsters challenged with SARS-CoV-2 significantly reduced viral load and histopathology score in the lungs. Moreover, the antibody interrupted monocyte infiltration into the lungs, which may have contributed to the reduction of disease severity by limiting immunopathological exacerbation. The use of this antibody could provide an important therapy for treatment of COVID-19 patients.
RESUMO
The crystal structures of the Fab' fragment of the anti-progesterone monoclonal antibody DB3 and its complexes with steroid haptens have shown that the D-JH junctional residue TrpH100 is a key contributor to binding site interactions with ligands. The indole group of TrpH100 also undergoes a significant conformational change between the bound and unliganded states, effectively opening and closing the combining site pocket. In order to explore the effect of substitutions at this position on steroid recognition, we have carried out mutagenesis on a construct encoding a three-domain single-chain fragment (VH/K) of DB3 expressed in Escherichia coli. TrpH100 was replaced by 13 different amino acids or deleted, and the functional and antigenic properties of the mutated fragments were analyzed. Most substitutions, including small, hydrophobic, hydrophilic, neutral, and negatively charged side chains, were reduced or abolished binding to free progesterone, although binding to progesterone-BSA was partially retained. The reduction in antigen binding was paralleled by alteration of the idiotype associated with the DB3 combining site. In contrast, the replacement of TrpH100 by Arg produced a mutant that retained wild-type antibody affinity and idiotype, but with altered specificity. Significant changes in this mutant included increased relative affinities of 10(4)-fold for progesterone-3-carboxymethyloxime and 10-fold for aetiocholanolone. Our results demonstrate an essential role for the junctional residue H100 in determining steroid-binding specificity and combining site idiotype and show that these properties can be changed by a single amino acid substitution at this position.
Assuntos
Anticorpos/química , Escherichia coli/imunologia , Mutação/fisiologia , Progesterona/imunologia , Sequência de Aminoácidos , Afinidade de Anticorpos , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Modelos Químicos , Dados de Sequência Molecular , Progesterona/química , Estrutura Secundária de Proteína , Sensibilidade e EspecificidadeRESUMO
Association between the class II major histocompatibility complex (MHC) and the class II invariant chain-associated peptide (CLIP) occurs naturally as an intermediate step in the MHC class II processing pathway. Here, we report the crystal structure of the murine class II MHC molecule I-A(b) in complex with human CLIP at 2.15A resolution. The structure of I-A(b) accounts, via the peptide-binding groove's unique physicochemistry, for the distinct peptide repertoire bound by this allele. CLIP adopts a similar conformation to peptides bound by other I-A alleles, reinforcing the notion that CLIP is presented as a conventional peptide antigen. When compared to the related HLA-DR3/CLIP complex structure, the CLIP peptide displays a slightly different conformation and distinct interaction pattern with residues in I-A(b). In addition, after examining the published sequences of peptides presented by I-A(b), we discuss the possibility of predicting peptide alignment in the I-A(b) binding groove using a simple scoring matrix.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Genes MHC da Classe II , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/metabolismo , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação de Linfócitos B/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Substâncias Macromoleculares , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
Peptides bind with high affinity to MHC class I molecules by anchoring certain side-chains (anchors) into specificity pockets in the MHC peptide-binding groove. Peptides that do not contain these canonical anchor residues normally have low affinity, resulting in impaired pMHC stability and loss of immunogenicity. Here, we report the crystal structure at 1.6 A resolution of an immunogenic, low-affinity peptide from the tumor-associated antigen MUC1, bound to H-2Kb. Stable binding is still achieved despite small, non-canonical residues in the C and F anchor pockets. This structure reveals how low-affinity peptides can be utilized in the design of novel peptide-based tumor vaccines. The molecular interactions elucidated in this non-canonical low-affinity peptide MHC complex should help uncover additional immunogenic peptides from primary protein sequences and aid in the design of alternative approaches for T-cell vaccines.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Mucina-1/química , Peptídeos/química , Animais , Apresentação de Antígeno , Sítios de Ligação , Desenho de Fármacos , Antígenos de Histocompatibilidade Classe I/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mucina-1/imunologia , Peptídeos/imunologia , Ligação Proteica , Conformação Proteica , VacinasRESUMO
The crystal structure of a non-standard peptide, YEA9, in complex with H-2Kb, at 1.5 A resolution demonstrates how YEA9 peptide can bind with surprisingly high affinity through insertion of alternative, long, non-canonical anchors into the B and E pockets. The use of "alternative pockets" represents a new mode of high affinity peptide binding, that should be considered when predicting peptide epitopes for MHC class I. These novel interactions encountered in this non-canonical high affinity peptide-MHC complex should help predict additional binding peptides from primary protein sequences and aid in the design of alternative approaches for peptide-based vaccines.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Peptídeos/química , Animais , Apresentação de Antígeno , Drosophila , Desenho de Fármacos , Proteínas Fúngicas/química , Proteínas Fúngicas/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Modelos Moleculares , Peptídeos/imunologia , Ligação Proteica , Conformação Proteica , VacinasRESUMO
γδ T cells bridge innate and adaptive immunity and function in immunosurveillance, immunoregulation, tumor cell recognition, and as first line of defense against microbial infection. Costimulation of epithelial γδ T cell activation by the JAML receptor can be induced by interaction with its endogenous ligand CAR or by binding of the stimulatory antibody HL4E10. We, therefore, determined the crystal structure of the JAML-HL4E10 Fab complex at 2.95 Šresolution. HL4E10 binds the membrane-proximal domain of JAML through hydrophobic interactions that account for nanomolar affinity and long half-life, contrasting with the fast kinetics and micromolar affinity of the hydrophilic CAR interaction with the membrane-distal JAML domain. Thus, despite different binding sites and mechanisms, JAML interaction with these two disparate ligands leads to the same functional outcome, namely JAML triggering and induction of cell signaling. Several characteristics of the HL4E10 antibody might then be harnessed in therapeutic applications, such as promoting healing of acute or chronic wounds.
Assuntos
Anticorpos Heterófilos/farmacologia , Anticorpos Monoclonais/farmacologia , Moléculas de Adesão Celular/metabolismo , Células de Langerhans/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/metabolismo , Animais , Anticorpos Heterófilos/química , Anticorpos Monoclonais/química , Sítios de Ligação de Anticorpos , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/imunologia , Linhagem Celular , Proliferação de Células , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Cricetinae , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Fragmentos Fab das Imunoglobulinas/química , Células de Langerhans/citologia , Camundongos , Fosfatidilinositol 3-Quinases/fisiologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Receptores Virais/química , Receptores Virais/metabolismo , Proteínas Recombinantes de Fusão/química , Propriedades de Superfície , Linfócitos T/imunologiaRESUMO
Genetic susceptibility to autoimmunity is frequently associated with specific MHC alleles. Diabetogenic MHC class II molecules, such as human HLA-DQ8 and mouse I-Ag7, typically have a small, uncharged amino acid residue at position 57 of their beta chain (beta57); this results in the absence of a salt bridge between beta57 and Argalpha76, which is adjacent to the P9 pocket of the peptide-binding groove. However, the influence of Argalpha76 on the selection of the TCR repertoire remains unknown, particularly when the MHC molecule binds a peptide with a neutral amino acid residue at position P9. Here, we have shown that diabetogenic MHC class II molecules bound to a peptide with a neutral P9 residue primarily selected and expanded cells expressing TCRs bearing a negatively charged residue in the first segment of their complementarity determining region 3beta. The crystal structure of one such TCR in complex with I-Ag7 bound to a peptide containing a neutral P9 residue revealed that a network of favorable long-range (greater than 4 A) electrostatic interactions existed among Argalpha76, the neutral P9 residue, and TCR, which supported the substantially increased TCR/peptide-MHC affinity. This network could be modulated or switched to a lower affinity interaction by the introduction of a negative charge at position P9 of the peptide. Our results support the existence of a switch at residue beta57 of the I-Ag7 and HLA-DQ8 class II molecules and potentially link normal thymic TCR selection with abnormal peripheral behavior.
Assuntos
Diabetes Mellitus Experimental/genética , Antígenos de Histocompatibilidade Classe II/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Separação Celular , Cristalografia por Raios X/métodos , Dimerização , Feminino , Teste de Complementação Genética , Predisposição Genética para Doença , Antígenos de Histocompatibilidade Classe II/fisiologia , Humanos , Hibridomas/patologia , Cinética , Camundongos , Conformação Molecular , Timo/metabolismoRESUMO
BACKGROUND: Cluster of differentiation 36 (CD36) is a transmembrane glycoprotein involved in many biological processes, such as platelet biology, angiogenesis and in the aetiopathology of atherosclerosis and cardiovascular diseases. Toll-like receptors (TLRs) are one of the most important receptors of the innate immune system. Their main function is the recognition of conserved structure of microorganisms. This recognition triggers signaling pathways that activate transcription of cytokines and co-stimulatory molecules which participate in the generation of an immune response against microbes. In particular, TLR2 has been shown to recognize a broad range of ligands. Recently, we showed that CD36 serves as a co-receptor for TLR2 and enhances recognition of specific diacylglycerides derived from bacteria. METHODOLOGY/ PRINCIPAL FINDINGS: Here, we investigate the mechanism by which CD36 contributes to ligand recognition and activation of TLR2 signaling pathway. We show that the ectodomain of murine CD36 (mCD36ED) directly interacts with negatively charged diacylglycerol ligands, which explains the specificity and selectivity of CD36 as a TLR2 co-receptor. We also show that mCD36ED amplifies the pro-inflammatory response to lipoteichoic acid in macrophages of wild-type mice and restores the pro-inflammatory response of macrophages from mice deficient in CD36 (oblivious), but not from mice deficient in cluster of differentiation 14 (CD14) (heedless). CONCLUSION/ SIGNIFICANCE: These data indicate that the CD36 ectodomain is the only relevant domain for activation of TLR2 signaling pathway and that CD36 and CD14 have a non-redundant role for loading ligands onto TLR2 in the plasma-membrane. The pro-inflammatory role of soluble CD36 can be relevant in the activation of the immune response against pathogens, as well as in the progression of chronic diseases. Therefore, an increased level of soluble forms of CD36, which has been reported to be increased in type II diabetic patients, could accelerate atherosclerosis by increasing the pro-inflammatory response to diacylglycerol ligands.
Assuntos
Antígenos CD36/biossíntese , Diglicerídeos/química , Receptor 2 Toll-Like/química , Animais , Diglicerídeos/metabolismo , Glicoproteínas/metabolismo , Sistema Imunitário , Ligantes , Receptores de Lipopolissacarídeos/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Polissacarídeos/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
The 1918 "Spanish" influenza pandemic represents the largest recorded outbreak of any infectious disease. The crystal structure of the uncleaved precursor of the major surface antigen of the extinct 1918 virus was determined at 3.0 angstrom resolution after reassembly of the hemagglutinin gene from viral RNA fragments preserved in 1918 formalin-fixed lung tissues. A narrow avian-like receptor-binding site, two previously unobserved histidine patches, and a less exposed surface loop at the cleavage site that activates viral membrane fusion reveal structural features primarily found in avian viruses, which may have contributed to the extraordinarily high infectivity and mortality rates observed during 1918.