RESUMEN
For many years, clinicians and scientists attempt to develop methods to stimulate the immune system to target malignant cells. Recent data suggest that effective cancer vaccination requires combination immunotherapies to overcome tumor immune evasion. Through presentation of both MHC-I and II molecules, DCs-based vaccine platforms are effective in generating detectable CD4 and CD8 T cell responses against tumor-associated antigens. Several platforms include DC transfection with mRNA of the desired tumor antigen. These DCs are then delivered to the host and elicit an immune response against the antigen of interest. We have recently established an mRNA genetic platform which induced specific CD8+ cytotoxic T cell response by DC vaccination against melanoma. In our study, an MHC-II mRNA DCs vaccine platform was developed to activate CD4+ T cells and to enhance the anti-tumor response. The invariant chain (Ii) was modified and the semi-peptide CLIP was replaced with an MHC-II binding peptide sequences of melanoma antigens. These chimeric MHC-II constructs are presented by DCs and induce proliferation of tumor specific CD4+ T cells. When administered in combination with the MHC-I platform into tumor bearing mice, these constructs were able to inhibit tumor growth, and improve mouse survival. Deciphering the immunological mechanism of action, we observed an efficient CTLs killing in addition to higher levels of Th1 and Th2 subsets in the groups immunized with a combination of the MHC-I and MHC-II constructs. These universal constructs can be applied in multiple combinations and offer an attractive opportunity to improve cancer treatment.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/inmunología , Vacunas contra el Cáncer/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Melanoma Experimental/inmunología , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/administración & dosificación , Línea Celular Tumoral , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Progresión de la Enfermedad , Inmunoterapia/métodos , Melanoma Experimental/genética , Melanoma Experimental/terapia , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Supervivencia , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
Recently, we have developed a novel genetic platform for improving dendritic cell (DC) induction of peptide-specific CD8 T cells, based on membrane-anchored ß2-microglobulin (ß2m) linked to a selected antigenic peptide at its N-terminus and to the cytosolic domain of toll-like receptor (TLR)4 C-terminally. In vitro transcribed mRNA transfection of antigen presenting cells resulted in polypeptides that efficiently coupled peptide presentation to cellular activation. In the present study, we evaluated the immunogenicity of such constructs in mRNA-transfected immature murine bone marrow-derived DCs. We show that the encoded peptide ß2m-TLR4 products were expressed at the cell surface up to 72 hours and stimulated the maturation of DCs. In vivo, these DCs prompted efficient peptide-specific T-cell activation and target cell killing which were superior to those induced by peptide-loaded, LPS-stimulated DCs. This superiority was also evident in the ability to protect mice from tumor progression following the administration of B16F10.9 melanoma cells and to inhibit the development of pre-established B16F10.9 tumors. Our results provide evidence that the products of two recombinant genes encoding for peptide-hß2m-TLR4 and peptide-hß2m-K(b) expressed from exogenous mRNA can cooperate to couple Major Histocompatibility Complex (MHC-I) peptide presentation to TLR-mediated signaling, offering a safe, economical and highly versatile genetic platform for a novel category of CTL-inducing vaccines.
Asunto(s)
Células Dendríticas/citología , Antígenos de Histocompatibilidad Clase I/metabolismo , Péptidos/química , ARN Mensajero/metabolismo , Receptor Toll-Like 4/metabolismo , Microglobulina beta-2/metabolismo , Animales , Presentación de Antígeno/inmunología , Células Presentadoras de Antígenos/citología , Antígenos de Neoplasias/química , Antineoplásicos/química , Células de la Médula Ósea/citología , Vacunas contra el Cáncer/inmunología , Membrana Celular/metabolismo , Clonación Molecular , Medios de Cultivo , Citosol/metabolismo , Femenino , Humanos , Lipopolisacáridos/química , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
Cryoablation is a low-invasive surgical procedure for management of malignant tumors. Tissue destruction is obtained by repeated deep freezing and thawing and results in coagulative necrosis and in apoptosis. This procedure induces the release of tumor-associated antigens and proinflammatory factors into the microenvironment. Local administration of immature dendritic cells (DCs) potentiates the immune response induced by cryoablation. To further augment the induction of long-lasting antitumor immunity, we investigated the clinical value of combining cryoimmunotherapy consisting of cryoablation and inoculation of immature DCs with administration of the immune adjuvant, CpG oligodeoxynucleotides. Injection of the murine Lewis lung carcinoma, D122-luc-5.5 that expresses the luciferase gene, results in spontaneous metastases, which can be easily monitored in vivo. The local tumor was treated by the combined treatment. The clinical outcome was assessed by monitoring tumor growth, metastasis in distant organs, overall survival, and protection from tumor recurrence. The nature of the induced T cell responses was analyzed. Combined cryoimmunotherapy results in reduced tumor growth, low metastasis and significantly prolonged survival. Moreover, this treatment induces antitumor memory that protected mice from rechallenge. The underlying suggested mechanisms are the generation of tumor-specific type 1 T cell responses, subsequent induction of cytotoxic T lymphocytes, and generation of systemic memory. Our data highlight the combined cryoimmunotherapy as a novel antitumor vaccine with promising preclinical results. Adjustment of this technique into practice will provide the therapeutic benefits of both, ablation of the primary tumor and induction of robust antitumor and antimetastatic immunity.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Carcinoma Pulmonar de Lewis/terapia , Islas de CpG , Criocirugía/métodos , Células Dendríticas/inmunología , Inmunoterapia/métodos , Neoplasias Pulmonares/terapia , Oligodesoxirribonucleótidos/uso terapéutico , Animales , Linfocitos T CD8-positivos/inmunología , Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/secundario , Carcinoma Pulmonar de Lewis/cirugía , Células Cultivadas , Terapia Combinada , Células Dendríticas/trasplante , Pie , Memoria Inmunológica , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/cirugía , Masculino , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Oligodesoxirribonucleótidos/administración & dosificación , Recurrencia , Organismos Libres de Patógenos Específicos , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Current HIV vaccines designed to stimulate CD8+ T cells have failed to induce immunologic control upon infection. The functions of vaccine-induced HIV-specific CD8+ T cells were investigated here in detail. Cytotoxic capacity was significantly lower than in HIV controllers and was not a consequence of low frequency or unaccumulated functional cytotoxic proteins. Low cytotoxic capacity was attributable to impaired degranulation in response to the low antigen levels present on HIV-infected targets. The vaccine-induced T cell receptor (TCR) repertoire was polyclonal and transduction of these TCRs conferred the same reduced functions. These results define a mechanism accounting for poor antiviral activity induced by these vaccines and suggest that an effective CD8+ T cell response may require a vaccination strategy that drives further TCR clonal selection.
Asunto(s)
Vacunas contra el SIDA , Degranulación de la Célula , Citotoxicidad Inmunológica , Infecciones por VIH , Linfocitos T Citotóxicos , Humanos , Vacunas contra el SIDA/inmunología , Células Clonales , Infecciones por VIH/prevención & control , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Citotóxicos/inmunología , Degranulación de la Célula/inmunologíaRESUMEN
Priming of naive CD8 T cells by dendritic cells (DCs) entails both effective antigen presentation on MHC class I products and co-stimulatory signaling. Their optimal coupling is a major goal in the development of CTL-inducing vaccines. We recently reported that a membranal derivative of the invariant MHC-I light chain, ß(2)-microglobulin (ß(2)m), markedly stabilizes MHC-I molecules and can serve as a universal platform for exceptional presentation of genetically linked peptides. To test whether it is possible to equip the resulting MHC-I complexes with an inherent ability to activate antigen-presenting cells, we engrafted the intracellular Toll/IL-1 receptor domain of mouse Toll-like receptor (TLR) 4 or TLR2 onto the peptide-ß(2)m scaffold. We evaluated the level of peptide presentation and status of cell activation conferred by such constructs in stably transfected mouse RAW264.7 macrophages and mRNA-transfected mouse DC2.4 DCs. We show that the encoded peptide-ß(2)m-TLR polypeptides are expressed at the cell surface, pair with endogenous heavy chains, stabilize MHC-I products, prompt efficient peptide-specific T-cell recognition and confer a constitutively activated phenotype on the transfected cells, as judged by the up-regulation of pro-inflammatory genes and surface co-stimulatory molecules. Our results provide evidence that the product of a single recombinant gene can couple MHC peptide presentation to TLR-mediated signaling and offer a safe, economical and highly versatile modality for a novel category of genetic CTL-inducing vaccines.
Asunto(s)
Presentación de Antígeno/inmunología , Células Presentadoras de Antígenos/inmunología , Antígenos H-2/inmunología , Péptidos/inmunología , Animales , Vectores Genéticos/genética , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Péptidos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Transfección , Microglobulina beta-2/genética , Microglobulina beta-2/inmunologíaRESUMEN
BACKGROUND: Soluble human leucocyte antigen (sHLA) molecules, released into the plasma, carry their original peptide cargo and provide insight into the protein synthesis and degradation schemes of their source cells and tissues. Other body fluids, such as pleural effusions, may also contain sHLA-peptide complexes, and can potentially serve as a source of tumor antigens since these fluids are drained from the tumor microenvironment. We explored this possibility by developing a methodology for purifying and analyzing large pleural effusion sHLA class I peptidomes of patients with malignancies or benign diseases. METHODS: Cleared pleural fluids, cell pellets present in the pleural effusions, and the primary tumor cells cultured from cancer patients' effusions, were used for immunoaffinity purification of the HLA molecules. The recovered HLA peptides were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and the resulting LC-MS/MS data were analyzed with the MaxQuant software tool. Selected tumor antigen peptides were tested for their immunogenicity potential with donor peripheral blood mononuclear cells (PBMCs) in an in vitro assay. RESULTS: Mass spectrometry analysis of the pleural effusions revealed 39,669 peptides attributable to 11,305 source proteins. The majority of peptides identified from the pleural effusions were defined as HLA ligands that fit the patients' HLA consensus sequence motifs. The membranal and soluble HLA peptidomes of each individual patient correlated to each other. Additionally, soluble HLA peptidomes from the same patient, obtained at different visits to the clinic, were highly similar. Compared with benign effusions, the soluble HLA peptidomes of malignant pleural effusions were larger and included HLA peptides derived from known tumor-associated antigens, including cancer/testis antigens, lung-related proteins, and vascular endothelial growth factor pathway proteins. Selected tumor-associated antigens that were identified by the immunopeptidomics were able to successfully prime CD8+ T cells. CONCLUSIONS: Pleural effusions contain sHLA-peptide complexes, and the pleural effusion HLA peptidome of patients with malignant tumors can serve as a rich source of biomarkers for tumor diagnosis and potential candidates for personalized immunotherapy.
Asunto(s)
Antígenos de Neoplasias , Derrame Pleural Maligno , Linfocitos T CD8-positivos , Cromatografía Liquida , Antígenos de Histocompatibilidad Clase I , Humanos , Leucocitos Mononucleares , Masculino , Péptidos , Espectrometría de Masas en Tándem , Microambiente Tumoral , Factor A de Crecimiento Endotelial VascularRESUMEN
The adoptive transfer of naturally occurring T cells that recognize cancer neoantigens has led to durable tumor regressions in select patients with cancer. However, it remains unknown whether such T cells can be isolated from and used to treat patients with glioblastoma, a cancer that is refractory to currently available therapies. To answer this question, we stimulated patient blood-derived memory T cells in vitro using peptides and minigenes that represented point mutations unique to patients' tumors (ie, candidate neoantigens) and then tested their ability to specifically recognize these mutations. In a cohort of five patients with glioblastoma, we found that circulating CD4+ memory T cells from one patient recognized a cancer neoantigen harboring a mutation in the EED gene (EEDH189N) that was unique to that patient's tumor. This finding suggests that neoantigen-reactive T cells could indeed be isolated from patients with glioblastoma, thereby providing a rationale for further efforts to develop neoantigen-directed adoptive T cell therapy for this disease.
Asunto(s)
Glioblastoma/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , HumanosRESUMEN
PURPOSE: Immunotherapies mediate the regression of human tumors through recognition of tumor antigens by immune cells that trigger an immune response. Mutations in the RAS oncogenes occur in about 30% of all patients with cancer. These mutations play an important role in both tumor establishment and survival and are commonly found in hotspots. Discovering T-cell receptors (TCR) that recognize shared mutated RAS antigens presented on MHC class I and class II molecules are thus promising reagents for "off-the-shelf" adoptive cell therapies (ACT) following insertion of the TCRs into lymphocytes. EXPERIMENTAL DESIGN: In this ongoing work, we screened for RAS antigen recognition in tumor-infiltrating lymphocytes (TIL) or by in vitro stimulation of peripheral blood lymphocytes (PBL). TCRs recognizing mutated RAS were identified from the reactive T cells. The TCRs were then reconstructed and virally transduced into PBLs and tested. RESULTS: Here, we detect and report multiple novel TCR sequences that recognize nonsynonymous mutant RAS hotspot mutations with high avidity and specificity and identify the specific class-I and -II MHC restriction elements involved in the recognition of mutant RAS. CONCLUSIONS: The TCR library directed against RAS hotspot mutations described here recognize RAS mutations found in about 45% of the Caucasian population and about 60% of the Asian population and represent promising reagents for "off-the-shelf" ACTs.
Asunto(s)
Inmunoterapia Adoptiva , Mutación , Neoplasias/genética , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/uso terapéutico , Proteínas ras/genética , HumanosRESUMEN
BACKGROUNDTherapeutic vaccinations against cancer have mainly targeted differentiation antigens, cancer-testis antigens, and overexpressed antigens and have thus far resulted in little clinical benefit. Studies conducted by multiple groups have demonstrated that T cells recognizing neoantigens are present in most cancers and offer a specific and highly immunogenic target for personalized vaccination.METHODSWe recently developed a process using tumor-infiltrating lymphocytes to identify the specific immunogenic mutations expressed in patients' tumors. Here, validated, defined neoantigens, predicted neoepitopes, and mutations of driver genes were concatenated into a single mRNA construct to vaccinate patients with metastatic gastrointestinal cancer.RESULTSThe vaccine was safe and elicited mutation-specific T cell responses against predicted neoepitopes not detected before vaccination. Furthermore, we were able to isolate and verify T cell receptors targeting KRASG12D mutation. We observed no objective clinical responses in the 4 patients treated in this trial.CONCLUSIONThis vaccine was safe, and potential future combination of such vaccines with checkpoint inhibitors or adoptive T cell therapy should be evaluated for possible clinical benefit in patients with common epithelial cancers.TRIAL REGISTRATIONPhase I/II protocol (NCT03480152) was approved by the IRB committee of the NIH and the FDA.FUNDINGCenter for Clinical Research, NCI, NIH.
Asunto(s)
Antígenos de Neoplasias , Vacunas contra el Cáncer , Neoplasias Gastrointestinales , Inmunidad Celular , Mutación Missense , Proteínas Proto-Oncogénicas p21(ras) , ARN Mensajero , Linfocitos T/inmunología , Sustitución de Aminoácidos , Antígenos de Neoplasias/administración & dosificación , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Femenino , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/inmunología , Neoplasias Gastrointestinales/terapia , Humanos , Masculino , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/inmunología , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , ARN Mensajero/inmunologíaRESUMEN
PURPOSE: The purpose of this study was to evaluate antigen experienced T cells in peripheral blood lymphocytes (PBL) for responses to p53 neoantigens. EXPERIMENTAL DESIGN: PBLs from patients with a mutated TP53 tumor were sorted for antigen-experienced T cells and in vitro stimulation (IVS) was performed with p53 neoantigens. The IVS cultures were stimulated with antigen-presenting cells expressing p53 neoantigens, enriched for 41BB/OX40 and grown with rapid expansion protocol. RESULTS: T-cell responses were not observed in the PBLs of 4 patients who did not have tumor-infiltrating lymphocyte (TIL) responses to mutated TP53. In contrast, 5 patients with TIL responses to mutated TP53 also had similar T-cell responses in their PBLs, indicating that the PBLs and TILs were congruent in p53 neoantigen reactivity. CD4+ and CD8+ T cells were specific for p53R175H, p53Y220C, or p53R248W neoantigens, including a 78% reactive T-cell culture against p53R175H and HLA-A*02:01. Tracking TCRB clonotypes (clonality, top ranked, and TP53 mutation-specific) supported the enrichment of p53 neoantigen-reactive T cells from PBLs. The same T-cell receptor (TCR) from the TIL was found in the IVS cultures in three cases and multiple unique TCRs were found in another patient. TP53 mutation-specific T cells also recognized tumor cell lines bearing the appropriate human leukocyte antigen restriction element and TP53 mutation, indicating these T cells could recognize processed and presented p53 neoantigens. CONCLUSIONS: PBL was a noninvasive source of T cells targeting TP53 mutations for cell therapy and can provide a window into intratumoral p53 neoantigen immune responses.See related commentary by Olivera et al., p. 1203.
Asunto(s)
Linfocitos T CD8-positivos , Proteína p53 Supresora de Tumor , Antígenos de Neoplasias/genética , Linfocitos T CD8-positivos/metabolismo , Humanos , Linfocitos Infiltrantes de Tumor/metabolismo , Oncogenes , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
T cells targeting shared oncogenic mutations can induce durable tumor regression in epithelial cancer patients. Such T cells can be detected in tumor infiltrating lymphocytes, but whether such cells can be detected in the peripheral blood of patients with the common metastatic epithelial cancer patients is unknown. Using a highly sensitive in vitro stimulation and cell enrichment of peripheral memory T cells from six metastatic cancer patients, we identified and isolated CD4+, and CD8+ memory T cells targeting the mutated KRASG12D and KRASG12V variants, respectively, in three patients. In an additional two metastatic colon cancer patients, we detected CD8+ neoantigen-specific cells targeting the mutated SMAD5 and MUC4 proteins. Therefore, memory T cells targeting unique as well as shared somatic mutations can be detected in the peripheral blood of epithelial cancer patients and can potentially be used for the development of effective personalized T cell-based cancer immunotherapy across multiple patients.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Neoplasias del Colon/inmunología , Regulación Neoplásica de la Expresión Génica , Mucina 4/inmunología , Proteínas Proto-Oncogénicas p21(ras)/inmunología , Proteína Smad5/inmunología , Presentación de Antígeno , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Separación Celular/métodos , Técnicas de Cocultivo , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Células Dendríticas/citología , Células Dendríticas/inmunología , Humanos , Memoria Inmunológica , Metástasis Linfática , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Terapia Molecular Dirigida , Mucina 4/genética , Mutación , Células Neoplásicas Circulantes/inmunología , Células Neoplásicas Circulantes/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal , Proteína Smad5/genética , Transducción GenéticaRESUMEN
Immunotherapies can mediate regression of human tumors with high mutation rates, but responses are rarely observed in patients with common epithelial cancers. This raises the question of whether patients with these common cancers harbor T lymphocytes that recognize mutant proteins expressed by autologous tumors that may represent ideal targets for immunotherapy. Using high-throughput immunologic screening of mutant gene products identified via whole-exome sequencing, we identified neoantigen-reactive tumor-infiltrating lymphocytes (TIL) from 62 of 75 (83%) patients with common gastrointestinal cancers. In total, 124 neoantigen-reactive TIL populations were identified, and all but one of the neoantigenic determinants were unique. The results of in vitro T-cell recognition assays demonstrated that 1.6% of the gene products encoded by somatic nonsynonymous mutations were immunogenic. These findings demonstrate that the majority of common epithelial cancers elicit immune recognition and open possibilities for cell-based immunotherapies for patients bearing these cancers. SIGNIFICANCE: TILs cultured from 62 of 75 (83%) patients with gastrointestinal cancers recognized neoantigens encoded by 1.6% of somatic mutations expressed by autologous tumor cells, and 99% of the neoantigenic determinants appeared to be unique and not shared between patients.This article is highlighted in the In This Issue feature, p. 983.
Asunto(s)
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Susceptibilidad a Enfermedades , Neoplasias Gastrointestinales/etiología , Neoplasias Gastrointestinales/metabolismo , Mutación , Biomarcadores de Tumor , Neoplasias Gastrointestinales/patología , Humanos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Receptores de Antígenos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patologíaRESUMEN
Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) targeting neoantigens can mediate tumor regression in selected patients with metastatic epithelial cancer. However, effectively identifying and harnessing neoantigen-reactive T cells for patient treatment remains a challenge and it is unknown whether current methods to detect neoantigen-reactive T cells are missing potentially clinically relevant neoantigen reactivities. We thus investigated whether the detection of neoantigen-reactive TILs could be enhanced by enriching T cells that express PD-1 and/or T cell activation markers followed by microwell culturing to avoid overgrowth of nonreactive T cells. In 6 patients with metastatic epithelial cancer, this method led to the detection of CD4+ and CD8+ T cells targeting 18 and 1 neoantigens, respectively, compared with 6 and 2 neoantigens recognized by CD4+ and CD8+ T cells, respectively, when using our standard TIL fragment screening approach. In 2 patients, no recognition of mutated peptides was observed using our conventional screen, while our high-throughput approach led to the identification of 5 neoantigen-reactive T cell receptors (TCRs) against 5 different mutations from one patient and a highly potent MHC class II-restricted KRASG12V-reactive TCR from a second patient. In addition, in a metastatic tumor sample from a patient with serous ovarian cancer, we isolated 3 MHC class II-restricted TCRs targeting the TP53G245S hot-spot mutation. In conclusion, this approach provides a highly sensitive platform to isolate clinically relevant neoantigen-reactive T cells or their TCRs for cancer treatment.
Asunto(s)
Separación Celular/métodos , Citometría de Flujo/métodos , Inmunoterapia Adoptiva/métodos , Linfocitos Infiltrantes de Tumor/trasplante , Neoplasias/terapia , Adulto , Anciano , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Carcinogénesis/genética , Femenino , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Masculino , Persona de Mediana Edad , Mutación , Neoplasias/genética , Neoplasias/inmunología , Oncogenes/genética , Oncogenes/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Sensibilidad y Especificidad , Células Tumorales CultivadasRESUMEN
Despite melanoma immunogenicity and remarkable therapeutic effects of negative immune checkpoint inhibitors, a significant fraction of patients does not respond to current treatments. This could be due to limitations in tumor immunogenicity and profound immunosuppression in the melanoma microenvironment. Moreover, insufficient tumor antigen processing and presentation by dendritic cells (DC) may hamper the development of tumor-specific T cells. Using two genetically engineered mouse melanoma models (RET and BRAFV600E transgenic mice), in which checkpoint inhibitor therapy alone is not efficacious, we performed proof-of-concept studies with an improved, multivalent DC vaccination strategy based on our recently developed genetic mRNA cancer vaccines. The in vivo expression of multiple chimeric MHC class I receptors allows a simultaneous presentation of several melanoma-associated shared antigens tyrosinase related protein (TRP)-1, tyrosinase, human glycoprotein 100 and TRP-2. The DC vaccine induced a significantly improved survival in both transgenic mouse models. Vaccinated melanoma-bearing mice displayed an increased CD8 T cell reactivity indicated by a higher IFN-γ production and an upregulation of activation marker expression along with an attenuated immunosuppressive pattern of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg). The combination of DC vaccination with ultra-low doses of paclitaxel or anti-PD-1 antibodies resulted in further prolongation of mouse survival associated with a stronger reduction of MDSC and Treg immunosuppressive phenotype. Our data suggest that an improved multivalent DC vaccine based on shared tumor antigens induces potent anti-tumor effects and could be combined with checkpoint inhibitors or targeting immunosuppressive cells to further improve their therapeutic efficiency.
RESUMEN
The interaction between the CD40 receptor on antigen-presenting cells (APCs) and its trimeric ligand on CD4 T cells is essential for the initiation and progression of the adaptive immune response. Here we undertook to endow CD40 with the capacity to trigger spontaneous APC activation through ligand-independent oligomerization. To this end we exploited the GCN4 yeast transcriptional activator, which contains a leucine zipper DNA-binding motif that induces homophilic interactions. We incorporated GCN4 variants forming homodimers, trimers, or tetramers at the intracellular domain of human and mouse CD40 and replaced the extracellular portion with peptide-ß2m or other peptide tags. In parallel we examined similarly truncated CD40 monomers lacking a GCN4 motif. The oligomeric products appeared to arrange in high-molecular-weight aggregates and were considerably superior to the monomer in their ability to trigger nuclear factor kB signaling, substantiating the anticipated constitutively active (ca) phenotype. Cumulative results in human and mouse APC lines transfected with caCD40 mRNA revealed spontaneous upregulation of CD80, IL-1ß, TNFα, IL-6, and IL-12, which could be further enhanced by caTLR4 mRNA. In mouse bone-marrow-derived dendritic cells caCD40 upregulated CD80, CD86, MHC-II, and IL-12 and in human monocyte-derived dendritic cells it elevated surface CD80, CD83 CD86, CCR7, and HLA-DR. Oligomeric products carrying the peptide-ß2m extracellular portion could support MHC-I presentation of the linked peptide up to 4 days post-mRNA transfection. These findings demonstrate that the expression of a single caCD40 derivative in APCs can exert multiple immunostimulatory effects, offering a new powerful tool in the design of gene-based cancer vaccines.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Antígenos CD40/metabolismo , Vacunas contra el Cáncer/genética , Células Dendríticas/fisiología , Saccharomyces cerevisiae/genética , Animales , Presentación de Antígeno , Antígenos CD/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Antígenos CD40/genética , Diferenciación Celular , Línea Celular , Citocinas/metabolismo , Ingeniería Genética , Antígenos de Histocompatibilidad/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Leucina Zippers/genética , Activación de Linfocitos , Ratones , FN-kappa B/metabolismo , Multimerización de Proteína/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Regulación hacia ArribaRESUMEN
Cytotoxic T lymphocytes (CTLs) are the major effector arm of the immune system against tumors. Many tumor-associated antigens (TAAs), known today as potential rejection antigens, were identified by their ability to induce CTL responses. CTLs utilize their clonotypic T cell receptor (TCR) to recognize short antigenic peptides presented on major histocompatibility complex (MHC)-I proteins. These consist of a membrane-attached α heavy chain, which forms the peptide binding pocket, and a noncovalently associated ß2m light chain, not anchored to the cell membrane. CTL activation requires that antigenic peptides be presented initially on professional antigen presenting cells (APCs), primarily dendritic cells (DCs). Autologous DCs are a powerful tool for the induction of antitumor responses and are thus widely explored as vehicles for cancer vaccines. Although encouraging evidence for the induction of tumor-specific CTLs by ex vivo-manipulated DCs came from numerous animal studies, reproducible objective clinical response in human trials is yet to be demonstrated.
Asunto(s)
Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismo , Animales , Vacunas contra el Cáncer/uso terapéutico , Diseño de Fármacos , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/uso terapéutico , Ratones , Ingeniería de Proteínas , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/uso terapéuticoRESUMEN
Identification and quantification of immunogenic peptides and tumor-derived epitopes presented on MHC-I molecules are essential for basic studies and vaccines generation. Although lymphocytes derived from transgenic mice can serve as sensitive detectors of processes of antigen presentation and recognition, they are not always available. The use of cell lines might be extremely useful. In this study, we generated a lacZ inducible CD8⺠hybridoma (BUSA14) capable of recognizing both human and mouse gp10025â33 melanoma antigens presented on dendritic and tumor cell lines. This hybridoma expresses a variety of membranal T cell markers and secretes IL-2 and TNFα. Thus, BUSA14 offers a quantifiable, cheap and straightforward tool for studying peptide presentation by MHC-I molecules on the cell surface.
Asunto(s)
Linfocitos T CD8-positivos/citología , Antígenos de Histocompatibilidad Clase I/metabolismo , Hibridomas/metabolismo , Antígeno gp100 del Melanoma/metabolismo , Animales , Línea Celular Tumoral , Células Dendríticas/metabolismo , Citometría de Flujo , Humanos , Hibridomas/citología , Interleucina-2/metabolismo , Operón Lac , Ratones , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The magnitude of response elicited by CTL-inducing vaccines correlates with the density of MHC class I (MHC-I)-peptide complexes formed on the APC membrane. The MHC-I L chain, beta2-microglobulin (beta2m), governs complex stability. We reasoned that genetically converting beta2m into an integral membrane protein should exert a marked stabilizing effect on the resulting MHC-I molecules and enhance vaccine efficacy. In the present study, we show that expression of membranal human beta2m (hbeta2m) in mouse RMA-S cells elevates MHC-I thermal stability. RMA-S transfectants bind an exogenous peptide at concentrations 10(4)- to 10(6)-fold lower than parental RMA-S, as detected by complex-specific Abs and by T cell activation. Moreover, saturation of the transfectants' MHC-I by exogenous peptide occurs within 1 min, as compared with approximately 1 h required for parental cells. At saturation, however, level of peptide bound by modified cells is only 3- to 5-fold higher. Expression of native hbeta2m only results in marginal effect on the binding profile. Soluble beta2m has no effect on the accelerated kinetics, but the kinetics of transfectants parallel that of parental cells in the presence of Abs to hbeta2m. Ab inhibition and coimmunoprecipitation analyses suggest that both prolonged persistence of peptide-receptive H chain/beta2m heterodimers and fast heterodimer formation via lateral diffusion may contribute to stabilization. In vivo, peptide-loaded transfectants are considerably superior to parental cells in suppressing tumor growth. Our findings support the role of an allosteric mechanism in determining ternary MHC-I complex stability and propose membranal beta2m as a novel scaffold for CTL induction.