RESUMO
Peptide-based cancer vaccines are widely investigated in the clinic but exhibit modest immunogenicity. One approach that has been explored to enhance peptide vaccine potency is covalent conjugation of antigens with cell-penetrating peptides (CPPs), linear cationic and amphiphilic peptide sequences designed to promote intracellular delivery of associated cargos. Antigen-CPPs have been reported to exhibit enhanced immunogenicity compared to free peptides, but their mechanisms of action in vivo are poorly understood. We tested eight previously described CPPs conjugated to antigens from multiple syngeneic murine tumor models and found that linkage to CPPs enhanced peptide vaccine potency in vivo by as much as 25-fold. Linkage of antigens to CPPs did not impact dendritic cell activation but did promote uptake of linked antigens by dendritic cells both in vitro and in vivo. However, T cell priming in vivo required Batf3-dependent dendritic cells, suggesting that antigens delivered by CPP peptides were predominantly presented via the process of cross-presentation and not through CPP-mediated cytosolic delivery of peptide to the classical MHC class I antigen processing pathway. Unexpectedly, we observed that many CPPs significantly enhanced antigen accumulation in draining lymph nodes. This effect was associated with the ability of CPPs to bind to lymph-trafficking lipoproteins and protection of CPP-antigens from proteolytic degradation in serum. These two effects resulted in prolonged presentation of CPP-peptides in draining lymph nodes, leading to robust T cell priming and expansion. Thus, CPPs can act through multiple unappreciated mechanisms to enhance T cell priming that can be exploited for cancer vaccines with enhanced potency.
Assuntos
Vacinas Anticâncer , Peptídeos Penetradores de Células , Imunogenicidade da Vacina , Linfonodos , Animais , Apresentação de Antígeno , Antígenos , Vacinas Anticâncer/imunologia , Peptídeos Penetradores de Células/farmacologia , Apresentação Cruzada , Células Dendríticas/imunologia , Linfonodos/imunologia , Camundongos , Linfócitos T/imunologia , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Phosphorodiamidate morpholino oligonucleotides (PMOs) make up a promising class of therapeutics for genetic disease. PMOs designed for "exon skipping" must be internalized into cells, reach the nucleus, and act on pre-mRNA to mediate their effects. One tactic for improving PMO delivery and exon skipping is to covalently conjugate PMOs to cell-penetrating peptides (CPPs). Here, we report the synthesis of PMOs conjugated to CPP chimeras, constructed by combining multiple CPPs into one sequence. The chimeric CPPs synergistically improve PMO activity up to 70-fold compared to that of the PMO alone and beyond the expected effects of each component peptide. By investigating the design space of CPP chimeras, we demonstrate that all components must be covalently attached, that the order of the two sequences matters, and that peptide identity can tune activity. We identified one chimera (pVEC-Bpep) to investigate in more detail and found that it engages mechanisms of endocytosis different from those of its parent peptides. We also examined the extent to which the beneficial effect comes from improved cellular uptake as opposed to the downstream steps required for exon skipping. Given the complexity of intracellular delivery, we anticipate this work will lead researchers to consider combining molecules with different physicochemical properties to aid in the delivery of biologic cargoes.
Assuntos
Peptídeos Penetradores de Células/farmacologia , Portadores de Fármacos/farmacologia , Morfolinos/administração & dosagem , Oligonucleotídeos Antissenso/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/genética , Sinergismo Farmacológico , Éxons/genética , Terapia Genética/métodos , Células HeLa , Humanos , Microscopia Intravital , Microscopia Confocal , Estudo de Prova de Conceito , Proteínas Recombinantes de Fusão/genéticaRESUMO
Exon-skipping antisense oligonucleotides are effective treatments for genetic diseases, yet exon-skipping activity requires that these macromolecules reach the nucleus. While cell-penetrating peptides can improve delivery, proteolytic instability often limits efficacy. It is hypothesized that the bicyclization of arginine-rich peptides would improve their stability and their ability to deliver oligonucleotides into the nucleus. Two methods were introduced for the synthesis of arginine-rich bicyclic peptides using cysteine perfluoroarylation chemistry. Then, the bicyclic peptides were covalently linked to a phosphorodiamidate morpholino oligonucleotide (PMO) and assayed for exon skipping activity. The perfluoroaryl cyclic and bicyclic peptides improved PMO activity roughly 14-fold over the unconjugated PMO. The bicyclic peptides exhibited increased proteolytic stability relative to the monocycle, demonstrating that perfluoroaryl bicyclic peptides are potent and stable delivery agents.
Assuntos
Compostos Bicíclicos com Pontes/química , Peptídeos Penetradores de Células/química , Sistemas de Liberação de Medicamentos , Fluorocarbonos/química , Oligonucleotídeos Antissenso/química , Peptídeos Penetradores de Células/isolamento & purificação , Portadores de Fármacos/química , Células HeLa , Humanos , Estrutura MolecularRESUMO
Determining whether a protein regulates its net electrostatic charge during electron transfer (ET) will deepen our mechanistic understanding of how polypeptides tune rates and free energies of ET (e.g., by affecting reorganization energy, and/or redox potential). Charge regulation during ET has never been measured for proteins because few tools exist to measure the net charge of a folded protein in solution at different oxidation states. Herein, we used a niche analytical tool (protein charge ladders analyzed with capillary electrophoresis) to determine that the net charges of myoglobin, cytochromeâ c, and azurin change by 0.62±0.06, 1.19±0.02, and 0.51±0.04 units upon single ET. Computational analysis predicts that these fluctuations in charge arise from changes in the pKa â values of multiple non-coordinating residues (predominantly histidine) that involve between 0.42-0.90â eV. These results suggest that ionizable residues can tune the reactivity of redox centers by regulating the net charge of the entire protein-cofactor-solvent complex.
Assuntos
Metaloproteínas/metabolismo , Azurina/química , Azurina/metabolismo , Citocromos c/química , Citocromos c/metabolismo , Transporte de Elétrons , Concentração de Íons de Hidrogênio , Metaloproteínas/química , Mioglobina/química , Mioglobina/metabolismo , Oxirredução , Eletricidade Estática , TermodinâmicaRESUMO
Personal neoantigen vaccines have been envisioned as an effective approach to induce, amplify and diversify antitumor T cell responses. To define the long-term effects of such a vaccine, we evaluated the clinical outcome and circulating immune responses of eight patients with surgically resected stage IIIB/C or IVM1a/b melanoma, at a median of almost 4 years after treatment with NeoVax, a long-peptide vaccine targeting up to 20 personal neoantigens per patient ( NCT01970358 ). All patients were alive and six were without evidence of active disease. We observed long-term persistence of neoantigen-specific T cell responses following vaccination, with ex vivo detection of neoantigen-specific T cells exhibiting a memory phenotype. We also found diversification of neoantigen-specific T cell clones over time, with emergence of multiple T cell receptor clonotypes exhibiting distinct functional avidities. Furthermore, we detected evidence of tumor infiltration by neoantigen-specific T cell clones after vaccination and epitope spreading, suggesting on-target vaccine-induced tumor cell killing. Personal neoantigen peptide vaccines thus induce T cell responses that persist over years and broaden the spectrum of tumor-specific cytotoxicity in patients with melanoma.
Assuntos
Antígenos de Neoplasias/genética , Vacinas Anticâncer/imunologia , Epitopos/imunologia , Memória Imunológica , Melanoma/imunologia , Humanos , Melanoma/patologiaRESUMO
High-throughput genome sequencing and computation have enabled rapid identification of targets for personalized medicine, including cancer vaccines. Synthetic peptides are an established mode of cancer vaccine delivery, but generating the peptides for each patient in a rapid and affordable fashion remains difficult. High-throughput peptide synthesis technology is therefore urgently needed for patient-specific cancer vaccines to succeed in the clinic. Previously, we developed automated flow peptide synthesis technology that greatly accelerates the production of synthetic peptides. Herein, we show that this technology permits the synthesis of high-quality peptides for personalized medicine. Automated flow synthesis produces 30-mer peptides in less than 35 minutes and 15- to 16-mer peptides in less than 20 minutes. The purity of these peptides is comparable with or higher than the purity of peptides produced by other methods. This work illustrates how automated flow synthesis technology can enable customized peptide therapies by accelerating synthesis and increasing purity. We envision that implementing this technology in clinical settings will greatly increase capacity to generate clinical-grade peptides on demand, which is a key step in reaching the full potential of personalized vaccines for the treatment of cancer and other diseases.
Assuntos
Antígenos de Neoplasias , Técnicas de Química Sintética/instrumentação , Técnicas de Química Sintética/métodos , Imunoterapia , Neoplasias/terapia , Peptídeos/síntese química , Medicina de Precisão , Automação , Vacinas Anticâncer , Humanos , Peptídeos/uso terapêuticoRESUMO
Cell-penetrating peptides (CPPs) can facilitate the intracellular delivery of large therapeutically relevant molecules, including proteins and oligonucleotides. Although hundreds of CPP sequences are described in the literature, predicting efficacious sequences remains difficult. Here, we focus specifically on predicting CPPs for the delivery of phosphorodiamidate morpholino oligonucleotides (PMOs), a compelling type of antisense therapeutic that has recently been FDA approved for the treatment of Duchenne muscular dystrophy. Using literature CPP sequences, 64 covalent PMO-CPP conjugates were synthesized and evaluated in a fluorescence-based reporter assay for PMO activity. Significant discrepancies were observed between the sequences that performed well in this assay and the sequences that performed well when conjugated to only a small-molecule fluorophore. As a result, we envisioned that our PMO-CPP library would be a useful training set for a computational model to predict CPPs for PMO delivery. We used the PMO activity data to fit a random decision forest classifier to predict whether or not covalent attachment of a given peptide would enhance PMO activity at least 3-fold. To validate the model experimentally, seven novel sequences were generated, synthesized, and tested in the fluorescence reporter assay. All computationally predicted positive sequences were positive in the assay, and one sequence performed better than 80% of the tested literature CPPs. These results demonstrate the power of machine learning algorithms to identify peptide sequences with particular functions and illustrate the importance of tailoring a CPP sequence to the cargo of interest.
RESUMO
Antitumor T-cell responses have the potential to be curative in cancer patients, but the induction of potent T-cell immunity through vaccination remains a largely unmet goal of immunotherapy. We previously reported that the immunogenicity of peptide vaccines could be increased by maximizing delivery to lymph nodes (LNs), where T-cell responses are generated. This was achieved by conjugating the peptide to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG (DSPE-PEG) to promote albumin binding, which resulted in enhanced lymphatic drainage and improved T-cell responses. Here, we expanded upon these findings and mechanistically dissected the properties that contribute to the potency of this amphiphile-vaccine (amph-vaccine). We found that multiple linkage chemistries could be used to link peptides with DSPE-PEG, and further, that multiple albumin-binding moieties conjugated to peptide antigens enhanced LN accumulation and subsequent T-cell priming. In addition to enhancing lymphatic trafficking, DSPE-PEG conjugation increased the stability of peptides in serum. DSPE-PEG peptides trafficked beyond immediate draining LNs to reach distal nodes, with antigen presented for at least a week in vivo, whereas soluble peptide presentation quickly decayed. Responses to amph-vaccines were not altered in mice deficient in the albumin-binding neonatal Fc receptor (FcRn), but required Batf3-dependent dendritic cells (DCs). Amph-peptides were processed by human DCs equivalently to unmodified peptides. These data define design criteria for enhancing the immunogenicity of molecular vaccines to guide the design of next-generation peptide vaccines. Cancer Immunol Res; 6(9); 1025-38. ©2018 AACR.
Assuntos
Vacinas Anticâncer/imunologia , Imunogenicidade da Vacina , Peptídeos/imunologia , Linfócitos T/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Animais , Células Dendríticas/imunologia , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoterapia , Linfonodos/imunologia , Camundongos Endogâmicos C57BL , Fosfatidiletanolaminas/metabolismo , Polietilenoglicóis/metabolismo , Receptores Fc/genética , Receptores Fc/imunologia , Albumina Sérica/metabolismo , Vacinas de Subunidades Antigênicas/químicaRESUMO
Retinoblastoma is the most common primary intraocular tumor in children. The first sign that is often reported by parents is the appearance of recurrent leukocoria (i.e., "white eye") in recreational photographs. A quantitative definition or scale of leukocoria--as it appears during recreational photography--has not been established, and the amount of clinical information contained in a leukocoric image (collected by a parent) remains unknown. Moreover, the hypothesis that photographic leukocoria can be a sign of early stage retinoblastoma has not been tested for even a single patient. This study used commercially available software (Adobe Photoshop®) and standard color space conversion algorithms (operable in Microsoft Excel®) to quantify leukocoria in actual "baby pictures" of 9 children with retinoblastoma (that were collected by parents during recreational activities i.e., in nonclinical settings). One particular patient with bilateral retinoblastoma ("Patient Zero") was photographed >7, 000 times by his parents (who are authors of this study) over three years: from birth, through diagnosis, treatment, and remission. This large set of photographs allowed us to determine the longitudinal and lateral frequency of leukocoria throughout the patient's life. This study establishes: (i) that leukocoria can emerge at a low frequency in early-stage retinoblastoma and increase in frequency during disease progression, but decrease upon disease regression, (ii) that Hue, Saturation and Value (i.e., HSV color space) are suitable metrics for quantifying the intensity of retinoblastoma-linked leukocoria; (iii) that different sets of intraocular retinoblastoma tumors can produce distinct leukocoric reflections; and (iv) the Saturation-Value plane of HSV color space represents a convenient scale for quantifying and classifying pupillary reflections as they appear during recreational photography.
Assuntos
Cor , Fotografação/métodos , Distúrbios Pupilares/diagnóstico , Neoplasias da Retina/diagnóstico , Retinoblastoma/diagnóstico , Adulto , Algoritmos , Pré-Escolar , Diagnóstico por Computador/métodos , Feminino , Humanos , Lactente , Estudos Longitudinais , Masculino , Distúrbios Pupilares/fisiopatologia , Distúrbios Pupilares/terapia , Reprodutibilidade dos Testes , Neoplasias da Retina/fisiopatologia , Neoplasias da Retina/terapia , Retinoblastoma/fisiopatologia , Retinoblastoma/terapia , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Is Janet Smith (reflections August 19) serious when she suggests that we help the NHS by giving back a day's pay? Perhaps as a clinical nurse specialist she has lost touch with how things really are in the NHS.