RESUMO
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 â¼4 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.
RESUMO
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.
Assuntos
Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/imunologia , Animais , Antibacterianos/química , Antibacterianos/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Humanos , Macrófagos/microbiologia , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Estudo de Prova de Conceito , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/metabolismo , Células RAW 264.7 , RatosRESUMO
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.
Assuntos
Proteínas de Bactérias/imunologia , Glicosiltransferases/imunologia , Interações Hospedeiro-Patógeno/imunologia , Staphylococcus aureus Resistente à Meticilina/fisiologia , Infecções Estafilocócicas/imunologia , Staphylococcus epidermidis/fisiologia , Fatores de Virulência/imunologia , Animais , Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/imunologia , Aderência Bacteriana/genética , Aderência Bacteriana/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catepsina G/genética , Catepsina G/imunologia , Catepsina G/metabolismo , Linhagem Celular Tumoral , Parede Celular/enzimologia , Parede Celular/genética , Parede Celular/imunologia , Epitopos/genética , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Camundongos , Sequências Repetitivas de Aminoácidos , Infecções Estafilocócicas/enzimologia , Infecções Estafilocócicas/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
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.
Assuntos
Glicosilfosfatidilinositóis/imunologia , Mastócitos/imunologia , Proteínas de Membrana/genética , Receptores de IgE/imunologia , Anafilaxia/genética , Anafilaxia/imunologia , Animais , Degranulação Celular , Células Cultivadas , Deleção de Genes , Imunoglobulina E/imunologia , Masculino , Mastócitos/citologia , Mastócitos/metabolismo , Proteínas de Membrana/imunologia , Camundongos , Fosforilação , Estabilidade Proteica , Transdução de SinaisRESUMO
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.
Assuntos
Glicosilfosfatidilinositóis/deficiência , Glicosilfosfatidilinositóis/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Antígenos/imunologia , Divisão Celular/genética , Divisão Celular/imunologia , Células Clonais , Concanavalina A/farmacologia , Feminino , Glicosilfosfatidilinositóis/fisiologia , Imunofenotipagem , Integrases/genética , Ativação Linfocitária/genética , Teste de Cultura Mista de Linfócitos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/imunologiaRESUMO
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.