RESUMEN

Treating patients who have cancer with vaccines that stimulate a targeted immune response is conceptually appealing, but cancer vaccine trials have not been successful in late-stage patients with treatment-refractory tumours1,2. We are testing melanoma FixVac (BNT111)-an intravenously administered liposomal RNA (RNA-LPX) vaccine, which targets four non-mutated, tumour-associated antigens that are prevalent in melanoma-in an ongoing, first-in-human, dose-escalation phase I trial in patients with advanced melanoma (Lipo-MERIT trial, ClinicalTrials.gov identifier NCT02410733). We report here data from an exploratory interim analysis that show that melanoma FixVac, alone or in combination with blockade of the checkpoint inhibitor PD1, mediates durable objective responses in checkpoint-inhibitor (CPI)-experienced patients with unresectable melanoma. Clinical responses are accompanied by the induction of strong CD4+ and CD8+ T cell immunity against the vaccine antigens. The antigen-specific cytotoxic T-cell responses in some responders reach magnitudes typically reported for adoptive T-cell therapy, and are durable. Our findings indicate that RNA-LPX vaccination is a potent immunotherapy in patients with CPI-experienced melanoma, and suggest the general utility of non-mutant shared tumour antigens as targets for cancer vaccination.

Asunto(s)

Antineoplásicos/uso terapéutico , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Melanoma/inmunología , Melanoma/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , ARN Neoplásico/genética , Linfocitos T/inmunología , Antígenos de Neoplasias/inmunología , Antineoplásicos/farmacología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/efectos adversos , Terapia Combinada , Humanos , Melanoma/tratamiento farmacológico , Melanoma/patología , Estadificación de Neoplasias , Linfocitos T/citología , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunología , Vacunación

RESUMEN

The quest for tumor-associated antigens (TAA) and neoantigens is a major focus of cancer immunotherapy. Here, we combine a neoantigen prediction pipeline and human leukocyte antigen (HLA) peptidomics to identify TAAs and neoantigens in 16 tumors derived from seven patients with melanoma and characterize their interactions with their tumor-infiltrating lymphocytes (TIL). Our investigation of the antigenic and T-cell landscapes encompassing the TAA and neoantigen signatures, their immune reactivity, and their corresponding T-cell identities provides the first comprehensive analysis of cancer cell T-cell cosignatures, allowing us to discover remarkable antigenic and TIL similarities between metastases from the same patient. Furthermore, we reveal that two neoantigen-specific clonotypes killed 90% of autologous melanoma cells, both in vitro and in vivo, showing that a limited set of neoantigen-specific T cells may play a central role in melanoma tumor rejection. Our findings indicate that combining HLA peptidomics with neoantigen predictions allows robust identification of targetable neoantigens, which could successfully guide personalized cancer immunotherapies.Significance: As neoantigen targeting is becoming more established as a powerful therapeutic approach, investigating these molecules has taken center stage. Here, we show that a limited set of neoantigen-specific T cells mediates tumor rejection, suggesting that identifying just a few antigens and their corresponding T-cell clones could guide personalized immunotherapy. Cancer Discov; 8(11); 1366-75. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333.

Asunto(s)

Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Linfocitos T/inmunología , Animales , Antígenos de Neoplasias/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Linfocitos T/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of ß2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.

Asunto(s)

Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Melanoma/inmunología , Melanoma/terapia , Mutación/genética , Medicina de Precisión/métodos , ARN/genética , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Antígeno B7-H1/inmunología , Antígenos CD8/inmunología , Vacunas contra el Cáncer/uso terapéutico , Epítopos/genética , Epítopos/inmunología , Humanos , Inmunoterapia/métodos , Melanoma/genética , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia/prevención & control , Nivolumab , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Linfocitos T/inmunología , Vacunación , Microglobulina beta-2/deficiencia

RESUMEN

Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard (51)Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the (51)Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies.

Asunto(s)

Células Presentadoras de Antígenos/inmunología , Citotoxicidad Inmunológica , Inmunidad Celular , Inmunidad Humoral , Inmunoensayo , Linfocitos T Citotóxicos/inmunología , Células Presentadoras de Antígenos/citología , Antígenos/genética , Antígenos/inmunología , Línea Celular Tumoral , Supervivencia Celular , Técnicas de Cocultivo , Genes Reporteros , Humanos , Luciferasas/genética , Luciferasas/inmunología , Plásmidos/química , Plásmidos/metabolismo , ARN Mensajero/genética , ARN Mensajero/inmunología , Linfocitos T Citotóxicos/citología , Transcripción Genética , Transfección

RESUMEN

Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/ß T-cell receptors (TCRα/ß). However, potential mispairing of introduced TCRα/ß-chains with endogenous ß/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vß-fragment to the TCR Cß-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/ß-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vß-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vß. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/ß-positive T-cells.

Asunto(s)

Complejo CD3/inmunología , Leucemia de Células T/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunología , Traslado Adoptivo , Animales , Biomarcadores de Tumor , Complejo CD3/genética , Membrana Celular , Proliferación Celular , Humanos , Inmunoterapia Adoptiva , Leucemia de Células T/genética , Leucemia de Células T/patología , Ratones , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Transducción de Señal , Linfocitos T/metabolismo , Células Tumorales Cultivadas

RESUMEN

Triple-negative breast cancer (TNBC) is a high medical need disease with limited treatment options. CD8+ T cell-mediated immunotherapy may represent an attractive approach to address TNBC. The objectives of this study were to assess the expression of CXorf61 in TNBCs and healthy tissues and to evaluate its capability to induce T cell responses. We show by transcriptional profiling of a broad comprehensive set of normal human tissue that CXorf61 expression is strictly restricted to testis. 53% of TNBC patients express this antigen in at least 30% of their tumor cells. In CXorf61-negative breast cancer cell lines CXorf61 expression is activated by treatment with the hypomethylating agent 5-aza-2'-deoxycytidine. By vaccination of HLA-A*02-transgenic mice with CXorf61 encoding RNA we obtained high frequencies of CXorf61-specific T cells. Cloning and characterization of T cell receptors (TCRs) from responding T cells resulted in the identification of the two HLA-A*0201-restricted T cell epitopes CXorf6166-74 and CXorf6179-87. Furthermore, by in vitro priming of human CD8+ T cells derived from a healthy donor recognizing CXorf6166-74 we were able to induce a strong antigen-specific immune response and clone a human TCR recognizing this epitope. In summary, our data confirms this antigen as promising target for T cell based therapies.

Asunto(s)

Antígenos de Neoplasias/administración & dosificación , Vacunas contra el Cáncer/administración & dosificación , Neoplasias de la Mama Triple Negativas/terapia , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/metabolismo , Clonación Molecular , Técnicas de Cocultivo , Metilación de ADN , Mapeo Epitopo , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Antígeno HLA-A2/metabolismo , Humanos , Esquemas de Inmunización , Células K562 , Linfocitos Infiltrantes de Tumor/inmunología , Ratones Transgénicos , Persona de Mediana Edad , ARN Mensajero/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transfección , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/metabolismo , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología

RESUMEN

We have developed a highly versatile platform for the systematic retrieval of T-cell receptors (TCRs) from single-antigen-reactive T cells and for characterization of their function and specificity. This approach enables rapid extraction of multiple TCRs from repertoires in individuals and not only broadens the diversity of TCRs suitable for clinical use, but also sets the stage for actively personalized immunotherapeutic strategies.

RESUMEN

The systematic assessment of the human immune system bears huge potential to guide rational development of novel immunotherapies and clinical decision making. Multiple assays to monitor the quantity, phenotype, and function of Ag-specific T cells are commonly used to unravel patients' immune signatures in various disease settings and during therapeutic interventions. When compared with tests measuring soluble analytes, cellular immune assays have a higher variation, which is a major technical factor limiting their broad adoption in clinical immunology. The key solution may arise from continuous control of assay performance using TCR-engineered reference samples. We developed a simple, stable, robust, and scalable technology to generate reference samples that contain defined numbers of functional Ag-specific T cells. First, we show that RNA-engineered lymphocytes, equipped with selected TCRs, can repetitively deliver functional readouts of a controlled size across multiple assay platforms. We further describe a concept for the application of TCR-engineered reference samples to keep assay performance within or across institutions under tight control. Finally, we provide evidence that these novel control reagents can sensitively detect assay variation resulting from typical sources of error, such as low cell quality, loss of reagent stability, suboptimal hardware settings, or inaccurate gating.

Asunto(s)

Pruebas Inmunológicas/métodos , Pruebas Inmunológicas/normas , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Expresión Génica , Ingeniería Genética , Antígenos HLA/química , Antígenos HLA/inmunología , Humanos , Inmunoterapia/métodos , Péptidos/química , Péptidos/inmunología , Multimerización de Proteína , Especificidad del Receptor de Antígeno de Linfocitos T/genética , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología

RESUMEN

The determination of the epitope specificity of disease-associated T-cell responses is relevant for the development of biomarkers and targeted immunotherapies against cancer, autoimmune, and infectious diseases. The lack of known T-cell epitopes and corresponding T-cell receptors (TCR) for novel antigens hinders the efficient development and monitoring of new therapies. We developed an integrated approach for the systematic retrieval and functional characterization of TCRs from single antigen-reactive T cells that includes the identification of epitope specificity. This is accomplished through the rapid cloning of full-length TCR-α and TCR-ß chains directly from single antigen-specific CD8(+) or CD4(+) T lymphocytes. The functional validation of cloned TCRs is conducted using in vitro-transcribed RNA transfer for expression of TCRs in T cells and HLA molecules in antigen-presenting cells. This method avoids the work and bias associated with repetitive cycles of in vitro T-cell stimulation, and enables fast characterization of antigen-specific T-cell responses. We applied this strategy to viral and tumor-associated antigens (TAA), resulting in the retrieval of 56 unique functional antigen-specific TCRs from human CD8(+) and CD4(+) T cells (13 specific for CMV-pp65, 16 specific for the well-known TAA NY-ESO-1, and 27 for the novel TAA TPTE), which are directed against 39 different epitopes. The proof-of-concept studies with TAAs NY-ESO-1 and TPTE revealed multiple novel TCR specificities. Our approach enables the rational development of immunotherapy strategies by providing antigen-specific TCRs and immunogenic epitopes.

Asunto(s)

Epítopos de Linfocito T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Antígenos de Neoplasias/inmunología , Línea Celular , Clonación Molecular , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/inmunología , Fosfoproteínas/inmunología , Unión Proteica , Receptores de Antígenos de Linfocitos T/genética , Proteínas de la Matriz Viral/inmunología