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1.
Front Immunol ; 13: 1032716, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36582233

RESUMEN

The presentation of virus-derived peptides by HLA class I molecules on the surface of an infected cell and the recognition of these HLA-peptide complexes by, and subsequent activation of, CD8+ cytotoxic T cells provides an important mechanism for immune protection against viruses. Recent advances in proteogenomics have allowed researchers to discover a growing number of unique HLA-restricted viral peptides, resulting in a rapidly expanding repertoire of targets for immunotherapeutics (i.e. bispecific antibodies, engineered T-cell receptors (TCRs), chimeric antigen receptor T-cells (CAR-Ts)) to infected tissues. However, genomic variability between viral strains, such as Hepatitis-B virus (HBV), in combination with differences in patient HLA alleles, make it difficult to develop therapeutics against these targets. To address this challenge, we developed a novel proteogenomics approach for generating patient-specific databases that enable the identification of viral peptides based on the viral transcriptomes sequenced from individual patient liver samples. We also utilized DNA sequencing of patient samples to identify HLA genotypes and assist in target selection. Liver samples from 48 HBV infected patients, primarily from Asia, were examined to reconstruct patient-specific HBV genomes, identify regions within the human chromosomes targeted by HBV integrations and obtain a comprehensive view of HBV peptide epitopes using our HLA class-I (HLA-I) immunopeptidomics discovery platform. Two previously reported HLA associated HBV-derived peptides, HLA-A02 binder FLLTRILTI (S194-202) from the large surface antigen and HLA-A11 binder STLPETTVVRR (C141-151) from the capsid protein were validated by our discovery platform, but both were detected at very low frequencies. In addition, we identified and validated, using heavy peptide analogues, novel strain-specific HBV-HLA associated peptides, such as GSLPQEHIVQK (P606-616) and variants. Overall, our novel approach can guide the development of bispecific antibody, TCR-T, or CAR-T based therapeutics for the treatment of HBV-related HCC and inform vaccine development.


Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteogenómica , Humanos , Virus de la Hepatitis B/genética , Carcinoma Hepatocelular/metabolismo , Linfocitos T CD8-positivos , Neoplasias Hepáticas/metabolismo , Péptidos , Genotipo
2.
J Proteome Res ; 17(11): 3932-3940, 2018 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-30277784

RESUMEN

The immune status of tumors critically influences their responsiveness to PD1 blockades and other immune-based therapies. Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) is a clinically validated predictive biomarker of response to checkpoint-inhibitor therapy in a limited number of clinical settings but is poorly predictive in most. With emerging evidence that multiple pathways and immune-checkpoint proteins may coordinately contribute to the adaptive immune resistance, the identification and quantitation of multiple immune markers in tumor tissue could help identify the controlling pathways in a given patient, guide the selection of optimal therapy, and monitor response to treatment. We developed and validated a sensitive and robust immuno-liquid chromatography-parallel reaction monitoring assay to simultaneously quantify the expression levels of six immune markers (CD8A, CD4, LAG3, PD1, PD-L1, and PD-L2) using as little as 1-2 mg of fresh frozen tissue. The lower limit of quantitation ranged from 0.07 ng/mg protein for PD1 to 1.0 ng/mg protein for CD4. The intrabatch accuracy was within -16.6% to 15.0% for all proteins at all concentrations, and the variation ranged from 0.8% to 14.7%, while interbatch accuracy was within -6.3% to 8.6%, and the variation ranged from 1.3% to 12.8%. The validated assay was then applied to quantify all six biomarkers in different tissues and was confirmed to have sufficient sensitivity (0.07-1.00 ng/mg protein) and reproducibility (variation ranged from 4.3 to 12.0%). In an analysis of 26 cervical tumors, CD8A and CD4 were detected in all tumors, followed by PD-L1 in 85%, LAG-3 in 65%, PD1 in 50%, and PD-L2 in 35%. The strongest correlations were observed between CD8A and CD4 ( r = 0.88) and CD8A and LAG-3 ( r = 0.86). PD1 was not significantly correlated with any of the other proteins tested. This method can be applied to survey the immune signatures across tumor types and tailored to incorporate additional markers as needed.


Asunto(s)
Biomarcadores de Tumor/genética , Cromatografía de Afinidad/normas , Cromatografía Liquida/normas , Péptidos/análisis , Espectrometría de Masas en Tándem/normas , Neoplasias del Cuello Uterino/diagnóstico , Secuencia de Aminoácidos , Antígenos CD/genética , Antígenos CD/inmunología , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Biomarcadores de Tumor/inmunología , Antígenos CD4/genética , Antígenos CD4/inmunología , Antígenos CD8/genética , Antígenos CD8/inmunología , Cromatografía de Afinidad/métodos , Cromatografía Liquida/métodos , Criopreservación/métodos , Femenino , Expresión Génica , Humanos , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Proteína 2 Ligando de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Espectrometría de Masas en Tándem/métodos , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/patología , Proteína del Gen 3 de Activación de Linfocitos
3.
Proteomics ; 16(14): 2019-27, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27214824

RESUMEN

Pharmacologic blockade of the myostatin (Mstn)/activin receptor pathway is being pursued as a potential therapy for several muscle wasting disorders. The functional benefits of blocking this pathway are under investigation, in particular given the findings that greater muscle hypertrophy results from Mstn deficiency arising from genetic ablation compared to post-developmental Mstn blockade. Using high-resolution MS coupled with SILAC mouse technology, we quantitated the relative proteomic changes in gastrocnemius muscle from Mstn knockout (Mstn(-/-) ) and mice treated for 2-weeks with REGN1033, an anti-Mstn antibody. Relative to wild-type animals, Mstn(-/-) mice had a two-fold greater muscle mass and a >1.5-fold change in expression of 12.0% of 1137 quantified muscle proteins. In contrast, mice treated with REGN1033 had minimal changes in muscle proteome (0.7% of 1510 proteins >1.5-fold change, similar to biological difference 0.5% of 1310) even though the treatment induced significant 20% muscle mass increase. Functional annotation of the altered proteins in Mstn(-/-) mice corroborates the mutiple physiological changes including slow-to-fast fiber type switch. Thus, the proteome-wide protein expression differs between Mstn(-/-) mice and mice subjected to specific Mstn blockade post-developmentally, providing molecular-level insights to inform mechanistic hypotheses to explain the observed functional differences.


Asunto(s)
Hipertrofia/genética , Músculo Esquelético/metabolismo , Enfermedades Musculares/genética , Miostatina/genética , Proteoma/genética , Animales , Anticuerpos Monoclonales/farmacología , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ontología de Genes , Humanos , Hipertrofia/metabolismo , Hipertrofia/patología , Marcaje Isotópico , Masculino , Ratones , Ratones Noqueados , Anotación de Secuencia Molecular , Fibras Musculares de Contracción Rápida/efectos de los fármacos , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Rápida/patología , Fibras Musculares de Contracción Lenta/efectos de los fármacos , Fibras Musculares de Contracción Lenta/metabolismo , Fibras Musculares de Contracción Lenta/patología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Miostatina/antagonistas & inhibidores , Miostatina/deficiencia , Tamaño de los Órganos , Proteoma/metabolismo
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