Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
MAbs ; 13(1): 1954136, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34313545

RESUMO

Inhibitors of programmed cell death protein 1 (PD-1) and its ligand (PD-L1) have dramatically changed the treatment landscape for patients with cancer. Clinical activity of anti-PD-(L)1 antibodies has resulted in increased median overall survival and durable responses in patients across selected tumor types. To date, 6 PD-1 and PD-L1, here collectively referred to as PD-(L)1, pathway inhibitors are approved by the US Food and Drug Administration for clinical use. The availability of multiple anti-PD-(L)1 antibodies provides treatment and dosing regimen choice for patients with cancer. Here, we describe the nonclinical characterization of dostarlimab (TSR-042), a humanized anti-PD-1 antibody, which binds with high affinity to human PD-1 and effectively inhibits its interaction with its ligands, PD-L1 and PD-L2. Dostarlimab enhanced effector T-cell functions, including cytokine production, in vitro. Since dostarlimab does not bind mouse PD-1, its single-agent antitumor activity was evaluated using humanized mouse models. In this model system, dostarlimab demonstrated antitumor activity as assessed by tumor growth inhibition, which was associated with increased infiltration of immune cells. Single-dose and 4-week repeat-dose toxicology studies in cynomolgus monkeys indicated that dostarlimab was well tolerated. In a clinical setting, based on data from the GARNET trial, dostarlimab (Jemperli) was approved for the treatment of adult patients with mismatch repair-deficient recurrent or advanced endometrial cancer that had progressed on or following prior treatment with a platinum-containing regimen. Taken together, these data demonstrate that dostarlimab is a potent anti-PD-1 receptor antagonist, with properties that support its continued clinical investigation in patients with cancer.


Assuntos
Anticorpos Monoclonais Humanizados , Antineoplásicos Imunológicos , Neoplasias Experimentais , Receptor de Morte Celular Programada 1 , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/farmacologia , Células CHO , Cricetulus , Humanos , Células Jurkat , Macaca fascicularis , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
J Biol Chem ; 289(48): 33557-67, 2014 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-25320089

RESUMO

During somatic hypermutation (SHM), deamination of cytidine by activation-induced cytidine deaminase and subsequent DNA repair generates mutations within immunoglobulin V-regions. Nucleotide insertions and deletions (indels) have recently been shown to be critical for the evolution of antibody binding. Affinity maturation of 53 antibodies using in vitro SHM in a non-B cell context was compared with mutation patterns observed for SHM in vivo. The origin and frequency of indels seen during in vitro maturation were similar to that in vivo. Indels are localized to CDRs, and secondary mutations within insertions further optimize antigen binding. Structural determination of an antibody matured in vitro and comparison with human-derived antibodies containing insertions reveal conserved patterns of antibody maturation. These findings indicate that activation-induced cytidine deaminase acting on V-region sequences is sufficient to initiate authentic formation of indels in vitro and in vivo and that point mutations, indel formation, and clonal selection form a robust tripartite system for antibody evolution.


Assuntos
Regiões Determinantes de Complementaridade/genética , Mutação INDEL , Hipermutação Somática de Imunoglobulina , Regiões Determinantes de Complementaridade/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Feminino , Humanos , Masculino
3.
Methods ; 65(1): 44-56, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23792919

RESUMO

Recent advances are described for the isolation and affinity maturation of antibodies that couple in vitro somatic hypermutation (SHM) with mammalian cell display, replicating key aspects of the adaptive immune system. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID). AID-directed SHM in vitro in non-B cells, combined with mammalian display of a library of human antibodies, initially naïve to SHM, can be used to isolate and affinity mature antibodies via iterative cycles of fluorescence-activated cell sorting (FACS) under increasingly stringent sort conditions. SHM observed in vitro closely resembles SHM observed in human antibodies in vivo in both mutation type and positioning in the antibody variable region. In addition, existing antibodies originating from mouse immunization, in vivo based libraries, or alternative display technologies such as phage can also be affinity matured in a similar manner. The display system has been developed to enable simultaneous high-level cell surface expression and secretion of the same protein through alternate splicing, where the displayed protein phenotype remains linked to genotype, allowing soluble secreted antibody to be simultaneously characterized in biophysical and cell-based functional assays. This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs.


Assuntos
Anticorpos Monoclonais/genética , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Antígenos/imunologia , Sequência de Bases , Separação Celular , Primers do DNA/genética , Evolução Molecular Direcionada , Descoberta de Drogas , Citometria de Fluxo , Biblioteca Gênica , Células HEK293 , Humanos , Dados de Sequência Molecular , Ligação Proteica , Engenharia de Proteínas
4.
J Biol Chem ; 288(27): 19861-9, 2013 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-23689374

RESUMO

A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.


Assuntos
Processamento Alternativo , Anticorpos Monoclonais/biossíntese , Afinidade de Anticorpos/genética , Evolução Molecular Direcionada , Expressão Gênica , Imunoglobulina G/biossíntese , Anticorpos Monoclonais/genética , Células HEK293 , Humanos , Imunoglobulina G/genética , Precursores de RNA/biossíntese , Precursores de RNA/genética
5.
J Biol Chem ; 288(11): 7688-7696, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23355464

RESUMO

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hßNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hßNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods.


Assuntos
Anticorpos/química , Regiões Determinantes de Complementaridade/metabolismo , Mutação , Animais , Anticorpos Monoclonais/química , Antígenos/química , Sequência de Bases , Ligação Competitiva , Separação Celular , Códon , Citocinas/metabolismo , Citometria de Fluxo , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Engenharia de Proteínas/métodos , Transdução de Sinais
6.
PLoS One ; 7(11): e49458, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23166676

RESUMO

A method has been developed for the rapid generation of high-affinity humanized antibodies from immunized animals without the need to make conventional hybridomas. Rearranged IgH D(J) regions were amplified from the spleen and lymph tissue of mice immunized with the human complement protein C5, fused with a limited repertoire of human germline heavy chain V-genes to form intact humanized heavy chains, and paired with a human light chain library. Completed heavy and light chains were assembled for mammalian cell surface display and transfected into HEK 293 cells co-expressing activation-induced cytidine deaminase (AID). Numerous clones were isolated by fluorescence-activated cell sorting, and affinity maturation, initiated by AID, resulted in the rapid evolution of high affinity, functional antibodies. This approach enables the efficient sampling of an immune repertoire and the direct selection and maturation of high-affinity, humanized IgGs.


Assuntos
Anticorpos Monoclonais Humanizados/biossíntese , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Proteínas Recombinantes/biossíntese , Hipermutação Somática de Imunoglobulina/imunologia , Animais , Complemento C5/imunologia , Citidina Desaminase/metabolismo , Descoberta de Drogas/métodos , Citometria de Fluxo , Células HEK293 , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Tecido Linfoide/imunologia , Camundongos , Hipermutação Somática de Imunoglobulina/genética , Baço/imunologia
7.
Proc Natl Acad Sci U S A ; 108(51): 20455-60, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22158898

RESUMO

A novel approach has been developed for the isolation and maturation of human antibodies that replicates key features of the adaptive immune system by coupling in vitro somatic hypermutation (SHM) with mammalian cell display. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID), and can be replicated in non-B cells through expression of recombinant AID. A library of human antibodies, based on germline V-gene segments with recombined human regions was used to isolate low-affinity antibodies to human ß nerve growth factor (hßNGF). These antibodies, initially naïve to SHM, were subjected to AID-directed SHM in vitro and selected using the same mammalian cell display system, as illustrated by the maturation of one of the antibodies to low pM K(D). This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs.


Assuntos
Anticorpos/química , Membrana Celular/metabolismo , Mutação , Hipermutação Somática de Imunoglobulina , Sequência de Aminoácidos , Linfócitos B/imunologia , Citometria de Fluxo/métodos , Glicosilação , Células HEK293 , Humanos , Imunoglobulina G/química , Imunoglobulina M/química , Cinética , Dados de Sequência Molecular , Fator de Crescimento Neural/química , Homologia de Sequência de Aminoácidos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...