RESUMEN
The central role of myeloid cells in driving autoimmune diseases and cancer has raised interest in manipulating their function or depleting them for therapeutic benefits. To achieve this, antibodies are used to antagonize differentiation, survival and polarization signals or to kill target cells, for example in the form of antibody-drug conjugates (ADC). The action of ADC in vivo can be hard to predict based on target expression pattern alone. The biology of the targeted receptor as well as its interplay with the ADC can have drastic effects on cell apoptosis versus survival. Here we investigated the efficacy of CD11b or Ly-6C/Ly-6G-specific variable fragments of camelid heavy chain-only antibodies (VHH) conjugated to Pseudomonas exotoxin A to deplete myeloid cells in vitro and in vivo. Our data highlight striking differences in cell killing in vivo, depending on the cell subset and organs targeted, but not antigen expression level or VHH affinity. We observed striking differences in depletion efficiency of monocytes versus granulocytes in mice. Despite similar binding of Ly-6C/Ly-6G-specific VHH immunotoxin to granulocytes and monocytes, granulocytes were significantly more sensitive than monocytes to immunotoxins treatment. Our results illustrate the need of early, thorough in vivo characterization of ADC candidates.
Asunto(s)
Antígenos Ly/inmunología , Epítopos/inmunología , Granulocitos/inmunología , Inmunotoxinas/farmacología , Monocitos/inmunología , Células Mieloides/inmunología , Anticuerpos de Dominio Único/inmunología , ADP Ribosa Transferasas/metabolismo , Animales , Toxinas Bacterianas/metabolismo , Antígeno CD11b/inmunología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Exotoxinas/metabolismo , Femenino , Granulocitos/efectos de los fármacos , Granulocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Especificidad de Órganos , Factores de Virulencia/metabolismo , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Immunotherapy using checkpoint-blocking antibodies against targets such as CTLA-4 and PD-1 can cure melanoma and non-small cell lung cancer in a subset of patients. The presence of CD8 T cells in the tumor correlates with improved survival. We show that immuno-positron emission tomography (immuno-PET) can visualize tumors by detecting infiltrating lymphocytes and, through longitudinal observation of individual animals, distinguish responding tumors from those that do not respond to therapy. We used 89Zr-labeled PEGylated single-domain antibody fragments (VHHs) specific for CD8 to track the presence of intratumoral CD8+ T cells in the immunotherapy-susceptible B16 melanoma model in response to checkpoint blockade. A 89Zr-labeled PEGylated anti-CD8 VHH detected thymus and secondary lymphoid structures as well as intratumoral CD8 T cells. Animals that responded to CTLA-4 therapy showed a homogeneous distribution of the anti-CD8 PET signal throughout the tumor, whereas more heterogeneous infiltration of CD8 T cells correlated with faster tumor growth and worse responses. To support the validity of these observations, we used two different transplantable breast cancer models, yielding results that conformed with predictions based on the antimelanoma response. It may thus be possible to use immuno-PET and monitor antitumor immune responses as a prognostic tool to predict patient responses to checkpoint therapies.
Asunto(s)
Linfocitos T CD8-positivos/fisiología , Antígeno CTLA-4/antagonistas & inhibidores , Neoplasias Mamarias Experimentales/terapia , Animales , Antígeno CTLA-4/fisiología , Femenino , Fragmentos de Inmunoglobulinas/inmunología , Fragmentos de Inmunoglobulinas/uso terapéutico , Inmunoterapia/métodos , Neoplasias Mamarias Experimentales/diagnóstico por imagen , Neoplasias Mamarias Experimentales/inmunología , Ratones , Trasplante de Neoplasias , Tomografía de Emisión de Positrones/métodos , Resultado del TratamientoRESUMEN
mAbs specific for surface proteins on APCs can serve as Ag-delivery vehicles that enhance immunogenicity. The practical use of such constructs is limited by the challenge of expressing and modifying full-sized mAbs. We generated single-domain Ab fragments (VHHs) specific for class II MHC (MHCII), CD11b, and CD36. VHH sequences were modified by inclusion of a C-terminal sortase motif to allow site-specific conjugation with various Ag payloads. We tested T cell activation using VHHs that target distinct APC populations; anti-MHCII adducts elicited strong activation of CD4+ T cells, whereas anti-CD11b showed CD8+ T cell activation superior to targeting via MHCII and CD36. Differences in Ag presentation among constructs were unrelated to dendritic cell subtype or routing to acidic compartments. When coupled to antigenic payloads, anti-MHCII VHH primed Ab responses against GFP, ubiquitin, an OVA peptide, and the α-helix of influenza hemagglutinin's stem; the last afforded protection against influenza infection. The versatility of the VHH scaffold and sortase-mediated covalent attachment of Ags suggests their broader application to generate desirable immune responses.
Asunto(s)
Complejo Antígeno-Anticuerpo/metabolismo , Células Dendríticas/fisiología , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Infecciones por Orthomyxoviridae/inmunología , Anticuerpos de Dominio Único/metabolismo , Animales , Presentación de Antígeno , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Camélidos del Nuevo Mundo , Células Cultivadas , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Gripe Humana/prevención & control , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Orthomyxoviridae/prevención & control , Anticuerpos de Dominio Único/inmunologíaRESUMEN
Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na(+)-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27(+) cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330-338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211-223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211-223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA.
Asunto(s)
Artritis Reactiva/inmunología , Proteínas Bacterianas/inmunología , Chlamydia trachomatis/inmunología , Epítopos de Linfocito T/inmunología , Antígeno HLA-B27/inmunología , Imitación Molecular/inmunología , Péptidos/inmunología , Artritis Reactiva/genética , Artritis Reactiva/microbiología , Artritis Reactiva/patología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Chlamydia trachomatis/química , Chlamydia trachomatis/genética , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Antígeno HLA-B27/química , Antígeno HLA-B27/genética , Humanos , Imitación Molecular/genética , Péptidos/química , Péptidos/genética , ProhibitinasRESUMEN
Standard genetic approaches allow the production of protein composites by fusion of polypeptides in head-to-tail fashion. Some applications would benefit from constructions that are genetically impossible, such as the site-specific linkage of proteins via their N or C termini, when a remaining free terminus is required for biological activity. We developed a method for the production of N-to-N and C-to-C dimers, with full retention of the biological activity of both fusion partners and without inflicting chemical damage on the proteins to be joined. We use sortase A to install on the N or C terminus of proteins of interest the requisite modifications to execute a strain-promoted copper-free cycloaddition and show that the ensuing ligation proceeds efficiently. Applied here to protein-protein fusions, the method reported can be extended to connecting proteins with any entity of interest.
Asunto(s)
Carbono/química , Complejos Multiproteicos/química , Nitrógeno/química , Ingeniería de Proteínas/métodos , Aminoaciltransferasas/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Endogámicos BALB C , Modelos Químicos , Estructura Molecular , Complejos Multiproteicos/farmacocinética , Multimerización de ProteínaRESUMEN
We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool.
Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Bacteriófago M13/metabolismo , Proteínas de la Cápside/metabolismo , Técnicas de Visualización de Superficie Celular/métodos , Cisteína Endopeptidasas/metabolismo , Animales , Bacteriófago M13/química , Bacteriófago M13/genética , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Ratones , Ratones Endogámicos C57BL , Propiedades de SuperficieRESUMEN
Chlamydia trachomatis triggers reactive arthritis, a spondyloarthropathy linked to the human major histocompatibility complex molecule HLA-B27, through an unknown mechanism that might involve molecular mimicry between chlamydial and self-derived HLA-B27 ligands. Chlamydia-specific CD8(+) T-cells are found in reactive arthritis patients, but the immunogenic epitopes are unknown. A previous screening of the chlamydial genome for putative HLA-B27 ligands predicted multiple peptides that were recognized in vitro by CD8(+) T-lymphocytes from patients. Here stable transfectants expressing bacterial fusion proteins in human cells were generated to investigate the endogenous processing and presentation by HLA-B27 of two such epitopes through comparative immunoproteomics of HLA-B27-bound peptide repertoires. A predicted T-cell epitope, from the CT610 gene product, was presented by HLA-B27. This is, to our knowledge, the first endogenously processed epitope involved in HLA-B27-restricted responses against C. trachomatis in reactive arthritis. A second predicted epitope, from the CT634 gene product, was not detected. Instead a non-predicted nonamer from the same protein was identified. Both bacterial peptides showed very high homology with human sequences containing the HLA-B27 binding motif. Thus, expression and intracellular processing of chlamydial proteins into human cells allowed us to identify two bacterial HLA-B27 ligands, including the first endogenous T-cell epitope from C. trachomatis involved in spondyloarthropathy. That human proteins contain sequences mimicking chlamydial T-cell epitopes suggests a basis for an autoimmune component of Chlamydia-induced HLA-B27-associated disease.
Asunto(s)
Presentación de Antígeno , Proteínas Bacterianas/inmunología , Epítopos/inmunología , Antígeno HLA-B27/inmunología , Linfocitos T/inmunología , Artritis Reactiva/inmunología , Artritis Reactiva/patología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Chlamydia trachomatis/genética , Chlamydia trachomatis/inmunología , Chlamydia trachomatis/metabolismo , Cromatografía Líquida de Alta Presión , Epítopos/análisis , Epítopos/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteómica/métodos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Linfocitos T/citología , Linfocitos T/metabolismo , TransfecciónRESUMEN
A strategy for the stable expression of proteins, or large protein fragments, from Chlamydia trachomatis into human cells was designed to identify bacterial epitopes endogenously processed and presented by HLA-B27. Fusion protein constructs in which the green fluorescent protein gene was placed at the 5'-end of the bacterial DNA primase gene or some of its fragments were transfected into B*2705-C1R cells. One of these constructs, including residues 90-450 of the bacterial protein, was stably and efficiently expressed. Mass spectrometry-based comparative analysis of HLA-B27-bound peptide pools led to identification of three HLA-B27 ligands differentially presented in the transfectant cells. Sequencing of these peptides confirmed that they were derived from the bacterial DNA primase. One of them, spanning residues 211-221, showed 55% sequence identity with a known self-ligand of HLA-B27 derived from its own molecule. The other two bacterial ligands, P-(112-121) and P-(112-122), were derived from the same region and differed in length by one residue at the C terminus. Both peptides showed >50% identity with multiple human protein sequences that possessed the optimal peptide motifs for HLA-B27 binding. Thus, expression of proteins from arthritogenic bacteria in HLA-B27-positive human cells allows identifying bacterial peptides that are endogenously processed and presented by HLA-B27 and show molecular mimicry with known self-ligands of this molecule and human proteins.
Asunto(s)
Presentación de Antígeno , Antígenos Bacterianos/análisis , Antígenos Bacterianos/química , Chlamydia trachomatis/química , Antígeno HLA-B27/química , Péptidos/análisis , Péptidos/química , Secuencia de Aminoácidos , Línea Celular , Chlamydia trachomatis/enzimología , ADN Primasa/química , ADN Primasa/metabolismo , Humanos , Ligandos , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TransfecciónRESUMEN
Many of the constitutive peptide ligands of HLA-B27, a molecule strongly associated with spondyloarthritis, are proteasome-independent. Stable isotope tagging, mass spectrometry, and epoxomicin-mediated inhibition were used to determine their percentage, structural features, and parental proteins. Of 104 molecular species examined, 29.8% were proteasome-independent, paralleling the level of HLA-B27 re-expression in the presence of epoxomicin after acid stripping. Proteasome-dependent and -independent ligands differed little in peptide motifs, flanking sequences, and cellular localization of the parental proteins. In contrast, whereas the former set arose from proteins whose size and isoelectric point distribution largely reflected those in the human proteome, proteasome-independent ligands, other than a few matching signal sequences, were almost totally derived from small (about 6-16.5 kDa) and basic proteins, which account for only 6.6% of the human proteome. Thus, a non-proteasomal proteolytic pathway with strong preference for small proteins is responsible for a significant fraction of the HLA-B27-bound peptide repertoire.