ABSTRACT
The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.
Subject(s)
Apoptosis , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/metabolism , Neoplasms/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/biosynthesis , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Cell Line, Tumor , Down-Regulation/genetics , Humans , Neoplasm Proteins/genetics , Neoplasms/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Ubiquitin-Protein Ligases/geneticsABSTRACT
Linker instability and impaired tumor targeting can affect the tolerability and efficacy of antibody-drug conjugates (ADCs). To improve these ADC characteristics, we recently described the use of a metal-organic linker, [ethylenediamineplatinum(II)]2+, herein called Lx Initial therapy studies in xenograft-bearing mice revealed that trastuzumab-Lx-auristatin F (AF) outperformed its maleimide benchmark trastuzumab-mal-AF and the Food and Drug Administration-approved ado-trastuzumab emtansine, both containing conventional linkers. In this study, we aimed to characterize Lx-based ADCs for in vivo stability and tumor targeting using 195mPt and 89Zr. Methods: The γ-emitter 195mPt was used to produce the radiolabeled Lx [195mPt]Lx89Zr-Desferrioxamine (89Zr-DFO) was conjugated to trastuzumab either via [195mPt]Lx (to histidine residues) or conventionally (to lysine residues) in order to monitor the biodistribution of antibody, payload, and linker separately. Linker stability was determined by evaluating the following ADCs for biodistribution in NCI-N87 xenograft-bearing nude mice 72 h after injection: trastuzumab-[195mPt]Lx-DFO-89Zr, trastuzumab-[195mPt]Lx-AF, and 89Zr-DFO-(Lys)trastuzumab (control), all having drug-to-antibody ratios (DARs) of 2.2-2.5. To assess the influence of DAR on biodistribution, 89Zr-DFO-(Lys)trastuzumab-Lx-AF with an AF-to-antibody ratio of 0, 2.6, or 5.2 was evaluated 96 h after injection. Results: Similar biodistributions were observed for trastuzumab-[195mPt]Lx-DFO-89Zr, trastuzumab-[195mPt]Lx-AF, and 89Zr-DFO-(Lys)trastuzumab irrespective of the isotope used for biodistribution assessment. The fact that Lx follows the antibody biodistribution indicates that the payload-Lx bond is stable in vivo. Uptake of the 3 conjugates, as percentage injected dose (%ID) per gram of tissue, was about 30 %ID/g in tumor tissue but less than 10 %ID/g in most healthy tissues. Trastuzumab-[195mPt]Lx-AF (DAR 2.2) showed a tendency toward faster blood clearance and an elevated liver uptake, which increased significantly to 28.1 ± 4.2 %ID/g at a higher DAR of 5.2, as revealed from the biodistribution and PET imaging studies. Conclusion: As shown by 195mPt/89Zr labeling, ADCs containing the Lx linker are stable in vivo. In the case of trastuzumab-Lx-AF (DARs 2.2 and 2.6), an unimpaired biodistribution was demonstrated.
Subject(s)
Immunoconjugates/chemistry , Isotopes/chemistry , Platinum/chemistry , Zirconium/chemistry , Animals , Deferoxamine/chemistry , Immunoconjugates/pharmacokinetics , Isotope Labeling , Mice , Tissue DistributionABSTRACT
Greater control is desirable in the stochastic conjugation technology used to synthesize antibody-drug conjugates (ADC). We have shown recently that a fluorescent dye can be stably conjugated to a mAb using a bifunctional platinum(II) linker. Here, we describe the general applicability of this novel linker technology for the preparation of stable and efficacious ADCs. The ethylenediamine platinum(II) moiety, herein called Lx, was coordinated to Desferal (DFO) or auristatin F (AF) to provide storable "semifinal" products, which were directly conjugated to unmodified mAbs. Conjugation resulted in ADCs with unimpaired mAb-binding characteristics, DAR in the range of 2.5 to 2.7 and approximately 85% payload bound to the Fc region, presumably to histidine residues. To evaluate the in vivo stability of Lx and its effect on pharmacokinetics and tumor targeting of an ADC, Lx-DFO was conjugated to the HER2 mAb trastuzumab, followed by radiolabeling with 89Zr. Trastuzumab-Lx-DFO-89Zr was stable in vivo and exhibited pharmacokinetic and tumor-targeting properties similar to parental trastuzumab. In a xenograft mouse model of gastric cancer (NCI-N87) or an ado-trastuzumab emtansine-resistant breast cancer (JIMT-1), a single dose of trastuzumab-Lx-AF outperformed its maleimide benchmark trastuzumab-Mal-AF and FDA-approved ado-trastuzumab emtansine. Overall, our findings show the potential of the Lx technology as a robust conjugation platform for the preparation of anticancer ADCs. Cancer Res; 77(2); 257-67. ©2016 AACR.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Immunoconjugates/pharmacology , Trastuzumab/pharmacology , Aminobenzoates , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Deferoxamine , Drug Design , Humans , Immunoconjugates/chemistry , Mice , Oligopeptides , Platinum Compounds , Radioactive Tracers , Stomach Neoplasms/pathology , Trastuzumab/chemistry , Xenograft Model Antitumor Assays , ZirconiumABSTRACT
Celiac disease (CD) is caused by intolerance to gluten. Oral supplementation with enzymes like Aspergillus niger propyl-endoprotease (AN-PEP), which can hydrolyse gluten, has been proposed to prevent the harmful effects of ingestion of gluten. The influence of meal composition on AN-PEP activity was investigated using an in vitro model that simulates stomach-like conditions. AN-PEP optimal dosage was 20 proline protease units (PPU)/g gluten. The addition of a carbonated drink strongly enhanced AN-PEP activity because of its acidifying effect. While fat did not affect gluten degradation by AN-PEP, the presence of food proteins slowed down gluten detoxification. Moreover, raw gluten was degraded more efficiently by AN-PEP than baked gluten. We conclude that the meal composition influences the amount of AN-PEP needed for gluten elimination. Therefore, AN-PEP should not be used to replace a gluten free diet, but rather to support digestion of occasional and/or inadvertent gluten consumption.
Subject(s)
Aspergillus niger/enzymology , Glutens/metabolism , Serine Endopeptidases/metabolism , Celiac Disease/therapy , Diet, Gluten-Free , Dietary Fats/metabolism , Dietary Proteins/metabolism , DigestionABSTRACT
Celiac disease is a T cell-mediated disease induced by dietary gluten, a component of which is gliadin. 95% of individuals with celiac disease carry the HLA (human leukocyte antigen)-DQ2 locus. Here we determined the T-cell receptor (TCR) usage and fine specificity of patient-derived T-cell clones specific for two epitopes from wheat gliadin, DQ2.5-glia-α1a and DQ2.5-glia-α2. We determined the ternary structures of four distinct biased TCRs specific for those epitopes. All three TCRs specific for DQ2.5-glia-α2 docked centrally above HLA-DQ2, which together with mutagenesis and affinity measurements provided a basis for the biased TCR usage. A non-germline encoded arginine residue within the CDR3ß loop acted as the lynchpin within this common docking footprint. Although the TCRs specific for DQ2.5-glia-α1a and DQ2.5-glia-α2 docked similarly, their interactions with the respective gliadin determinants differed markedly, thereby providing a basis for epitope specificity.
Subject(s)
Celiac Disease/immunology , Epitopes, T-Lymphocyte/chemistry , Gliadin/chemistry , HLA-DQ Antigens/chemistry , Receptors, Antigen, T-Cell/chemistry , Gliadin/immunology , Humans , Immunogenetic Phenomena , Models, Molecular , Molecular Conformation , TriticumABSTRACT
Celiac disease is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins, including the α-gliadins. It has been shown that α-gliadins harbor several major epitopes involved in the disease pathogenesis. A major step towards elimination of gluten toxicity for celiac disease patients would thus be the elimination of such epitopes from α-gliadins. We have analyzed over 3,000 expressed α-gliadin sequences from 11 bread wheat cultivars to determine whether they encode for peptides potentially involved in celiac disease. All identified epitope variants were synthesized as peptides and tested for binding to the disease-associated HLA-DQ2 and HLA-DQ8 molecules and for recognition by patient-derived α-gliadin specific T cell clones. Several specific naturally occurring amino acid substitutions were identified for each of the α-gliadin derived peptides involved in celiac disease that eliminate the antigenic properties of the epitope variants. Finally, we provide proof of principle at the peptide level that through the systematic introduction of such naturally occurring variations α-gliadins genes can be generated that no longer encode antigenic peptides. This forms a crucial step in the development of strategies to modify gluten genes in wheat so that it becomes safe for celiac disease patients. It also provides the information to design and introduce safe gluten genes in other cereals, which would exhibit improved quality while remaining safe for consumption by celiac disease patients.
Subject(s)
Celiac Disease/metabolism , Gliadin/chemistry , Peptides/chemistry , Bread , Cell Proliferation , Epitopes/chemistry , Expressed Sequence Tags , Genetic Variation , HLA-DQ Antigens/metabolism , Humans , Lymphocytes/cytology , Phylogeny , Protein Structure, Tertiary , TriticumABSTRACT
B*2704 is strongly associated to ankylosing spondylitis in Asian populations. It differs from the main HLA-B27 allotype, B*2705, in three amino acid changes. We analyzed the influence of tapasin, TAP, and immunoproteasome induction on maturation, surface expression, and T cell allorecognition of B*2704 and compared some of these features with B*2705 and B*2706, allotypes not associated to disease. In the tapasin-deficient .220 cell line, this chaperone significantly influenced the extent of folding of B*2704 and B*2705, but not their egress from the endoplasmic reticulum. In contrast, B*2706 showed faster folding and no accumulation in the endoplasmic reticulum in the absence of tapasin. Surface expression of B*2704 was more tapasin dependent than B*2705. However, expression of free H chain decreased in the presence of this chaperone for B*2705 but not B*2704, suggesting that more suboptimal ligands were loaded on B*2705 in the absence of tapasin. Despite its influence on surface expression, tapasin had little effect on allorecognition of B*2704. Both surface expression and T cell recognition of B*2704 were critically dependent on TAP, as established with TAP-deficient and TAP-proficient T2 cells. Both immunoproteasome and surface levels of B*2704 were induced by IFN-gamma, but this had little effect on allorecognition. Thus, except for the differential effects of tapasin on surface expression, the tapasin, TAP, and immunoproteasome dependency of B*2704 for maturation, surface expression, and T cell recognition are similar to B*2705, indicating that basic immunological features are shared by the two major HLA-B27 allotypes associated to ankylosing spondylitis in human populations.
Subject(s)
ATP-Binding Cassette Transporters/physiology , Antigen Presentation/immunology , HLA-B Antigens/physiology , Membrane Transport Proteins/physiology , Proteasome Endopeptidase Complex/physiology , Spondylitis, Ankylosing/immunology , Alleles , Cell Line , Cell Membrane/immunology , Cell Membrane/metabolism , HLA-B Antigens/biosynthesis , HLA-B Antigens/metabolism , HLA-B27 Antigen , Humans , Protein Folding , Spondylitis, Ankylosing/geneticsABSTRACT
HLA-B*2707 is associated with ankylosing spondylitis in most populations. Like the non-associated allotypes B*2706 and B*2709, it lacks Asp116 and shows preference for peptides with nonpolar C-terminal residues. The relationships between the peptide specificity of B*2707 and those of the disease-associated B*2705 and the non-associated subtypes were analyzed by determining the overlap between the corresponding peptide repertoires, the sequence of shared and differential ligands, and by comparing allospecific T cell epitopes with peptide sharing. The B*2707-bound repertoire was as different from that of B*2705 as from those of B*2706, B*2709, or the two latter subtypes from each other. Differences between B*2707 and B*2705 were based on their C-terminal residue specificity and a subtle modulation at other positions. Differential usage of secondary anchor residues explained the disparity between the B*2707-, B*2706-, and B*2709-bound repertoires. Similar differences in residue usage were found between B*2707 and both B*2704 and B*2706, as expected from the high peptide overlap between the two latter subtypes. T cell cross-reaction paralleled peptide sharing, suggesting that many shared ligands conserve their alloantigenic features on distinct subtypes. Our results indicate that association of HLA-B27 subtypes with ankylosing spondylitis does not correlate with higher peptide sharing among disease-associated subtypes or with obvious peptide motifs.
Subject(s)
Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/metabolism , HLA-B Antigens/metabolism , HLA-B27 Antigen/metabolism , Peptides/metabolism , Spondylarthritis/immunology , Amino Acid Motifs , Cell Line , HLA-B Antigens/immunology , HLA-B27 Antigen/immunology , Humans , Peptides/immunology , Protein Binding , Spondylarthritis/metabolism , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolismABSTRACT
The peptide specificity of HLA-B*1403, an allotype associated with ankylosing spondylitis (Lopez-Larrea, C., Mijiyawa, M., Gonzalez, S., Fernandez-Morera, J. L., Blanco-Gelaz, M. A., Martinez-Borra, J., and Lopez-Vazquez, A. (2002) Arthritis Rheum. 46, 2968-2971) was compared with those of the non-associated B*1402 and the prototypic disease-associated B*2705 allotypes. Although differing by a single residue (L156R), B*1402 and B*1403 shared only 32-35% of their peptide repertoires. Subtype-related differences observed in multiple peptide positions, including P3 and P7, were largely explained by a direct effect of the L156R change on peptide specificity. The HLA-B14 subtypes shared only approximately 3% of their peptide repertoires with B*2705. This was due to distinct residue usage at most positions, as revealed by statistical comparison of B*1402, B*1403, and B*2705-bound nonamers. Nevertheless, shared ligands between B*2705 and B*1403 were formally identified, although ligands common to B*2705 and B*1403, but absent from B*1402, were not found. Alloreactive T-cells were used as a tool to analyze epitope sharing among B*1402, B*1403, and B*2705. The percentage of cross-reactive T-cell clones closely paralleled peptide overlap, suggesting that shared ligands tend to maintain their antigenic features when bound to the different allotypes. Our results indicate that B*1403 and B*2705 can present common peptides. However, both the disparity of their peptide repertoires and the lack of binding features shared by these two allotypes, but not B*1402, argue against, although do not exclude, a mechanism of spondyloarthritis mediated by specific ligands of B*2705 and B*1403.
Subject(s)
Epitopes, T-Lymphocyte/chemistry , HLA-B Antigens/physiology , HLA-B27 Antigen/physiology , Spondylitis, Ankylosing/genetics , Spondylitis, Ankylosing/metabolism , Amino Acid Sequence , Antibodies, Monoclonal/chemistry , Antigen Presentation , Cell Line , DNA/metabolism , Epitopes/chemistry , Flow Cytometry , HLA-B Antigens/chemistry , HLA-B14 Antigen , HLA-B27 Antigen/chemistry , Humans , Ligands , Mass Spectrometry , Molecular Sequence Data , Peptides/chemistry , Protein Binding , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , T-Lymphocytes, Cytotoxic/metabolism , TransfectionABSTRACT
HLA-B*2702, B*2704, and B*2705 are strongly associated with spondyloarthritis, whereas B*2706 is not. Subtypes differ among each other by a few amino acid changes and bind overlapping peptide repertoires. In this study we asked whether differential subtype association with disease is related to differentially bound peptides or to altered antigenicity of shared ligands. Alloreactive CTL raised against B*2704 were analyzed for cross-reaction with B*2705, B*2702, B*2706, and mutants mimicking subtype changes. These CTL are directed against many alloantigen-bound peptides and can be used to analyze the antigenicity of HLA-B27 ligands on different subtypes. Cross-reaction of anti-B*2704 CTL with B*2705 and B*2702 correlated with overlap of their peptidic anchor motifs, suggesting that many shared ligands have similar antigenic features on these three subtypes. Moreover, the percent of anti-B*2704 CTL cross-reacting with B*2706 was only slightly lower than the overlap between the corresponding peptide repertoires, suggesting that most shared ligands have similar antigenic features on these two subtypes. Cross-reaction with B*2705 or mutants mimicking changes between B*2704 and B*2705 was donor-dependent. In contrast, cross-reaction with B*2702 or B*2706 was less variable among individuals. Conservation of antigenic properties among subtypes has implications for allorecognition, as it suggests that shared peptides may determine cross-reaction across exposed amino acid differences in the MHC molecules and that the antigenic distinctness of closely related allotypes may differ among donors. Our results also suggest that differential association of HLA-B27 subtypes with spondyloarthritis is more likely related to differentially bound peptides than to altered antigenicity of shared ligands.
Subject(s)
Conserved Sequence , Epitopes, T-Lymphocyte/immunology , HLA-B27 Antigen/immunology , Peptide Fragments/immunology , Peptide Fragments/metabolism , Spondylarthritis/immunology , Amino Acid Substitution/genetics , Amino Acid Substitution/immunology , Cell Line , Cell Line, Transformed , Clone Cells , Conserved Sequence/genetics , Cytotoxicity Tests, Immunologic , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/metabolism , HLA-B Antigens/genetics , HLA-B Antigens/immunology , HLA-B Antigens/metabolism , HLA-B27 Antigen/genetics , HLA-B27 Antigen/metabolism , Humans , Ligands , Molecular Mimicry/genetics , Molecular Mimicry/immunology , Mutagenesis, Site-Directed , Peptide Fragments/genetics , Polymorphism, Genetic/immunology , Spondylarthritis/genetics , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolismABSTRACT
HLA-B*2704 is strongly associated with ankylosing spondylitis. B*2706, which differs from B*2704 by two amino acid changes, is not associated with this disease. A systematic comparison of the B*2704- and B*2706-bound peptide repertoires was carried out to elucidate their overlap and differential features and to correlate them with disease susceptibility. Both subtypes shared about 90% of their peptide repertoires, consisting of peptides with Arg(2) and C-terminal aliphatic or Phe residues. B*2706 polymorphism influenced specificity at three anchor positions: it favored basic residues at P3 and POmega-2 and impaired binding of Tyr and Arg at POmega. Thus, the main structural feature of peptides differentially bound to B*2704 was the presence of C-terminal Tyr or Arg, together with a strong preference for aliphatic/aromatic P3 residues. This is the only known feature of B*2704 and B*2706 that correlates to their differential association with spondyloarthropathy. The concomitant presence of basic P3 and POmega-2 residues was observed only among peptides differentially bound to B*2706, suggesting that it impairs binding to B*2704. Similarity between peptide overlap and the degree of cross-reaction with alloreactive T lymphocytes suggested that the majority of shared ligands maintain unaltered antigenic features in the context of both subtypes.