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1.
J Biol Chem ; 293(25): 9880-9891, 2018 06 22.
Article in English | MEDLINE | ID: mdl-29720399

ABSTRACT

4-1BBL is a member of the tumor necrosis factor (TNF) superfamily and is the ligand for the TNFR superfamily receptor, 4-1BB. 4-1BB plays an immunomodulatory role in T cells and NK cells, and agonists of this receptor have garnered strong attention as potential immunotherapy agents. Broadly speaking, the structural features of TNF superfamily members, their receptors, and ligand-receptor complexes are similar. However, a published crystal structure of human 4-1BBL suggests that it may be unique in this regard, exhibiting a three-bladed propeller-like trimer assembly that is distinctly different from that observed in other family members. This unusual structure also suggests that the human 4-1BB/4-1BBL complex may be structurally unique within the TNF/TNFR superfamily, but to date no structural data have been reported. Here we report the crystal structure of the human 4-1BB/4-1BBL complex at 2.4-Å resolution. In this structure, 4-1BBL does not adopt the unusual trimer assembly previously reported, but instead forms a canonical bell-shaped trimer typical of other TNF superfamily members. The structure of 4-1BB is also largely canonical as is the 4-1BB/4-1BBL complex. Mutational data support the 4-1BBL structure reported here as being biologically relevant, suggesting that the previously reported structure is not. Together, the data presented here offer insight into structure/function relationships in the 4-1BB/4-1BBL system and improve our structural understanding of the TNF/TNFR superfamily more broadly.


Subject(s)
4-1BB Ligand/chemistry , 4-1BB Ligand/metabolism , Tumor Necrosis Factor Receptor Superfamily, Member 9/chemistry , Tumor Necrosis Factor Receptor Superfamily, Member 9/metabolism , Binding Sites , Crystallography, X-Ray , HEK293 Cells , Humans , Ligands , Protein Binding , Protein Conformation , Protein Multimerization
2.
J Control Release ; 234: 104-14, 2016 07 28.
Article in English | MEDLINE | ID: mdl-27212104

ABSTRACT

Receptor clustering is important for signaling among the therapeutically relevant TNFR superfamily of receptors. In nature, this clustering is driven by trimeric ligands often presented in large numbers as cell surface proteins. Molecules capable of driving similar levels of clustering could make good agonists and hold therapeutic value. However, recapitulating such extensive clustering using typical biotherapeutic formats, such as antibodies, is difficult. Consequently, generating effective agonists of TNFR superfamily receptors is challenging. Toward addressing this challenge we have used lipid- and polyion complex-based micelles as platforms for presenting receptor-binding biologics in a multivalent format that facilitates receptor clustering and imparts strong agonist activity. We show that receptor-binding scFvs or small antibody mimetics that have no agonist activity on their own can be transformed into potent agonists through multivalent presentation on a micelle surface and that the activity of already active multivalent agonists can be enhanced. Using this strategy, we generated potent agonists against two different TNFR superfamily receptors and mouse tumor model studies demonstrate that these micellar agonists have therapeutic efficacy in vivo. Due to its ease of implementation and applicability independent of agonist molecular format, we anticipate that this strategy could be useful for developing agonists to a variety of receptors that rely on clustering to signal.


Subject(s)
Antineoplastic Agents/administration & dosage , Maleimides/chemistry , Nanoparticles/chemistry , Phosphatidylethanolamines/chemistry , Polyethylene Glycols/chemistry , Receptors, Tumor Necrosis Factor/agonists , Single-Chain Antibodies/administration & dosage , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Humans , Jurkat Cells , Mice , Micelles , Protein Binding , Single-Chain Antibodies/chemistry , Xenograft Model Antitumor Assays
3.
MAbs ; 8(6): 1118-25, 2016.
Article in English | MEDLINE | ID: mdl-27210548

ABSTRACT

Fusion of proteins to the Fc region of IgG is widely used to express cellular receptors and other extracellular proteins, but cleavage of the fusion partner is sometimes required for downstream applications. Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) is a protease with exquisite specificity for human IgG, and it can also cleave Fc-fusion proteins at a single site in the N-terminal region of the CH2 domain. However, the site of IdeS cleavage results in the disulfide-linked hinge region partitioning with the released protein, complicating downstream usage of the cleaved product. To tailor the Fc fragment for release of partner proteins by IdeS treatment, we investigated the effect of deleting regions of IgG-derived sequence that are upstream of the cleavage site. Elimination of the IgG-derived hinge sequence along with several residues of the CH2 domain had negligible effects on expression and purity of the fusion protein, while retaining efficient processing by IdeS. An optimal Fc fragment comprising residues 235-447 of the human IgG1 heavy chain sufficed for efficient production of fusion proteins and minimized the amount of residual Ig-derived sequence on the cleavage product following IdeS treatment. Pairing of this truncated Fc fragment with IdeS cleavage enables highly specific cleavage of Fc-fusion proteins, thus eliminating the need to engineer extraneous cleavage sequences. This system should be helpful for producing Fc-fusion proteins requiring downstream cleavage, particularly those that are sensitive to internal miscleavage if treated with alternative proteases.


Subject(s)
Bacterial Proteins/chemistry , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin G/chemistry , Proteolysis , Recombinant Fusion Proteins/chemistry , Chromatography, Gel , Chromatography, Liquid , Hinge Exons , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin G/genetics , Mass Spectrometry , Protein Domains , Recombinant Fusion Proteins/genetics , Substrate Specificity
4.
J Pharm Sci ; 102(11): 3920-4, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24105735

ABSTRACT

Glycine-serine (GS) linkers are commonly used in recombinant proteins to connect domains. Here, we report the posttranslational O-glycosylation of a GS linker in a novel fusion protein. The structure of the O-glycan moiety is a xylose-based core substituted with hexose and sulfated hexauronic acid residues. The total level of O-xylosylation was approximately 30% in the material expressed in HEK-293 cell lines. There was an approximate 10-fold reduction in O-xylosylation levels when the material was expressed in Chinese hamster ovary cell lines. Similar O-glycan structures have been reported for human urinary thrombomodulin and represent the initial building block for proteoglycans such as chondroitin sulfate and heparin. The sites of attachment, determined by electron transfer dissociation mass spectrometry, were localized to serine in the linker regions of the recombinant fusion protein. This attachment could be attributed, in part, to the inherent xylosyltransferase motif present in GS linkers. Elimination of the O-glycan moiety was achieved with modified linkers containing only glycine residues. The aggregation and fragmentation behavior of the GGG construct were comparable to the GSG-linked material during thermal stress. The O-xylosylation reported has implications for the manufacturing consistency of recombinant proteins containing GS linkers.


Subject(s)
Glycine/chemistry , Protein Processing, Post-Translational , Recombinant Fusion Proteins/metabolism , Serine/chemistry , Tenascin/metabolism , Xylose/metabolism , Amino Acid Sequence , Animals , CHO Cells , Carbohydrate Sequence , Cricetinae , Cricetulus , Glycine/metabolism , Glycosylation , Humans , Molecular Sequence Data , Protein Engineering , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Serine/metabolism , Tenascin/chemistry , Xylose/chemistry
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