RESUMO
The affinity of T-cell receptor (TCR) determines the efficacy of TCR-based immunotherapy. By using human leucocyte antigen (HLA)-A*02 transgenic mice, a TCR was generated previously specific for human tumour testis antigen peptide MAGE-A3112-120 (KVAELVHFL) HLA-A*02 complex. We developed an approach to humanize the murine TCR by replacing the mouse framework with sequences of folding optimized human TCR variable domains for retaining binding affinity. The resultant humanized TCR exhibited higher affinity and conferred better anti-tumour activity than its parent murine MAGE-A3 TCR (SRm1). In addition, the affinity of humanized TCR was enhanced further to achieve improved T-cell activation. Our studies demonstrated that the human TCR variable domain frameworks could provide support for complementarity-determining regions from a murine TCR, and retain the original binding activity. It could be used as a generic approach of TCR humanization.
Assuntos
Antígeno HLA-A2/imunologia , Imunoterapia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Sobrevivência Celular , Células Cultivadas , Antígeno HLA-A2/genética , Antígeno HLA-A2/isolamento & purificação , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/isolamento & purificaçãoRESUMO
The inhibitory checkpoint molecule programmed death (PD)-1 plays a vital role in maintaining immune homeostasis upon binding to its ligands, PD-L1 and PD-L2. Several recent studies have demonstrated that soluble PD-1 (sPD-1) can block the interaction between membrane PD-1 and PD-L1 to enhance the antitumor capability of T cells. However, the affinity of natural sPD-1 binding to PD-L1 is too low to permit therapeutic applications. Here, a PD-1 variant with approximately 3000-fold and 70-fold affinity increase to bind PD-L1 and PD-L2, respectively, was generated through directed molecular evolution and phage display technology. Structural analysis showed that mutations at amino acid positions 124 and 132 of PD-1 played major roles in enhancing the affinity of PD-1 binding to its ligands. The high-affinity PD-1 mutant could compete with the binding of antibodies specific to PD-L1 or PD-L2 on cancer cells or dendritic cells, and it could enhance the proliferation and IFN-γ release of activated lymphocytes. These features potentially qualify the high-affinity PD-1 variant as a unique candidate for the development of a new class of PD-1 immune-checkpoint blockade therapeutics.
Assuntos
Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Aminoácidos/metabolismo , Proliferação de Células/fisiologia , Técnicas de Visualização da Superfície Celular/métodos , Células Dendríticas/metabolismo , Humanos , Interferon gama/metabolismo , Ligantes , Ativação Linfocitária/fisiologia , Ligação Proteica/fisiologia , Linfócitos T/metabolismoRESUMO
Hepatitis C virus (HCV) readily establishes chronic infection, which is characterized by failure of virus-specific CD8+ T cells. HCV uses epitope mutation and T-cell exhaustion to escape from the host immune response. Previously, we engineered high-affinity T-cell receptors (HATs) targeting human immunodeficiency virus escape mutants. In this study, the affinity of a T-cell receptor specific for the HLA-A2-restricted HCV immunodominant epitope NS3 1406-1415 (KLVALGINAV) was improved from a KD of 6.6 µM to 40 pM. These HATs could also target HCV NS3 naturally occurring variants, including an escape variant vrt1 (KLVVLGINAV), with high affinities. The HATs can be used as high-affinity targeting molecules at the centre of the immune synapse for the HLA-restricted NS3 antigen. By fusing the HAT with a T-cell activation molecule, an anti-CD3 single-chain variable fragment, we constructed a molecule called high-affinity T-cell activation core (HATac), which can redirect functional CTLs possessing any specificity to recognize and kill cells presenting HCV NS3 antigens. This capability was verified with T2 cells loaded with prototype or variant peptides and HepG2 cells expressing the truncated NS3 prototype or variant proteins. The results indicate that HATac targeting the HLA-restricted NS3 antigen may provide a useful tool for circumventing immune escape mutants and T-cell exhaustion caused by HCV infection.
Assuntos
Epitopos de Linfócito T/imunologia , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Epitopos Imunodominantes/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas não Estruturais Virais/imunologia , Complexo CD3/imunologia , Epitopos de Linfócito T/genética , Variação Genética , Células HEK293 , Antígeno HLA-A2/imunologia , Células Hep G2 , Antígenos de Hepatite/genética , Antígenos de Hepatite/imunologia , Humanos , Epitopos Imunodominantes/genética , Receptores de Antígenos de Linfócitos T/genética , Anticorpos de Cadeia Única/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas não Estruturais Virais/genéticaRESUMO
BACKGROUND: Plasmid-based overexpression of genes has been the principal strategy for metabolic engineering. However, for biotechnological applications, plasmid-based expression systems are not suitable because of genetic instability, and the requirement for constant selective pressure to ensure plasmid maintenance. RESULTS: To overcome these drawbacks, we constructed an Escherichia coli lycopene production strain that does not carry a plasmid or an antibiotic marker. This was achieved using triclosan-induced chromosomal evolution, a high gene copy expression system. The engineered strain demonstrated high genetic stability in the absence of the selective agent during fermentation. The replacement of native appY promoter with a T5 promoter, and the deletion of the iclR gene in E. coli CBW 12241 further improved lycopene production. The resulting strain, E. coli CBW 12241(ΔiclR, PT5-appY), produced lycopene at 33.43 mg per gram of dry cell weight. CONCLUSIONS: A lycopene hyper-producer E. coli strain that does not carry a plasmid or antibiotic marker was constructed using triclosan-induced chromosomal evolution. The methods detailed in this study can be used to engineer E. coli to produce other metabolites.
Assuntos
Carotenoides/biossíntese , Cromossomos/metabolismo , Escherichia coli/metabolismo , Evolução Molecular , Biomassa , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Licopeno , Plasmídeos/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Transativadores/genética , Triclosan/farmacologiaRESUMO
The activated T cells can be suppressed by programed death-1 (PD-1) axis through low affinity interaction between PD-1 and PD-ligand 1 (PD-L1) in solution or on antigen presenting cells. In clinic, the concentration of soluble PD-L1 in peripheral blood negatively correlates with cancer prognosis. However, there is little information about the relation between the affinity of PD-1/PD-L1 interaction and the suppressive capacity of PD-1 axis. In this study, we analyzed inhibitory roles of high affinity soluble human PD-L1 (hPD-L1) variants, which were generated with directed molecular evolution. Resultant two clones L3C7-hPD-L1 and L3B3-hPD-L1 showed over 20 folds greater affinity than that of native hPD-L1. We found that L3B3-hPD-L1 and L3C7-hPD-L1 could compete with an anti-PD-1 antibody (EH12.1) for binding to hPD-1. More importantly, although native soluble hPD-L1 can induce suppressive effects on activated T cells, we found L3B3-hPD-L1 and L3C7-hPD-L1 attenuated the strength of PD-1 axis for suppressing the proliferation and interferon γ (IFN-γ) secretion of PBMC. In conclusion, our data provide direct evidence in which immune checkpoint receptor-ligand interactive strength can alter the the suppressive function, in particular, the suppressive capacity of PD-1 axis could be decreased with enhanced affinity of soluble PD-L1 and PD-1 interaction. Our study might provide a new direction for manipulating immune checkpoints.