Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
Add more filters










Publication year range
1.
Chem Sci ; 13(11): 3147-3160, 2022 Mar 16.
Article in English | MEDLINE | ID: mdl-35414872

ABSTRACT

The antibody-drug conjugate (ADC) is a well-validated modality for the cell-specific delivery of small molecules with impact expanding rapidly beyond their originally-intended purpose of treating cancer. However, antibody-mediated delivery (AMD) remains inefficient, limiting its applicability to targeting highly potent payloads to cells with high antigen expression. Maximizing the number of payloads delivered per antibody is one key way in which delivery efficiency can be improved, although this has been challenging to carry out; with few exceptions, increasing the drug-to-antibody ratio (DAR) above ∼4 typically destroys the biophysical properties and in vivo efficacy for ADCs. Herein, we describe the development of a novel bioconjugation platform combining cysteine-engineered (THIOMAB) antibodies and recombinant XTEN polypeptides for the unprecedented generation of homogeneous, stable "TXCs" with DAR of up to 18. Across three different bioactive payloads, we demonstrated improved AMD to tumors and Staphylococcus aureus bacteria for high-DAR TXCs relative to conventional low-DAR ADCs.

2.
mBio ; 12(3): e0020221, 2021 06 29.
Article in English | MEDLINE | ID: mdl-34061593

ABSTRACT

Pseudomonas aeruginosa causes life-threatening infections that are associated with antibiotic failure. Previously, we identified the antibiotic G2637, an analog of arylomycin, targeting bacterial type I signal peptidase, which has moderate potency against P. aeruginosa. We hypothesized that an antibody-antibiotic conjugate (AAC) could increase its activity by colocalizing P. aeruginosa bacteria with high local concentrations of G2637 antibiotic in the intracellular environment of phagocytes. Using a novel technology of screening for hybridomas recognizing intact bacteria, we identified monoclonal antibody 26F8, which binds to lipopolysaccharide O antigen on the surface of P. aeruginosa bacteria. This antibody was engineered to contain 6 cysteines and was conjugated to the G2637 antibiotic via a lysosomal cathepsin-cleavable linker, yielding a drug-to-antibody ratio of approximately 6. The resulting AAC delivered a high intracellular concentration of free G2637 upon phagocytosis of AAC-bound P. aeruginosa by macrophages, and potently cleared viable P. aeruginosa bacteria intracellularly. The molar concentration of AAC-associated G2637 antibiotic that resulted in elimination of bacteria inside macrophages was approximately 2 orders of magnitude lower than the concentration of free G2637 required to eliminate extracellular bacteria. This study demonstrates that an anti-P. aeruginosa AAC can locally concentrate antibiotic and kill P. aeruginosa inside phagocytes, providing additional therapeutic options for antibiotics that are moderately active or have an unfavorable pharmacokinetics or toxicity profile. IMPORTANCE Antibiotic treatment of life-threatening P. aeruginosa infections is associated with low clinical success, despite the availability of antibiotics that are active in standard microbiological in vitro assays, affirming the need for new therapeutic approaches. Antibiotics often fail in the preclinical stage due to insufficient efficacy against P. aeruginosa. One potential strategy is to enhance the local concentration of antibiotics with limited inherent anti-P. aeruginosa activity. This study presents proof of concept for an antibody-antibiotic conjugate, which releases a high local antibiotic concentration inside macrophages upon phagocytosis, resulting in potent intracellular killing of phagocytosed P. aeruginosa bacteria. This approach may provide new therapeutic options for antibiotics that are dose limited.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antibodies, Monoclonal/pharmacology , Macrophages/drug effects , Macrophages/immunology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/immunology , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Drug Delivery Systems/methods , Humans , Macrophages/microbiology , Mice , Microbial Viability/drug effects , Phagocytosis/drug effects , Proof of Concept Study , Pseudomonas Infections/drug therapy , Pseudomonas Infections/immunology , Pseudomonas aeruginosa/metabolism , RAW 264.7 Cells , Rats
4.
MAbs ; 10(7): 979-991, 2018 10.
Article in English | MEDLINE | ID: mdl-30102105

ABSTRACT

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a growing health threat worldwide. Efforts to identify novel antibodies that target S. aureus cell surface antigens are a promising direction in the development of antibiotics that can halt MRSA infection. We biochemically and structurally characterized three patient-derived MRSA-targeting antibodies that bind to wall teichoic acid (WTA), which is a polyanionic surface glycopolymer. In S. aureus, WTA exists in both α- and ß-forms, based on the stereochemistry of attachment of a N-acetylglucosamine residue to the repeating phosphoribitol sugar unit. We identified a panel of antibodies cloned from human patients that specifically recognize the α or ß form of WTA, and can bind with high affinity to pathogenic wild-type strains of S. aureus bacteria. To investigate how the ß-WTA specific antibodies interact with their target epitope, we determined the X-ray crystal structures of the three ß-WTA specific antibodies, 4462, 4497, and 6078 (Protein Data Bank IDs 6DWI, 6DWA, and 6DW2, respectively), bound to a synthetic WTA epitope. These structures reveal that all three of these antibodies, while utilizing distinct antibody complementarity-determining region sequences and conformations to interact with ß-WTA, fulfill two recognition principles: binding to the ß-GlcNAc pyranose core and triangulation of WTA phosphate residues with polar contacts. These studies reveal the molecular basis for targeting a unique S. aureus cell surface epitope and highlight the power of human patient-based antibody discovery techniques for finding novel pathogen-targeting therapeutics.


Subject(s)
Anti-Bacterial Agents/chemistry , Antibodies, Bacterial/chemistry , Antigen-Antibody Complex/metabolism , Cell Wall/chemistry , Complementarity Determining Regions/chemistry , Methicillin-Resistant Staphylococcus aureus/physiology , Staphylococcal Infections/immunology , Teichoic Acids/chemistry , Anti-Bacterial Agents/blood , Antibodies, Bacterial/blood , Antigen-Antibody Complex/genetics , Cell Wall/metabolism , Crystallography, X-Ray/methods , Humans , Immunity, Humoral , Protein Binding , Protein Conformation , Staphylococcal Infections/therapy , Structure-Activity Relationship , Teichoic Acids/metabolism , Transgenes/genetics
5.
mBio ; 7(5)2016 09 06.
Article in English | MEDLINE | ID: mdl-27601569

ABSTRACT

UNLABELLED: The type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo IMPORTANCE: The type I signal peptidase of Staphylococcus aureus (SpsB) enables the secretion of numerous proteins by cleavage of the signal peptide. We synthesized an SpsB inhibitor with potent activity against various clinical S. aureus strains. The predominant S. aureus strain USA300 develops resistance to this inhibitor by mutations in a novel transcriptional repressor (cro/cI), causing overexpression of a putative ABC transporter. This mechanism promotes the cleavage and secretion of various proteins independently of SpsB and compensates for the requirement of SpsB for viability in vitro However, bacteria overexpressing the ABC transporter and lacking SpsB secrete reduced levels of virulence-associated proteins and are unable to infect mice. This study describes a bacterial resistance mechanism that provides novel insights into the biology of bacterial secretion.


Subject(s)
ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Staphylococcus aureus/enzymology , Staphylococcus aureus/genetics , Animals , Anti-Bacterial Agents/pharmacology , Disease Models, Animal , Drug Resistance, Bacterial , Gene Expression , Membrane Proteins/antagonists & inhibitors , Mice , Microbial Sensitivity Tests , Mutation , Selection, Genetic , Staphylococcal Infections/microbiology , Staphylococcus aureus/drug effects , Staphylococcus aureus/metabolism , Virulence
6.
J Infect Dis ; 209(10): 1542-50, 2014 May 15.
Article in English | MEDLINE | ID: mdl-24286981

ABSTRACT

Little is known about the expression of methicillin-resistant Staphylococcus aureus (MRSA) genes during infection conditions. Here, we described the transcriptome of the clinical MRSA strain USA300 derived from human cutaneous abscesses, and compared it with USA300 bacteria derived from infected kidneys in a mouse model. Remarkable similarity between the transcriptomes allowed us to identify genes encoding multiple proteases and toxins, and iron- and peptide-transporter molecules, which are upregulated in both infections and are likely important for establishment of infection. We also showed that disruption of the global transcriptional regulators agr and sae prevents in vivo upregulation of many toxins and proteases, protecting mice from lethal infection dose, and hinting at the role of these transcriptional regulators in the pathology of MRSA infection.


Subject(s)
Gene Expression Regulation, Bacterial/physiology , Methicillin-Resistant Staphylococcus aureus/metabolism , Transcriptome , Abscess/microbiology , Animals , Humans , Methicillin-Resistant Staphylococcus aureus/classification , Methicillin-Resistant Staphylococcus aureus/genetics , Methicillin-Resistant Staphylococcus aureus/pathogenicity , Mice , Protein Array Analysis , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , Skin Diseases, Bacterial/microbiology , Virulence
7.
PLoS Pathog ; 9(10): e1003653, 2013.
Article in English | MEDLINE | ID: mdl-24130480

ABSTRACT

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.


Subject(s)
Bacterial Proteins/immunology , Glycosyltransferases/immunology , Host-Pathogen Interactions/immunology , Methicillin-Resistant Staphylococcus aureus/physiology , Staphylococcal Infections/immunology , Staphylococcus epidermidis/physiology , Virulence Factors/immunology , Animals , Antibodies, Bacterial/genetics , Antibodies, Bacterial/immunology , Bacterial Adhesion/genetics , Bacterial Adhesion/immunology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cathepsin G/genetics , Cathepsin G/immunology , Cathepsin G/metabolism , Cell Line, Tumor , Cell Wall/enzymology , Cell Wall/genetics , Cell Wall/immunology , Epitopes/genetics , Epitopes/immunology , Epitopes/metabolism , Female , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Host-Pathogen Interactions/genetics , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Mice , Repetitive Sequences, Amino Acid , Staphylococcal Infections/enzymology , Staphylococcal Infections/genetics , Virulence Factors/genetics , Virulence Factors/metabolism
8.
Blood ; 118(16): 4377-83, 2011 Oct 20.
Article in English | MEDLINE | ID: mdl-21865342

ABSTRACT

A key event and potential therapeutic target in allergic and asthmatic diseases is signaling by the IgE receptor FcεRI, which depends on its interactions with Src family kinases (SFK). Here we tested the hypothesis that glycosylphosphatidylinositiol-anchored proteins (GPI-AP) are involved in FcεRI signaling, based on previous observations that GPI-AP colocalize with and mediate activation of SFK. We generated mice with a hematopoietic cell-specific GPI-AP deficiency by targeted disruption of the GPI biosynthesis gene PigA. In these mice, IgE-mediated passive cutaneous anaphylaxis was largely abolished. PigA-deficient mast cells cultured from these mice showed impaired degranulation in response to stimulation with IgE and antigen in vitro, despite normal IgE binding and antigen-induced FcεRI aggregation. On stimulation of these cells with IgE and antigen, coprecipitation of the FcεRI α-chain with the γ-chain and ß-chain was markedly reduced. As a result, IgE/antigen-induced FcεRI-Lyn association and γ-chain tyrosine phosphorylation were both impaired in PigA-deficient cells. These data provide genetic evidence for an unanticipated key role of GPI-AP in FcεRI interchain interactions and early FcεRI signaling events, necessary for antigen-induced mast cell degranulation.


Subject(s)
Glycosylphosphatidylinositols/immunology , Mast Cells/immunology , Membrane Proteins/genetics , Receptors, IgE/immunology , Anaphylaxis/genetics , Anaphylaxis/immunology , Animals , Cell Degranulation , Cells, Cultured , Gene Deletion , Immunoglobulin E/immunology , Male , Mast Cells/cytology , Mast Cells/metabolism , Membrane Proteins/immunology , Mice , Phosphorylation , Protein Stability , Signal Transduction
9.
J Immunol ; 173(6): 3810-5, 2004 Sep 15.
Article in English | MEDLINE | ID: mdl-15356128

ABSTRACT

The functions of GPI-anchored proteins in T lymphocyte activation have been controversial. This issue was addressed by studying the responses of T lymphocytes from T lymphocyte-specific GPI anchor-deficient mice to different stimuli that normally allow coligation of TCR and GPI-anchored proteins. Stimulation of GPI anchor-deficient T lymphocytes with ConA induced 2-fold higher proliferative responses than did normal cells. In response to allogeneic stimulation, proliferation of GPI anchor-deficient T lymphocytes was enhanced 2- to 3-fold. The response to ConA of a GPI anchor-deficient anti-OVA T lymphocyte clone generated from these mice was approximately 3-fold higher than that of cells from the same clone in which GPI anchor expression was restored by retroviral transduction. The response of the GPI anchor-deficient cloned anti-OVA T lymphocytes to antigenic stimulation was similar to that of the retrovirally restored cells. These results indicate that coligation with GPI-anchored proteins counteracts the response to TCR stimulation by ConA or alloantigen but not protein Ag.


Subject(s)
Glycosylphosphatidylinositols/deficiency , Glycosylphosphatidylinositols/genetics , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Animals , Antigens/immunology , Cell Division/genetics , Cell Division/immunology , Clone Cells , Concanavalin A/pharmacology , Female , Glycosylphosphatidylinositols/physiology , Immunophenotyping , Integrases/genetics , Lymphocyte Activation/genetics , Lymphocyte Culture Test, Mixed , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Male , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Ovalbumin/immunology
10.
Blood ; 104(9): 2825-31, 2004 Nov 01.
Article in English | MEDLINE | ID: mdl-15238423

ABSTRACT

Signaling by transmembrane immunoglobulin G (IgG)-Fc receptors (FcgammaRs) in response to ligand involves association with membrane microdomains that contain glycosyl phosphatidylinositol (GPI)-anchored proteins. Recent in vitro studies showed enhancement of FcgammaR signaling by forced monoclonal antibody-mediated cocrosslinking with various GPI-anchored proteins. Here, the possibility that GPI-anchored proteins are involved in normal physiologic FcgammaR effector functions in response to a model ligand was studied using myeloid-specific GPI-anchor-deficient mice, generated by Cre-loxP conditional targeting. GPI-anchor-deficient primary myeloid cells exhibited normal FcgammaR expression and binding or endocytosis of IgG-immune complexes (IgG-ICs). Strikingly, after stimulation with IgG-ICs, tumor necrosis factor-alpha release, dendritic cell maturation, and antigen presentation were strongly reduced by GPI-anchor deficiency. Tyrosine phosphorylation of the FcR gamma-chain in response to IgG-IC was impaired in GPI-anchor-deficient cells. Myeloid GPI-anchor deficiency resulted in attenuated in vivo inflammatory processes during IgG-IC-mediated alveolitis. This study provides the first genetic evidence for an essential role of GPI-anchored proteins in physiologic FcgammaR effector functions in vitro and in vivo.


Subject(s)
Glycosylphosphatidylinositols/deficiency , Myeloid Cells/chemistry , Receptors, IgG/physiology , Animals , Antigen Presentation , Antigen-Antibody Complex , Dendritic Cells/cytology , Glycosylphosphatidylinositols/physiology , Immunoglobulin G , Inflammation , Ligands , Mice , Mice, Knockout , Myeloid Cells/physiology , Phosphorylation , Tumor Necrosis Factor-alpha/metabolism , Tyrosine
SELECTION OF CITATIONS
SEARCH DETAIL
...