RESUMEN
Novel chimeric antigen receptor (CAR) T-cell approaches are needed to improve therapeutic efficacy in solid tumors. High-risk neuroblastoma is an aggressive pediatric solid tumor that expresses cell-surface GPC2 and GD2 with a tumor microenvironment infiltrated by CD16a-expressing innate immune cells. Here we engineer T-cells to express a GPC2-directed CAR and simultaneously secrete a bispecific innate immune cell engager (BiCE) targeting both GD2 and CD16a. In vitro, GPC2.CAR-GD2.BiCE T-cells induce GPC2-dependent cytotoxicity and secrete GD2.BiCE that promotes GD2-dependent activation of antitumor innate immunity. In vivo, GPC2.CAR-GD2.BiCE T-cells locally deliver GD2.BiCE and increase intratumor retention of NK-cells. In mice bearing neuroblastoma patient-derived xenografts and reconstituted with human CD16a-expressing immune cells, GD2.BiCEs enhance GPC2.CAR antitumor efficacy. A CAR.BiCE strategy should be considered for tumor histologies where antigen escape limits CAR efficacy, especially for solid tumors like neuroblastoma that are infiltrated by innate immune cells.
Asunto(s)
Gangliósidos , Inmunidad Innata , Inmunoterapia Adoptiva , Células Asesinas Naturales , Neuroblastoma , Receptores Quiméricos de Antígenos , Linfocitos T , Neuroblastoma/inmunología , Neuroblastoma/terapia , Neuroblastoma/patología , Animales , Humanos , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Ratones , Gangliósidos/inmunología , Línea Celular Tumoral , Inmunoterapia Adoptiva/métodos , Células Asesinas Naturales/inmunología , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Glipicanos/inmunología , Glipicanos/metabolismo , Microambiente Tumoral/inmunología , FemeninoRESUMEN
Peptide-centric chimeric antigen receptors (PC-CARs) recognize oncoprotein epitopes displayed by cell-surface human leukocyte antigens (HLAs) and offer a promising strategy for targeted cancer therapy. We have previously developed a PC-CAR targeting a neuroblastoma-associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes. Here, we determine the 2.1-angstrom crystal structure of the PC-CAR-PHOX2B-HLA-A*24:02-ß2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). This PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactive group, covering a combined global population frequency of up to 46.7%. Biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation, and CAR T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.
Asunto(s)
Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/genética , Péptidos/química , Epítopos , Antígenos de NeoplasiasRESUMEN
The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.
Asunto(s)
Antígenos de Neoplasias , Neuroblastoma , Proteínas Oncogénicas , Péptidos , Receptores Quiméricos de Antígenos , Animales , Humanos , Ratones , África/etnología , Alelos , Secuencia de Aminoácidos , Carcinogénesis , Reacciones Cruzadas , Antígenos HLA-A/química , Antígenos HLA-A/inmunología , Neuroblastoma/genética , Neuroblastoma/inmunología , Neuroblastoma/terapia , Proteínas Oncogénicas/antagonistas & inhibidores , Proteínas Oncogénicas/inmunología , Péptidos/antagonistas & inhibidores , Péptidos/química , Péptidos/inmunología , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/uso terapéuticoRESUMEN
Peptide-Centric Chimeric Antigen Receptors (PC-CARs), which recognize oncoprotein epitopes displayed by human leukocyte antigens (HLAs) on the cell surface, offer a promising strategy for targeted cancer therapy 1 . We have previously developed a PC-CAR targeting a neuroblastoma- associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes 2 . Here, we determine the 2.1 Å structure of the PC-CAR:PHOX2B/HLA-A*24:02/ß2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). The PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactivity group, covering a combined American population frequency of up to 25.2%. Comprehensive characterization using biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation and CAR-T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.
RESUMEN
The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.
Asunto(s)
Antígenos de Neoplasias/inmunología , Antígenos HLA/inmunología , Inmunoterapia , Neoplasias/inmunología , Neoplasias/terapia , Proteínas Oncogénicas/inmunología , Receptores Quiméricos de Antígenos/inmunología , Animales , Antígenos de Neoplasias/metabolismo , Línea Celular , Línea Celular Tumoral , Reacciones Cruzadas , Reactividad Cruzada , Femenino , Antígenos HLA/metabolismo , Proteínas de Homeodominio/inmunología , Proteínas de Homeodominio/metabolismo , Humanos , Interferón gamma/inmunología , Ratones , Neoplasias/metabolismo , Proteínas Oncogénicas/antagonistas & inhibidores , Proteínas Oncogénicas/metabolismo , Linfocitos T/inmunología , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismoRESUMEN
Here we propose a SARS-CoV-2 vaccine design concept based on identification of highly conserved regions of the viral genome and newly acquired adaptations, both predicted to generate epitopes presented on major histocompatibility complex (MHC) class I and II across the vast majority of the population. We further prioritize genomic regions that generate highly dissimilar peptides from the human proteome and are also predicted to produce B cell epitopes. We propose sixty-five 33-mer peptide sequences, a subset of which can be tested using DNA or mRNA delivery strategies. These include peptides that are contained within evolutionarily divergent regions of the spike protein reported to increase infectivity through increased binding to the ACE2 receptor and within a newly evolved furin cleavage site thought to increase membrane fusion. Validation and implementation of this vaccine concept could specifically target specific vulnerabilities of SARS-CoV-2 and should engage a robust adaptive immune response in the vast majority of the population.
RESUMEN
Here we propose a vaccination strategy for SARS-CoV-2 based on identification of both highly conserved regions of the virus and newly acquired adaptations that are presented by MHC class I and II across the vast majority of the population, are highly dissimilar from the human proteome, and are predicted B cell epitopes. We present 65 peptide sequences that we expect to result in a safe and effective vaccine which can be rapidly tested in DNA, mRNA, or synthetic peptide constructs. These include epitopes that are contained within evolutionarily divergent regions of the spike protein reported to increase infectivity through increased binding to the ACE2 receptor, and within a novel furin cleavage site thought to increase membrane fusion. This vaccination strategy specifically targets unique vulnerabilities of SARS-CoV-2 and should engage a robust adaptive immune response in the vast majority of the human population.
RESUMEN
Here we propose a vaccination strategy for SARS-CoV-2 based on identification of both highly conserved regions of the virus and newly acquired adaptations that are presented by MHC class I and II across the vast majority of the population, are highly dissimilar from the human proteome, and are predicted B cell epitopes. We present 65 peptide sequences that we expect to result in a safe and effective vaccine which can be rapidly tested in DNA, mRNA, or synthetic peptide constructs. These include epitopes that are contained within evolutionarily divergent regions of the spike protein reported to increase infectivity through increased binding to the ACE2 receptor, and within a novel furin cleavage site thought to increase membrane fusion. This vaccination strategy specifically targets unique vulnerabilities of SARS-CoV-2 and should engage a robust adaptive immune response in the vast majority of the human population.
RESUMEN
Peptide exchange technologies are essential for the generation of pMHC-multimer libraries used to probe diverse, polyclonal TCR repertoires in various settings. Here, using the molecular chaperone TAPBPR, we develop a robust method for the capture of stable, empty MHC-I molecules comprising murine H2 and human HLA alleles, which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Alternatively, catalytic amounts of TAPBPR can be used to exchange placeholder peptides with high affinity peptides of interest. Using the same system, we describe high throughput assays to validate binding of multiple candidate peptides on empty MHC-I/TAPBPR complexes. Combined with tetramer-barcoding via a multi-modal cellular indexing technology, ECCITE-seq, our approach allows a combined analysis of TCR repertoires and other T cell transcription profiles together with their cognate antigen specificities in a single experiment. The new approach allows TCR/pMHC interactions to be interrogated easily at large scale.
Asunto(s)
Proteínas Portadoras/química , Antígenos de Histocompatibilidad Clase I/química , Proteínas de Transporte de Membrana/química , Chaperonas Moleculares/química , Péptidos/química , Dominios y Motivos de Interacción de Proteínas , Alelos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Biblioteca de Genes , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunidad Celular , Inmunoquímica , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Linfocitos TRESUMEN
Despite recent advances in cancer immunotherapy, the process of immunoediting early in tumorigenesis remains obscure. Here, we employ a mathematical model that utilizes the Cancer Genome Atlas (TCGA) data to elucidate the contribution of individual mutations and HLA alleles to the immunoediting process. We find that common cancer mutations including BRAF-V600E and KRAS-G12D are predicted to bind none of the common HLA alleles, and are thus "immunogenically silent" in the human population. We identify regions of proteins that are not presented by HLA at a population scale, coinciding with frequently mutated hotspots in cancer, and other protein regions broadly presented across the population in which few mutations occur. We also find that 9/29 common HLA alleles contribute disproportionately to the immunoediting of early oncogenic mutations. These data provide insights into immune evasion of common driver mutations and a molecular basis for the association of particular HLA genotypes with cancer susceptibility.
Asunto(s)
Antígenos HLA/genética , Antígenos HLA/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Alelos , Carcinogénesis/genética , Transformación Celular Neoplásica/genética , Humanos , Inmunogenicidad Vacunal , Inmunoterapia , Mutación , Neoplasias/genéticaRESUMEN
Carcinogen-induced cancers typically have high mutation burdens and an inflamed microenvironment and thus are poised to respond to immune checkpoint inhibitors (ICIs). However, cancers with loss-of-function mutations in the SWI/SNF complex have few additional mutations yet also have an inflamed immunophenotype and should respond to ICI therapy.
Asunto(s)
Ensamble y Desensamble de Cromatina/inmunología , Inmunoterapia , Proteínas Nucleares/inmunología , Tumor Rabdoide/tratamiento farmacológico , ADN Helicasas/genética , Humanos , Inmunoterapia/métodos , Mutación/genética , Proteínas Nucleares/genética , Tumor Rabdoide/genética , Factores de Transcripción/genéticaRESUMEN
High-risk neuroblastomas show a paucity of recurrent somatic mutations at diagnosis. As a result, the molecular basis for this aggressive phenotype remains elusive. Recent progress in regulatory network analysis helped us elucidate disease-driving mechanisms downstream of genomic alterations, including recurrent chromosomal alterations. Our analysis identified three molecular subtypes of high-risk neuroblastomas, consistent with chromosomal alterations, and identified subtype-specific master regulator proteins that were conserved across independent cohorts. A 10-protein transcriptional module-centered around a TEAD4-MYCN positive feedback loop-emerged as the regulatory driver of the high-risk subtype associated with MYCN amplification. Silencing of either gene collapsed MYCN-amplified (MYCNAmp) neuroblastoma transcriptional hallmarks and abrogated viability in vitro and in vivo Consistently, TEAD4 emerged as a robust prognostic marker of poor survival, with activity independent of the canonical Hippo pathway transcriptional coactivators YAP and TAZ. These results suggest novel therapeutic strategies for the large subset of MYCN-deregulated neuroblastomas.Significance: Despite progress in understanding of neuroblastoma genetics, little progress has been made toward personalized treatment. Here, we present a framework to determine the downstream effectors of the genetic alterations sustaining neuroblastoma subtypes, which can be easily extended to other tumor types. We show the critical effect of disrupting a 10-protein module centered around a YAP/TAZ-independent TEAD4-MYCN positive feedback loop in MYCNAmp neuroblastomas, nominating TEAD4 as a novel candidate for therapeutic intervention. Cancer Discov; 8(5); 582-99. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Musculares/genética , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/genética , Aciltransferasas , Proteínas de Ciclo Celular , Línea Celular Tumoral , Biología Computacional/métodos , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Humanos , Proteínas Musculares/metabolismo , Proteína Proto-Oncogénica N-Myc/metabolismo , Estadificación de Neoplasias , Neuroblastoma/diagnóstico , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Interferencia de ARN , Factores de Transcripción de Dominio TEA , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
The identification of recurrent human leukocyte antigen (HLA) neoepitopes driving T cell responses against tumors poses a significant bottleneck in the development of approaches for precision cancer therapeutics. Here, we employ a bioinformatics method, Prediction of T Cell Epitopes for Cancer Therapy, to analyze sequencing data from neuroblastoma patients and identify a recurrent anaplastic lymphoma kinase mutation (ALK R1275Q) that leads to two high affinity neoepitopes when expressed in complex with common HLA alleles. Analysis of the X-ray structures of the two peptides bound to HLA-B*15:01 reveals drastically different conformations with measurable changes in the stability of the protein complexes, while the self-epitope is excluded from binding due to steric hindrance in the MHC groove. To evaluate the range of HLA alleles that could display the ALK neoepitopes, we used structure-based Rosetta comparative modeling calculations, which accurately predict several additional high affinity interactions and compare our results with commonly used prediction tools. Subsequent determination of the X-ray structure of an HLA-A*01:01 bound neoepitope validates atomic features seen in our Rosetta models with respect to key residues relevant for MHC stability and T cell receptor recognition. Finally, MHC tetramer staining of peripheral blood mononuclear cells from HLA-matched donors shows that the two neoepitopes are recognized by CD8+ T cells. This work provides a rational approach toward high-throughput identification and further optimization of putative neoantigen/HLA targets with desired recognition features for cancer immunotherapy.
Asunto(s)
Quinasa de Linfoma Anaplásico/genética , Quinasa de Linfoma Anaplásico/inmunología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Epítopos/genética , Epítopos/inmunología , Mutación , Alelos , Secuencia de Aminoácidos , Quinasa de Linfoma Anaplásico/metabolismo , Antígenos de Neoplasias/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Biología Computacional/métodos , Epítopos/química , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Modelos Moleculares , Péptidos/genética , Péptidos/inmunología , Péptidos/metabolismo , Conformación Proteica , Multimerización de Proteína , Relación Estructura-ActividadRESUMEN
Inconsistent detection of plant-based dietary small RNAs in circulation has thwarted the use of dietary RNA therapeutics. Here we demonstrate mice consuming diets rich in vegetables displayed enhanced serum levels of the plant specific small RNA MIR2911. Differential centrifugation, size-exclusion chromatography, and proteinase K treatment of plant extracts suggest this RNA resides within a proteinase K-sensitive complex. Plant derived MIR2911 was more bioavailable than the synthetic RNA. Furthermore, MIR2911 exhibited unusual digestive stability compared with other synthetic plant microRNAs. The characteristics of circulating MIR2911 were also unusual as it was not associated with exosomes and fractionated as a soluble complex that was insensitive to proteinase K treatment, consistent with MIR2911 being stabilized by modifications conferred by the host. These results indicate that intrinsic stability and plant-based modifications orchestrate consumer uptake of this anomalous plant based small RNA and invite revisiting plant-based microRNA therapeutic approaches.
Asunto(s)
Magnoliopsida/genética , MicroARNs/sangre , MicroARNs/farmacocinética , Alimentación Animal , Animales , Disponibilidad Biológica , Brassica/química , Digestión , Endopeptidasa K/metabolismo , Exosomas , Magnoliopsida/metabolismo , Masculino , Ratones Endogámicos ICR , Nanopartículas , Estabilidad del ARNRESUMEN
Glycosylation is an important post-translational modification during protein production in eukaryotic cells, and it is essential for protein structure, stability, half-life, and biological functions. In this study, we produced various monoclonal antibody (mAb) glycoforms from Chinese hamster ovary (CHO) cells, including the natively glycosylated antibody, the enriched G0 form, the deglycosylated form, the afucosylated form, and the high mannose form, and we compared their intrinsic properties, side-by-side, through biophysical and biochemical approaches. Spectroscopic analysis indicates no measureable secondary or tertiary structural changes after in vitro or in vivo modification of the glycosylation pattern. Thermal unfolding experiments show that the high mannose and deglycosylated forms have reduced thermal stability of the CH2 domain compared with the other tested glycoforms. We also observed that the individual domain's thermal stability could be pH dependent. Proteolysis analysis indicates that glycosylation plays an important role in stabilizing mAbs against proteases. The stability of antibody glycoforms at the storage condition (2-8 °C) and at accelerated conditions (30 and 40 °C) was evaluated, and the results indicate that glycosylation patterns do not substantially affect the storage stability of the antibody we studied.