RESUMEN
We report the development of a novel platform to enhance the efficacy and safety of follicular lymphoma (FL) treatment. Since lymphoma is a clonal malignancy of a diversity system, every tumor has a different antibody on its cell surface. Combinatorial autocrine-based selection is used to rapidly identify specific ligands for these B cell receptors on the surface of FL tumor cells. The selected ligands are used in a chimeric antigen receptor T cell (CAR-T) format for redirection of human cytotoxic T lymphocytes. Essentially, the format is the inverse of the usual CAR-T protocol. Instead of being a guide molecule, the antibody itself is the target. Thus, these studies raise the possibility of personalized treatment of lymphomas using a private antibody binding ligand that can be obtained in a few weeks.
Asunto(s)
Linfoma de Células B/terapia , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Comunicación Autocrina , Femenino , Humanos , Ligandos , Linfoma de Células B/inmunología , Linfoma de Células B/metabolismo , Ratones Endogámicos NOD , Ratones SCID , Fragmentos de Péptidos/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Design of drug with prolonged therapeutic action is one of the rapid developing fields of modern medical science and required implementation of different methods of protein chemistry and molecular biology. There are several therapeutic proteins needing increasing of their stability, pharmacokinetic, and pharmacodynamics parameters. To make long-live DNA-encoded drug PEGylation was proposed. Alternatively polysialic (colominic) acid, extracted from the cell wall of E. coli, fractionated to the desired size by anion-exchange chromatography and chemically activated to the amine-reactive aldehyde form, may be chemically attached to the polypeptide chain. Conjugates of proteins and polysialic acid generally resemble properties of protein-PEG conjugates, but possess significant negative net charge and are thought to be fully degradable after endocytosis due to the presence of intracellular enzymes, hydrolyzing the polysialic acid. Complete biodegradation of the polysialic acid moiety makes this kind of conjugates preferable for creation of drugs, intended for chronic use. Here, we describe two different protocols of chemical polysialylation. First protocol was employed for the CHO-derived human butyrylcholinesterase with optimized for recovery of specific enzyme activity. Polysialic acid moieties are attached at various lysine residues. Another protocol was developed for high-yield conjugation of human insulin; major conjugation point is the N-terminal residue of the insulin's light chain. These methods may allow to produce polysialylated conjugates of various proteins or polypeptides with reasonable yield and without significant loss of functional activity.
Asunto(s)
Proteínas Recombinantes/metabolismo , Ácidos Siálicos/metabolismo , Animales , Butirilcolinesterasa/metabolismo , Células CHO , Línea Celular , Cricetulus , Escherichia coli/metabolismo , Humanos , Insulina/metabolismo , Lisina/metabolismo , Péptidos/metabolismo , Polisacáridos/metabolismoRESUMEN
The creation of effective bioscavengers as a pretreatment for exposure to nerve agents is a challenging medical objective. We report a recombinant method using chemical polysialylation to generate bioscavengers stable in the bloodstream. Development of a CHO-based expression system using genes encoding human butyrylcholinesterase and a proline-rich peptide under elongation factor promoter control resulted in self-assembling, active enzyme multimers. Polysialylation gives bioscavengers with enhanced pharmacokinetics which protect mice against 4.2 LD(50) of S-(2-(diethylamino)ethyl) O-isobutyl methanephosphonothioate without perturbation of long-term behavior.
Asunto(s)
Butirilcolinesterasa/química , Butirilcolinesterasa/farmacocinética , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacocinética , Secuencia de Aminoácidos , Animales , Butirilcolinesterasa/administración & dosificación , Butirilcolinesterasa/genética , Células CHO , Sustancias para la Guerra Química/toxicidad , Cricetinae , Cricetulus , Humanos , Dosificación Letal Mediana , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Fármacos Neuroprotectores/administración & dosificación , Compuestos Organotiofosforados/antagonistas & inhibidores , Compuestos Organotiofosforados/toxicidad , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacocinética , Ácidos Siálicos/químicaRESUMEN
Multiple sclerosis (MS) is a severe inflammatory and neurodegenerative disease with an autoimmune background. Despite the variety of therapeutics available against MS, the development of novel approaches to its treatment is of high importance in modern pharmaceutics. In this study, experimental autoimmune encephalomyelitis (EAE) in Dark Agouti rats has been treated with immunodominant peptides of the myelin basic protein (MBP) encapsulated in mannosylated small unilamellar vesicles. The results show that liposome-encapsulated MBP(46-62) is the most effective in reducing maximal disease score during the first attack, while MBP(124-139) and MBP(147-170) can completely prevent the development of the exacerbation stage. Both mannosylation of liposomes and encapsulation of peptides are critical for the therapeutic effect, since neither naked peptides nor nonmannosylated liposomes, loaded or empty, have proved effective. The liposome-mediated synergistic effect of the mixture of 3 MBP peptides significantly suppresses the progression of protracted EAE, with the median cumulative disease score being reduced from 22 to 14 points, compared to the placebo group; prevents the production of circulating autoantibodies; down-regulates the synthesis of Th1 cytokines; and induces the production of brain-derived neurotrophic factor in the central nervous system. Thus, the proposed formulation ameliorates EAE, providing for a less severe first attack and rapid recovery from exacerbation, and offers a promising therapeutic modality in MS treatment.
Asunto(s)
Encefalitis/prevención & control , Hipersensibilidad/prevención & control , Liposomas , Péptidos/uso terapéutico , Animales , Western Blotting , Encefalitis/etiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Hipersensibilidad/complicaciones , Ratones , Ratas , Resonancia por Plasmón de SuperficieRESUMEN
Tremendous efforts to produce an efficient vaccine for HIV infection have been unsuccessful. The ability of HIV to utilize sophisticated mechanisms to escape killing by host immune system rises dramatic problems in the development of antiviral therapeutics. The HIV infection proceeds by interaction of coat viral glycoprotein gp120 trimer with CD4(+) receptor of the lymphocyte. Thus this surface antigen may be regarded as a favorable target for immunotherapy. In the present study, we have developed three different strategies to produce gp120-specific response in autoimmune prone mice (SJL strain) as potential tools for production "catalytic vaccine". Therefore (i) reactive immunization by peptidylphosphonate, structural part of the coat glycoprotein, (ii) immunization by engineered fused epitopes of gp120 and encephalogenic peptide, a part of myelin basic protein, and (iii) combined vaccination by DNA and corresponding gp120 fragments incorporated into liposomes were investigated. In the first two cases monoclonal antibodies and their recombinant fragments with amidolytic and gp120-specific proteolytic activities were characterized. In the last case, catalytic antibodies with virus neutralizing activity proved in cell line models were harvested.