RESUMEN
Bispecific T-cell engagers (BiTEs) bring together tumour cells and cytotoxic T cells by binding to specific cell-surface tumour antigens and T-cell receptors, and have been clinically successful for the treatment of B-cell malignancies. Here we show that a BiTE-sialidase fusion protein enhances the susceptibility of solid tumours to BiTE-mediated cytolysis of tumour cells via targeted desialylation-that is, the removal of terminal sialic acid residues on glycans-at the BiTE-induced T-cell-tumour-cell interface. In xenograft and syngeneic mouse models of leukaemia and of melanoma and breast cancer, and compared with the parental BiTE molecules, targeted desialylation via the BiTE-sialidase fusion proteins enhanced the formation of immunological synapses, T-cell activation and T-cell-mediated tumour-cell cytolysis in the presence of the target antigen. The targeted desialylation of tumour cells may enhance the potency of therapies relying on T-cell engagers.
Asunto(s)
Neuraminidasa , Animales , Neuraminidasa/metabolismo , Humanos , Ratones , Línea Celular Tumoral , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Linfocitos T/inmunología , Femenino , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Activación de Linfocitos , Ácido N-Acetilneuramínico/metabolismo , Ácido N-Acetilneuramínico/química , Ensayos Antitumor por Modelo de Xenoinjerto , Linfocitos T Citotóxicos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Triple-negative breast cancer (TNBC) is a highly diverse group of malignant neoplasms which tend to have poor outcomes, and the development of new targets and strategies to treat these cancers is sorely needed. Antibody-drug conjugate (ADC) therapy has been shown to be a promising targeted therapy for treating many cancers, but has only rarely been tried in patients with TNBC. A major reason the efficacy of ADC therapy in the setting of TNBC has not been more fully investigated is the lack of appropriate target molecules. In this work we were able to identify an effective TNBC target for use in immunotherapy. We were guided by our previous observation that in some breast cancer patients the protein tropomyosin receptor kinase B cell surface protein (TrkB) had become immunogenic, suggesting that it was somehow sufficiently chemically different enough (presumably by mutation) to escaped immune tolerance. We postulated that this difference might well offer a means for selective targeting by antibodies. We engineered site-specific ADCs using a dual variable domain (DVD) format which combines anti-TrkB antibody with the h38C2 catalytic antibody. This format enables rapid, one-step, and homogeneous conjugation of ß-lactam-derivatized drugs. Following conjugation to ß-lactam-derivatized monomethyl auristatin F, the TrkB-targeting DVD-ADCs showed potency against multiple breast cancer cell lines, including TNBC cell lines. In addition, our isolation of antibody that specifically recognized the breast cancer-associated mutant form of TrkB, but not the wild type TrkB, indicates the possibility of further refining the selectivity of anti-TrkB DVD-ADCs, which should enhance their therapeutic index. These results confirmed our supposition that TrkB is a potential target for immunotherapy for TNBC, as well as for other cancers with mutated cell surface proteins.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Inmunoconjugados/uso terapéutico , Inmunoterapia/métodos , Glicoproteínas de Membrana/inmunología , Oligopéptidos/uso terapéutico , Receptor trkB/inmunología , Neoplasias de la Mama Triple Negativas/terapia , Línea Celular Tumoral , Femenino , Humanos , Proteínas de la Membrana , Terapia Molecular Dirigida , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Host cell surface glycans play critical roles in influenza virus A (IVA) infection ranging from modulation of IVA attachment to membrane fusion and host tropism. Approaches for quick and sensitive profile of viral avidity toward a specific type of host cell glycan can contribute to the understanding of tropism switching among different IVA strains. Here, we developed a method based on chemoenzymatic glycan engineering to investigate the possible involvement of α1-2-fucosides in IVA infections. Using a truncated human fucosyltransferase 1 (hFUT1), we created α1-2-fucosides in situ on host cells to assess their influence on the host cell binding to IVA hemagglutinin and the susceptibility of host cells toward IVA-induced killing. We discovered that the newly created α1-2-fucosides on host cells enhanced the infection of several human pandemic IVA subtypes either directly or indirectly. These findings suggest that glycan epitopes other than sialic acid should also be considered for assessing the human pandemic risk of this viral pathogen.
Asunto(s)
Fucosa/química , Fucosiltransferasas/química , Glicósidos/metabolismo , Virus de la Influenza A/patogenicidad , Gripe Humana/etiología , Técnicas Microbiológicas/métodos , Animales , Células CHO , Ingeniería Celular , Supervivencia Celular , Cricetulus , Perros , Epítopos/química , Epítopos/metabolismo , Glicósidos/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Interacciones Huésped-Patógeno , Humanos , Virus de la Influenza A/metabolismo , Virus de la Influenza A/fisiología , Células de Riñón Canino Madin Darby , Tropismo Viral , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
Herein we present a concept in cancer where an immune response is detrimental rather than helpful. In the cancer setting, the immune system is generally considered to be helpful in curtailing the initiation and progression of tumors. In this work we show that a patient's immune response to their tumor can, in fact, either enhance or inhibit tumor cell growth. Two closely related autoantibodies to the growth factor receptor TrkB were isolated from cancer patients' B cells. Although highly similar in sequence, one antibody was an agonist while the other was an antagonist. The agonist antibody was shown to increase breast cancer cell growth both in vitro and in vivo, whereas the antagonist antibody inhibited growth. From a mechanistic point of view, we showed that binding of the agonist antibody to the TrkB receptor was functional in that it initiated downstream signaling identical to its natural growth factor ligand, brain-derived neurotrophic factor (BDNF). Our study shows that individual autoantibodies may play a role in cancer patients.
Asunto(s)
Autoanticuerpos/inmunología , Autoantígenos/inmunología , Neoplasias de la Mama/patología , Glicoproteínas de Membrana/inmunología , Metástasis de la Neoplasia/inmunología , Receptor trkB/inmunología , Animales , Autoanticuerpos/sangre , Autoanticuerpos/aislamiento & purificación , Autoanticuerpos/metabolismo , Autoantígenos/sangre , Autoantígenos/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Factor Neurotrófico Derivado del Encéfalo/inmunología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Neoplasias de la Mama/sangre , Neoplasias de la Mama/inmunología , Línea Celular Tumoral , Movimiento Celular/inmunología , Proliferación Celular , Femenino , Humanos , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/sangre , Ratones , Receptor trkB/agonistas , Receptor trkB/antagonistas & inhibidores , Receptor trkB/sangre , Transducción de Señal/inmunologíaRESUMEN
In the original version of this article the author name Xiaolei Chen was published incorrectly. This has been corrected to Xiao Lei Chen.
RESUMEN
Chemoenzymatic modification of cell-surface glycan structures has emerged as a complementary approach to metabolic oligosaccharide engineering. Here, we identify Pasteurella multocida α2-3-sialyltransferase M144D mutant, Photobacterium damsela α2-6-sialyltransferase, and Helicobacter mustelae α1-2-fucosyltransferase, as efficient tools for live-cell glycan modification. Combining these enzymes with Helicobacter pylori α1-3-fucosyltransferase, we develop a host-cell-based assay to probe glycan-mediated influenza A virus (IAV) infection including wild-type and mutant strains of H1N1 and H3N2 subtypes. At high NeuAcα2-6-Gal levels, the IAV-induced host-cell death is positively correlated with haemagglutinin (HA) binding affinity to NeuAcα2-6-Gal. Remarkably, an increment of host-cell-surface sialyl Lewis X (sLeX) exacerbates the killing by several wild-type IAV strains and a previously engineered mutant HK68-MTA. Structural alignment of HAs from HK68 and HK68-MTA suggests formation of a putative hydrogen bond between Trp222 of HA-HK68-MTA and the C-4 hydroxyl group of the α1-3-linked fucose of sLeX, which may account for the enhanced host cell killing of that mutant.
Asunto(s)
Proteínas Bacterianas/metabolismo , Glicosiltransferasas/metabolismo , Hemaglutininas/inmunología , Interacciones Huésped-Patógeno/inmunología , Gripe Humana/inmunología , Oligosacáridos/metabolismo , Animales , Proteínas Bacterianas/genética , Bioensayo/métodos , Células CHO , Cricetulus , Perros , Glicosiltransferasas/genética , Voluntarios Sanos , Helicobacter mustelae/genética , Helicobacter mustelae/metabolismo , Hemaglutininas/metabolismo , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/inmunología , Gripe Humana/virología , Microscopía Intravital/métodos , Luciferasas de la Bacteria/genética , Luciferasas de la Bacteria/metabolismo , Pulmón/patología , Células de Riñón Canino Madin Darby , Ingeniería Metabólica/métodos , Microscopía Fluorescente/métodos , Imagen Molecular/métodos , Oligosacáridos/inmunología , Pasteurella multocida/genética , Pasteurella multocida/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Antígeno Sialil Lewis X , Coloración y Etiquetado/métodosRESUMEN
The diverse physiological roles of the neurotrophin family have long prompted exploration of their potential as therapeutic agents for nerve injury and neurodegenerative diseases. To date, clinical trials of one family member, brain-derived neurotrophic factor (BDNF), have disappointingly failed to meet desired endpoints. Contributing to these failures is the fact that BDNF is pharmaceutically a nonideal biologic drug candidate. It is a highly charged, yet is a net hydrophobic molecule with a low molecular weight that confers a short t1/2 in man. To circumvent these shortcomings of BDNF as a drug candidate, we have employed a function-based cellular screening assay to select activating antibodies of the BDNF receptor TrkB from a combinatorial human short-chain variable fragment antibody library. We report here the successful selection of several potent TrkB agonist antibodies and detailed biochemical and physiological characterization of one such antibody, ZEB85. By using a human TrkB reporter cell line and BDNF-responsive GABAergic neurons derived from human ES cells, we demonstrate that ZEB85 is a full agonist of TrkB, comparable in potency to BDNF toward human neurons in activation of TrkB phosphorylation, canonical signal transduction, and mRNA transcriptional regulation.
Asunto(s)
Comunicación Autocrina , Neuronas GABAérgicas/metabolismo , Biblioteca de Genes , Glicoproteínas de Membrana/agonistas , Receptor trkB/agonistas , Transducción de Señal/efectos de los fármacos , Anticuerpos de Cadena Única , Transcripción Genética/efectos de los fármacos , Línea Celular , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Fosforilación/efectos de los fármacos , Receptor trkB/genética , Receptor trkB/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/farmacologíaRESUMEN
Influenza A virus hemagglutinin (HA) initiates viral entry by engaging host receptor sialylated glycans via its receptor-binding site (RBS). The amino acid sequence of the RBS naturally varies across avian and human influenza virus subtypes and is also evolvable. However, functional sequence diversity in the RBS has not been fully explored. Here, we performed a large-scale mutational analysis of the RBS of A/WSN/33 (H1N1) and A/Hong Kong/1/1968 (H3N2) HAs. Many replication-competent mutants not yet observed in nature were identified, including some that could escape from an RBS-targeted broadly neutralizing antibody. This functional sequence diversity is made possible by pervasive epistasis in the RBS 220-loop and can be buffered by avidity in viral receptor binding. Overall, our study reveals that the HA RBS can accommodate a much greater range of sequence diversity than previously thought, which has significant implications for the complex evolutionary interrelationships between receptor specificity and immune escape.
Asunto(s)
Sitios de Unión/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Evasión Inmune , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Receptores Virales/genética , Secuencia de Aminoácidos , Anticuerpos Antivirales/inmunología , Variación Antigénica/genética , Variación Antigénica/inmunología , Antígenos Virales/genética , Secuencia de Bases , Sitios de Unión/inmunología , Cristalografía por Rayos X/instrumentación , Evolución Molecular , Células HEK293 , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Gripe Humana/virología , Mutación , Conformación Proteica , Receptores Virales/inmunología , Análisis de Secuencia , Internalización del VirusRESUMEN
The relatively recent discovery and characterization of human broadly neutralizing antibodies (bnAbs) against influenza virus provide valuable insights into antiviral and vaccine development. However, the factors that influence the evolution of high-affinity bnAbs remain elusive. We therefore explore the functional sequence space of bnAb C05, which targets the receptor-binding site (RBS) of influenza haemagglutinin (HA) via a long CDR H3. We combine saturation mutagenesis with yeast display to enrich for C05 variants of CDR H3 that bind to H1 and H3 HAs. The C05 variants evolve up to 20-fold higher affinity but increase specificity to each HA subtype used in the selection. Structural analysis reveals that the fine specificity is strongly influenced by a highly conserved substitution that regulates receptor binding in different subtypes. Overall, this study suggests that subtle natural variations in the HA RBS between subtypes and species may differentially influence the evolution of high-affinity bnAbs.