RESUMO
Viral escape from CD8+ cytotoxic T lymphocyte responses correlates with disease progression and represents a significant challenge for vaccination. Here, we demonstrate that CD8+ T cell recognition of the naturally occurring MHC-I-restricted LCMV-associated immune escape variant Y4F is restored following vaccination with a proline-altered peptide ligand (APL). The APL increases MHC/peptide (pMHC) complex stability, rigidifies the peptide and facilitates T cell receptor (TCR) recognition through reduced entropy costs. Structural analyses of pMHC complexes before and after TCR binding, combined with biophysical analyses, revealed that although the TCR binds similarly to all complexes, the p3P modification alters the conformations of a very limited amount of specific MHC and peptide residues, facilitating efficient TCR recognition. This approach can be easily introduced in peptides restricted to other MHC alleles, and can be combined with currently available and future vaccination protocols in order to prevent viral immune escape.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vírus da Coriomeningite Linfocítica/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Antivirais/metabolismo , Linfócitos T CD8-Positivos/fisiologia , Proteínas de Ligação a DNA/imunologia , Epitopos/imunologia , Epitopos de Linfócito T/imunologia , Genes RAG-1/imunologia , Ligantes , Ativação Linfocitária/imunologia , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/patogenicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeos/metabolismo , Prolina/metabolismo , Ligação Proteica , Linfócitos T Citotóxicos/imunologia , Vacinação/métodosRESUMO
MHC-I epitope presentation to CD8+ T cells is directly dependent on peptide loading and selection during antigen processing. However, the exact molecular bases underlying peptide selection and binding by MHC-I remain largely unknown. Within the peptide-loading complex, the peptide editor tapasin is key to the selection of MHC-I-bound peptides. Here, we have determined an ensemble of crystal structures of MHC-I in complex with the peptide exchange-associated dipeptide GL, as well as the tapasin-associated scoop loop, alone or in combination with candidate epitopes. These results combined with mutation analyses allow us to propose a molecular model underlying MHC-I peptide selection by tapasin. The N termini of bound peptides most probably bind first in the N-terminal and middle region of the MHC-I peptide binding cleft, upon which the peptide C termini are tested for their capacity to dislodge the tapasin scoop loop from the F pocket of the MHC-I cleft. Our results also indicate important differences in peptide selection between different MHC-I alleles.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Cristalografia por Raios X , Células HEK293 , Humanos , Leucina/genética , Proteínas de Membrana Transportadoras/química , Camundongos Knockout , Modelos Moleculares , Mutação/genética , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
Antibody-drug conjugates (ADCs) have demonstrated great therapeutic potential due to their ability to target the delivery of potent cytotoxins. However, the heterogeneous nature of conventional drug conjugation strategies can affect the safety, efficacy, and stability of ADCs. Site-specific conjugations can resolve these issues, but often require genetic modification of Immunoglobulin G (IgG), which can impact yield or cost of production, or require undesirable chemical linkages. Here, we describe a near-traceless conjugation method that enables the efficient modification of native IgG, without the need for genetic engineering or glycan modification. This method utilizes engineered variants of sortase A to catalyze noncanonical isopeptide ligation. Sortase A was fused to an antibody-binding domain to improve ligation efficiency. Antibody labeling is limited to five lysine residues on the heavy chain and one on the light chain of human IgG1. The ADCs exhibit conserved antigen and Fc-receptor interactions, as well as potent cytolytic activity.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Imunoglobulina G/química , Peptídeos/química , Biocatálise , Humanos , Coloração e RotulagemRESUMO
Herein we disclose the transformation of maleimides into water-soluble tris(2-carboxyethyl)phosphonium ylides and their subsequent application in the bioconjugation of protein- and peptide-linked aldehydes. The new entry into Wittig bioconjugate chemistry proceeds under mild conditions and relies on highly water soluble reagents, which are likely already part of most biochemists' inventory.
Assuntos
MaleimidasRESUMO
We review drug conjugates combining a tumor-selective moiety with a cytotoxic agent as cancer treatments. Currently, antibody-drug conjugates (ADCs) are the most common drug conjugates used clinically as cancer treatments. While providing both efficacy and favorable tolerability, ADCs have limitations due to their size and complexity. Peptides as tumor-targeting carriers in peptide-drug conjugates (PDCs) offer a number of benefits. Melphalan flufenamide (melflufen) is a highly lipophilic PDC that takes a novel approach by utilizing increased aminopeptidase activity to selectively increase the release and concentration of cytotoxic alkylating agents inside tumor cells. The only other PDC approved currently for clinical use is 177Lu-dotatate, a targeted form of radiotherapy combining a somatostatin analog with a radionuclide. It is approved as a treatment for gastroenteropancreatic neuroendocrine tumors. Results with other PDCs combining synthetic analogs of natural peptide ligands with cytotoxic agents have been mixed. The field of drug conjugates as drug delivery systems for the treatment of cancer continues to advance with the application of new technologies. Melflufen provides a paradigm for rational PDC design, with a targeted mechanism of action and the potential for deepening responses to treatment, maintaining remissions, and eradicating therapy-resistant stem cells.
Assuntos
Antineoplásicos/uso terapêutico , Citotoxinas/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Imunoconjugados/uso terapêutico , Neoplasias/terapia , Peptídeos/uso terapêutico , Radioterapia/métodos , Portadores de Fármacos/química , Desenho de Fármacos , Humanos , Melfalan/análogos & derivados , Melfalan/uso terapêutico , Peptídeos/química , Preparações Farmacêuticas/química , Fenilalanina/análogos & derivados , Fenilalanina/uso terapêutico , Somatostatina/uso terapêuticoRESUMO
The immunogenicity of H-2D(b) (D(b)) restricted epitopes can be significantly increased by substituting peptide position 3 to a proline (p3P). The p3P modification enhances MHC stability without altering the conformation of the modified epitope allowing for T-cell cross-reactivity with the native peptide. The present study reveals how specific interactions between p3P and the highly conserved MHC heavy chain residue Y159 increase the stability of D(b) in complex with an optimized version of the melanoma-associated epitope gp10025-33 . Furthermore, the p3P modification directly increased the affinity of the D(b)/gp10025-33 -specific T-cell receptor (TCR) pMel. Surprisingly, the enhanced TCR binding was independent from the observed increased stability of the optimized D(b)/gp10025-33 complex and from the interactions formed between p3P and Y159, indicating a direct effect of the p3P modification on TCR recognition.
Assuntos
Antígeno de Histocompatibilidade H-2D/imunologia , Prolina/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Antígeno gp100 de Melanoma/imunologia , Substituição de Aminoácidos , Animais , Cristalografia por Raios X , Epitopos de Linfócito T/imunologia , Antígeno de Histocompatibilidade H-2D/química , Antígeno de Histocompatibilidade H-2D/ultraestrutura , Camundongos , Simulação de Dinâmica Molecular , Prolina/genética , Conformação Proteica , Ressonância de Plasmônio de Superfície , Linfócitos T Citotóxicos/metabolismo , Antígeno gp100 de Melanoma/genéticaRESUMO
Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.
Assuntos
Materiais Biocompatíveis/química , Proteínas Recombinantes de Fusão/química , Seda/química , Animais , Materiais Biocompatíveis/isolamento & purificação , Materiais Biocompatíveis/metabolismo , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Estrutura Terciária de Proteína , Coelhos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Seda/isolamento & purificação , Seda/metabolismo , Aranhas/química , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Heterozygous loss-of-function mutations in the GRN gene are a common cause of frontotemporal dementia. Such mutations lead to decreased plasma and cerebrospinal fluid levels of progranulin (PGRN), a neurotrophic factor with lysosomal functions. Sortilin is a negative regulator of extracellular PGRN levels and has shown promise as a therapeutic target for frontotemporal dementia, enabling increased extracellular PGRN levels through inhibition of sortilin-mediated PGRN degradation. Here we report the development of a high-affinity sortilin-binding affibody-peptide fusion construct capable of increasing extracellular PGRN levels in vitro. By genetic fusion of a sortilin-binding affibody generated through phage display and a peptide derived from the progranulin C-terminus, an affinity protein (A3-PGRNC15*) with 185-pM affinity for sortilin was obtained. Treating PGRN-secreting and sortilin-expressing human glioblastoma U-251 cells with the fusion protein increased extracellular PGRN levels up to 2.5-fold, with an EC50 value of 1.3 nM. Our results introduce A3-PGRNC15* as a promising new agent with therapeutic potential for the treatment of frontotemporal dementia. Furthermore, the work highlights means to increase binding affinity through synergistic contribution from two orthogonal polypeptide units.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Progranulinas , Proteólise , Proteínas Recombinantes de Fusão , Progranulinas/metabolismo , Progranulinas/genética , Humanos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Linhagem Celular Tumoral , Ligação Proteica , Demência Frontotemporal/metabolismo , Demência Frontotemporal/genética , Peptídeos/farmacologia , Peptídeos/metabolismoRESUMO
Affinity protein-oligonucleotide conjugates are increasingly being explored as diagnostic and therapeutic tools. Despite growing interest, these probes are typically constructed using outdated, non-selective chemistries, and little has been done to investigate how conjugation to oligonucleotides influences the function of affinity proteins. Herein, we report a novel site-selective conjugation method for furnishing affinity protein-oligonucleotide conjugates in a 93% yield within fifteen minutes. Using SPR, we explore how the choice of affinity protein, conjugation strategy, and DNA length impact target binding and reveal the deleterious effects of non-specific conjugation methods. Furthermore, we show that these adverse effects can be minimised by employing our site-selective conjugation strategy, leading to improved performance in an immuno-PCR assay. Finally, we investigate the interactions between affinity protein-oligonucleotide conjugates and live cells, demonstrating the benefits of site-selective conjugation. This work provides critical insight into the importance of conjugation strategy when constructing affinity protein-oligonucleotide conjugates.
RESUMO
The increasing availability of antibodies toward human proteins enables broad explorations of the proteomic landscape in cells, tissues, and body fluids. This includes assays with antibody suspension bead arrays that generate protein profiles of plasma samples by flow cytometer analysis. However, antibody selectivity is context dependent so it is necessary to corroborate on-target detection over off-target binding. To address this, we describe a concept to directly verify interactions from antibody-coupled beads by analysis of their eluates by Western blots and MS. We demonstrate selective antibody binding in complex samples with antibodies toward a set of chosen proteins with different abundance in plasma and serum, and illustrate the need to adjust sample and bead concentrations accordingly. The presented approach will serve as an important tool for resolving differential protein profiles from antibody arrays within plasma biomarker discoveries.
Assuntos
Anticorpos Imobilizados/química , Proteínas Sanguíneas/metabolismo , Proteínas de Fase Aguda , Especificidade de Anticorpos , Quimiocinas CC/sangue , Bases de Dados de Proteínas , Humanos , Lipocalina-2 , Lipocalinas/sangue , Proteômica , Proteínas Proto-Oncogênicas/sangueRESUMO
The molecular basis underlying T-cell recognition of MHC molecules presenting altered peptide ligands is still not well-established. A hierarchy of T-cell activation by MHC class I-restricted altered peptide ligands has been defined using the T-cell receptor P14 specific for H-2D(b) in complex with the immunodominant lymphocytic choriomeningitis virus peptide gp33 (KAVYNFATM). While substitution of tyrosine to phenylalanine (Y4F) or serine (Y4S) abolished recognition by P14, the TCR unexpectedly recognized H-2D(b) in complex with the alanine-substituted semiagonist Y4A, which displayed the most significant structural modification. The observed functional hierarchy gp33 > Y4A > Y4S = Y4F was neither due to higher stabilization capacity nor to differences in structural conformation. However, thermodynamic analysis demonstrated that while recognition of the full agonist H-2D(b) /gp33 was strictly enthalpy driven, recognition of the weak agonist H-2D(b) /Y4A was instead entropy driven with a large reduction in the favorable enthalpy term. The fourfold larger negative heat capacity derived for the interaction of P14 with H-2D(b) /gp33 compared with H-2D(b) /Y4A can possibly be explained by higher water entrapment at the TCR/MHC interface, which is also consistent with the measured opposite entropy contributions for the interactions of P14 with both MHCs. In conclusion, this study demonstrates that P14 makes use of different strategies to adapt to structural modifications in the MHC/peptide complex.
Assuntos
Antígenos Virais/química , Glicoproteínas/química , Antígenos H-2/química , Fragmentos de Peptídeos/química , Peptídeos/química , Proteínas/química , Receptores de Antígenos de Linfócitos T/química , Proteínas Virais/química , Animais , Antígenos Virais/imunologia , Dicroísmo Circular , Cristalografia por Raios X , Glicoproteínas/imunologia , Antígenos H-2/imunologia , Cinética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fragmentos de Peptídeos/imunologia , Peptídeos/imunologia , Proteínas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Organismos Livres de Patógenos Específicos , Ressonância de Plasmônio de Superfície , Termodinâmica , Proteínas Virais/imunologiaRESUMO
Herein, we describe a general protocol for the selection of target-binding affinity protein molecules from a phagemid-encoded library. The protocol is based on our experience with phage display selections of non-immunoglobulin affibody affinity proteins but can in principle be applied to perform biopanning experiments from any phage-displayed affinity protein library available in a similar phagemid vector. The procedure begins with an amplification of the library from frozen bacterial glycerol stocks via cultivation and helper phage superinfection, followed by a step-by-step instruction of target protein preparation, selection cycles, and post-selection analyses. The described procedures in this standard protocol are relatively conservative and rely on ordinary reagents and equipment available in most molecular biology laboratories.
Assuntos
Bacteriófagos , Biologia Molecular , Bacteriófagos/genética , Bioprospecção , Técnicas de Visualização da Superfície Celular , Biblioteca GênicaRESUMO
We describe the development and characterization of the (to date) smallest Natural Killer (NK) cell re-directing human B Cell Maturation Antigen (hBCMA) x CD16 dual engagers for potential treatment of multiple myeloma, based on combinations of small 58 amino acid, non-immunoglobulin, affibody affinity proteins. Affibody molecules to human CD16a were selected from a combinatorial library by phage display resulting in the identification of three unique binders with affinities (KD) for CD16a in the range of 100 nM-3 µM. The affibody exhibiting the highest affinity demonstrated insensitivity towards the CD16a allotype (158F/V) and did not interfere with IgG (Fc) binding to CD16a. For the construction of hBCMA x CD16 dual engagers, different CD16a binding arms, including bi-paratopic affibody combinations, were genetically fused to a high-affinity hBCMA-specific affibody. Such 15-23 kDa dual engager constructs showed simultaneous hBCMA and CD16a binding ability and could efficiently activate resting primary NK cells and trigger specific lysis of a panel of hBCMA-positive multiple myeloma cell lines. Hence, we report a novel class of uniquely small NK cell engagers with specific binding properties and potent functional profiles.
RESUMO
BACKGROUND: Enhanced levels of a dipeptide, WG-am, have been reported among elite controllers - patients who spontaneously control their HIV-1 infection. This study aimed to evaluate anti-HIV-1 activity and mechanism of action of WG-am. METHODS: Drug sensitivity assays in TZM.bl cells, PBMCs and ACH-2 cells using WT and mutated HIV-1 strainswere performed to evaluate the antiviral mechanism of WG-am. Mass spectrometry-based proteomics and Real-time PCR analysis of reverse transcription steps were performed to unravel the second anti-HIV-1 mechanism of WG-am. RESULTS: The data suggest that WG-am binds to the CD4 binding pocket of HIV-1 gp120 and blocks its binding to the host cell receptors. Additionally, the time course assay showed that WG-am also inhibited HIV-1 at 4-6 hours post-infection, suggesting a second antiviral mechanism. Drug sensitivity assays under acidic wash conditions confirmed the ability of WG-am to internalise into the host cell in an HIV independent manner. Proteomic studies showed a clustering of all samples treated with WG-am independent of the number of doses or presence or absence of HIV-1. Differentially expressed proteins due to the WG-am treatment indicated an effect on HIV-1 reverse transcription, which was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). CONCLUSION: Naturally occurring in HIV-1 elite controllers, WG-am stands out as a new kind of antiviral compound with two independent inhibitory mechanisms of action on HIV-1 replication. WG-am halts HIV-1 entry to the host cell by binding to HIV-1 gp120, thereby blocking the binding of HIV-1 to the host cell. WG-am also exerts a post-entry but pre-integration antiviral effect related to RT-activity.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Dipeptídeos , Proteômica , Infecções por HIV/tratamento farmacológico , Antivirais , Controladores de Elite , Replicação ViralRESUMO
G3BP is the central node within stress-induced protein-RNA interaction networks known as stress granules (SGs). The SG-associated proteins Caprin-1 and USP10 bind mutually exclusively to the NTF2 domain of G3BP1, promoting and inhibiting SG formation, respectively. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1-derived short linear motif (SLiM). Caprin-1 interacts with His-31 and His-62 within a third NTF2-binding site outside those covered by USP10, as confirmed using biochemical and biophysical-binding assays. Nano-differential scanning fluorimetry revealed reduced thermal stability of G3BP1-NTF2 at acidic pH. This destabilization was counterbalanced significantly better by bound USP10 than Caprin-1. The G3BP1/USP10 complex immunoprecipated from human U2OS cells was more resistant to acidic buffer washes than G3BP1/Caprin-1. Acidification of cellular condensates by approximately 0.5 units relative to the cytosol was detected by ratiometric fluorescence analysis of pHluorin2 fused to G3BP1. Cells expressing a Caprin-1/FGDF chimera with higher G3BP1-binding affinity had reduced Caprin-1 levels and slightly reduced condensate sizes. This unexpected finding may suggest that binding of the USP10-derived SLiM to NTF2 reduces the propensity of G3BP1 to enter condensates.
Assuntos
DNA Helicases , Grânulos de Estresse , Humanos , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Concentração de Íons de Hidrogênio , Ubiquitina TiolesteraseRESUMO
The chemical reactions used to make antibody-DNA conjugates in many immunoassays diminish antigen-binding activity and yield heterogeneous products. Here, we address these issues by developing an antibody-based rolling circle amplification (RCA) strategy using a fusion of φX174 gene A* protein and Z(mab25) (A*-Zmab). The φX174 gene A* protein is an enzyme that can covalently link with DNA, while the Z(mab25) protein moiety can bind to specific species of antibodies. The DNA in an A*-Zmab conjugate was attached to the A* protein at a site chosen to not interfere with protein function, as determined by enzyme-linked immunosorbent assay (ELISA) and gel mobility shift analysis. The novel A*-Zmab-DNA conjugate retained its binding capabilities to a specific class of murine immunoglobulin γ1 (IgG1) but not to rabbit IgG. This indicates the generality of the A*-Zmab-based immuno-RCA assay that can be used in-sandwich ELISA format. Moreover, the enzymatic covalent method dramatically increased the yields of A*-Zmab-DNA conjugates up to 80% after a 15 min reaction. Finally, sensitive detection of human interferon-γ (IFN-γ) was achieved by immuno-RCA using our fusion protein in sandwich ELISA format. This new approach of the use of site-specific enzymatic DNA conjugation to proteins should be applicable to fabrication of novel immunoassays for biosensing.
Assuntos
Anticorpos Monoclonais/análise , Antígenos/análise , Imunoglobulina G/análise , Interferon gama/análise , Proteínas Virais/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Bacteriófagos/química , Técnicas Biossensoriais , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoensaio , Imunoconjugados/química , Imunoglobulina G/química , Interferon gama/imunologia , Camundongos , Coelhos , Proteínas Recombinantes de Fusão/química , Especificidade da EspécieRESUMO
Targeting the signaling pathway of growth differentiation factor 8 (GDF8), also known as myostatin, has been regarded as a promising strategy to increase muscle mass in the elderly and in patients. Accumulating evidence in animal models and clinical trials has indicated that a rational approach is to inhibit a limited number of transforming growth factor ß (TGF-ß) family ligands, including GDF8 and activin A, without affecting other members. Here, we focused on one of the endogenous antagonists against TGF-ß family ligands, follistatin-like 3 (FSTL3), which mainly binds and neutralizes activins, GDF8, and GDF11. Although bivalent human FSTL3 Fc-fusion protein was rapidly cleared from mouse circulation similar to follistatin (FST)-Fc, monovalent FSTL3-Fc (mono-FSTL3-Fc) generated with the knobs-into-holes technology exhibited longer serum half-life. Systemic administration of mono-FSTL3-Fc in mice induced muscle fiber hypertrophy and increased muscle mass in vivo. Our results indicate that the monovalent FSTL3-based therapy overcomes the difficulties of current anti-GDF8 therapies.
RESUMO
Due to the wide scale of inflammatory processes in different types of disease, more sensitive and specific biomarkers are required to improve prevention and treatment. Cluster of differentiation 69 (CD69) is one of the earliest cell surface proteins expressed by activated leukocytes. Here we characterize and optimize potential new imaging probes, Affibody molecules targeting CD69 for imaging of activated immune cells. Analysis of candidates isolated in a previously performed selection from a Z variant E. coli library to the recombinant extracellular domain of human CD69, identified one cross-reactive Z variant with affinity to murine and human CD69. Affinity maturation was performed by randomization of the primary Z variant, followed by selections from the library. The resulting Z variants were evaluated for affinity towards human and murine CD69 and thermal stability. The in vivo biodistribution was assessed by SPECT/CT in rats following conjugation of the Z variants by a DOTA chelator and radiolabeling with Indium-111. A primary Z variant with a Kd of approximately 50 nM affinity to human and murine CD69 was identified. Affinity maturation generated 5 additional Z variants with improved or similar affinity. All clones exhibited suitable stability. Radiolabeling and in vivo biodistribution in rat demonstrated rapid renal clearance for all variants, while the background uptake and washout varied. The variant ZCD69:4 had the highest affinity for human and murine CD69 (34 nM) as well as the lowest in vivo background binding. In summary, we describe the discovery, optimization and evaluation of novel Affibody molecules with affinity for CD69. Affibody molecule ZCD69:4 is suitable for further development for imaging of activated immune cells.
Assuntos
Imunoconjugados/farmacocinética , Lectinas Tipo C/antagonistas & inibidores , Imagem Molecular/métodos , Compostos Radiofarmacêuticos/farmacocinética , Proteínas Recombinantes de Fusão/farmacocinética , Animais , Afinidade de Anticorpos , Antígenos CD , Antígenos de Diferenciação de Linfócitos T , Reações Cruzadas , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/química , Radioisótopos de Índio , Injeções Intravenosas , Masculino , Camundongos , Estabilidade Proteica , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/química , Ratos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Distribuição TecidualRESUMO
The introduction of different methodologies for the construction and screening of complex protein libraries has provided powerful means in protein engineering for the development of molecules with desired traits. A challenge faced in many situations is to adapt a given methodology for efficient and rapid identification of the most interesting variants present in a library. In the present study, the concept of Darwinian selection based on a growth advantage for clones having the desired trait has been investigated. Using a beta-lactamase-based PCA (protein fragment complementation assay), affinity maturation of a TNFalpha (tumour necrosis factor alpha)-binding Affibody molecule with an initial 2 nM affinity for the target has been performed. Initial characterization of the PCA system, based on the affinity-driven reconstitution of beta-lactamase activity in the periplasm of cells harbouring a library member showing affinity for a co-expressed target protein, showed that the system was responsive to promoter induction level, interaction affinity and applied selection pressure. Using combinatorial protein engineering principles, a 107 library of second-generation Affibody molecules was constructed and subjected to selection of improved variants by library growth in liquid culture. The results show that, after a pre-selection step on semi-solid medium to eliminate non-binding variants, present in the majority, two rounds of selection in liquid culture resulted in an enrichment for binders showing up to 8-fold higher affinity for the TNFalpha target than the ancestral variant. Biosensor analyses showed that the major factor for the improved affinity could be attributed to reduced off-rate constants.
Assuntos
Seleção Genética , Fator de Necrose Tumoral alfa/metabolismo , Técnicas Biossensoriais , Técnicas de Química CombinatóriaRESUMO
The pneumococcal serine rich repeat protein (PsrP) is displayed on the surface of Streptococcus pneumoniae with a suggested role in colonization in the human upper respiratory tract. Full-length PsrP is a 4000 residue-long multi-domain protein comprising a positively charged functional binding region (BR) domain for interaction with keratin and extracellular DNA during pneumococcal adhesion and biofilm formation, respectively. The previously determined crystal structure of the BR domain revealed a flat compressed barrel comprising two sides with an extended ß-sheet on one side, and another ß-sheet that is distorted by loops and ß-turns on the other side. Crystallographic B-factors indicated a relatively high mobility of loop regions that were hypothesized to be important for binding. Furthermore, the crystal structure revealed an inter-molecular ß-sheet formed between edge strands of two symmetry-related molecules, which could promote bacterial aggregation during biofilm formation. Here we report the near complete 15N/13C/1H backbone resonance assignment of the BR domain of PsrP, revealing a secondary structure profile that is almost identical to the X-ray structure. Dynamic 15N-T1, T2 and NOE data suggest a monomeric and rigid structure of BR with disordered residues only at the N- and C-termini. The presented peak assignment will allow us to identify BR residues that are crucial for ligand binding.