RESUMEN
Mutations in KRAS are some of the most common across multiple cancer types and are thus attractive targets for therapy. Recent studies demonstrated that mutant KRAS generates immunogenic neoantigens that are targetable by adoptive T-cell therapy in metastatic diseases. To expand mutant KRAS-specific immunotherapies, it is critical to identify additional HLA-I allotypes that can present KRAS neoantigens and their cognate T-cell receptors (TCR). Here, we identified a murine TCR specific to a KRAS-G12V neoantigen (7VVVGAVGVGK16) using a vaccination approach with transgenic mice expressing HLA-A*03:01 (HLA-A3). This TCR demonstrated exquisite specificity for mutant G12V and not WT KRAS peptides. To investigate the molecular basis for neoantigen recognition by this TCR, we determined its structure in complex with HLA-A3(G12V). G12V-TCR CDR3ß and CDR1ß formed a hydrophobic pocket to interact with p6 Val of the G12V but not the WT KRAS peptide. To improve the tumor sensitivity of this TCR, we designed rational substitutions to improve TCR:HLA-A3 contacts. Two substitutions exhibited modest improvements in TCR binding avidity to HLA-A3 (G12V) but did not sufficiently improve T-cell sensitivity for further clinical development. Our study provides mechanistic insight into how TCRs detect neoantigens and reveals the challenges in targeting KRAS-G12V mutations.
RESUMEN
BACKGROUND: Tumor-specific mutated proteins can create immunogenic non-self, mutation-containing 'neoepitopes' that are attractive targets for adoptive T-cell therapies. To avoid the complexity of defining patient-specific, private neoepitopes, there has been major interest in targeting common shared mutations in driver genes using off-the-shelf T-cell receptors (TCRs) engineered into autologous lymphocytes. However, identifying the precise naturally processed neoepitopes to pursue is a complex and challenging process. One method to definitively demonstrate whether an epitope is presented at the cell surface is to elute peptides bound to a specific major histocompatibility complex (MHC) allele and analyze them by mass spectrometry (MS). These MS data can then be prospectively applied to isolate TCRs specific to the neoepitope. METHODS: We created mono-allelic cell lines expressing one class I HLA allele and one common mutated oncogene in order to eliminate HLA deconvolution requirements and increase the signal of recovered peptides. MHC-bound peptides on the surface of these cell lines were immunoprecipitated, purified, and analyzed using liquid chromatography-tandem mass spectrometry, producing a list of mutation-containing minimal epitopes. To validate the immunogenicity of these neoepitopes, HLA-transgenic mice were vaccinated using the minimal peptides identified by MS in order to generate neoepitope-reactive TCRs. Specificity of these candidate TCRs was confirmed by peptide titration and recognition of transduced targets. RESULTS: We identified precise neoepitopes derived from mutated isoforms of KRAS, EGFR, BRAF, and PIK3CA presented by HLA-A*03:01 and/or HLA-A*11:01 across multiple biological replicates. From our MS data, we were able to successfully isolate murine TCRs that specifically recognize four HLA-A*11:01 restricted neoepitopes (KRAS G13D, PIK3CA E545K, EGFR L858R and BRAF V600E) and three HLA-A*03:01 restricted neoepitopes (KRAS G12V, EGFR L858R and BRAF V600E). CONCLUSIONS: Our data show that an MS approach can be used to demonstrate which shared oncogene-derived neoepitopes are processed and presented by common HLA alleles, and those MS data can rapidly be used to develop TCRs against these common tumor-specific antigens. Although further characterization of these neoepitope-specific murine TCRs is required, ultimately, they have the potential to be used clinically for adoptive cell therapy.
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Neoplasias , Proteínas Proto-Oncogénicas B-raf , Humanos , Ratones , Animales , Proteínas Proto-Oncogénicas p21(ras) , Antígenos de Neoplasias , Antígenos de Histocompatibilidad , Receptores de Antígenos de Linfocitos T/genética , Péptidos , Epítopos , Proteínas de Neoplasias , Antígenos HLA-A , Receptores ErbBRESUMEN
Adoptive cell transfer of tumor-infiltrating lymphocytes (TIL) can mediate durable complete responses in some patients with common epithelial cancers but does so infrequently. A better understanding of T-cell responses to neoantigens and tumor-related immune evasion mechanisms requires having the autologous tumor as a reagent. We investigated the ability of patient-derived tumor organoids (PDTO) to fulfill this need and evaluated their utility as a tool for selecting T-cells for adoptive cell therapy. PDTO established from metastases from patients with colorectal, breast, pancreatic, bile duct, esophageal, lung, and kidney cancers underwent whole exomic sequencing (WES), to define mutations. Organoids were then evaluated for recognition by autologous TIL or T-cells transduced with cloned T-cell receptors recognizing defined neoantigens. PDTO were also used to identify and clone TCRs from TIL targeting private neoantigens and define those tumor-specific targets. PDTO were successfully established in 38/47 attempts. 75% were available within 2 months, a timeframe compatible with screening TIL for clinical administration. These lines exhibited good genetic fidelity with their parental tumors, especially for mutations with higher clonality. Immunologic recognition assays demonstrated instances of HLA allelic loss not found by pan-HLA immunohistochemistry and in some cases WES of fresh tumor. PDTO could also be used to show differences between TCRs recognizing the same antigen and to find and clone TCRs recognizing private neoantigens. PDTO can detect tumor-specific defects blocking T-cell recognition and may have a role as a selection tool for TCRs and TIL used in adoptive cell therapy.
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Neoplasias , Linfocitos T , Humanos , Antígenos de Neoplasias , Neoplasias/metabolismo , Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T , Linfocitos Infiltrantes de TumorRESUMEN
A common theme across multiple successful immunotherapies for cancer is the recognition of tumor-specific mutations (neoantigens) by T cells. The rapid discovery of such antigen responses could lead to improved therapies through the adoptive transfer of T cells engineered to express neoantigen-reactive T cell receptors (TCRs). Here, through CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) and TCR-seq of non-small cell lung cancer (NSCLC) tumor-infiltrating lymphocytes (TILs), we develop a neoantigen-reactive T cell signature based on clonotype frequency and CD39 protein and CXCL13 mRNA expression. Screening of TCRs selected by the signature allows us to identify neoantigen-reactive TCRs with a success rate of 45% for CD8+ and 66% for CD4+ T cells. Because of the small number of samples analyzed (4 patients), generalizability remains to be tested. However, this approach can enable the quick identification of neoantigen-reactive TCRs and expedite the engineering of personalized neoantigen-reactive T cells for therapy.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Antígenos de Neoplasias , Linfocitos T CD8-positivos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Linfocitos Infiltrantes de Tumor , Receptores de Antígenos de Linfocitos T , Linfocitos TRESUMEN
The accurate identification of antitumor T cell receptors (TCRs) represents a major challenge for the engineering of cell-based cancer immunotherapies. By mapping 55 neoantigen-specific TCR clonotypes (NeoTCRs) from 10 metastatic human tumors to their single-cell transcriptomes, we identified signatures of CD8+ and CD4+ neoantigen-reactive tumor-infiltrating lymphocytes (TILs). Neoantigen-specific TILs exhibited tumor-specific expansion with dysfunctional phenotypes, distinct from blood-emigrant bystanders and regulatory TILs. Prospective prediction and testing of 73 NeoTCR signature-derived clonotypes demonstrated that half of the tested TCRs recognized tumor antigens or autologous tumors. NeoTCR signatures identified TCRs that target driver neoantigens and nonmutated viral or tumor-associated antigens, suggesting a common metastatic TIL exhaustion program. NeoTCR signatures delineate the landscape of TILs across metastatic tumors, enabling successful TCR prediction based purely on TIL transcriptomic states for use in cancer immunotherapy.
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Antígenos de Neoplasias/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Metástasis de la Neoplasia , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Transcriptoma , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Redes Reguladoras de Genes , Humanos , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , RNA-Seq , Análisis de la Célula IndividualRESUMEN
Tumor neoepitopes presented by major histocompatibility complex (MHC) class I are recognized by tumor-infiltrating lymphocytes (TIL) and are targeted by adoptive T-cell therapies. Identifying which mutant neoepitopes from tumor cells are capable of recognition by T cells can assist in the development of tumor-specific, cell-based therapies and can shed light on antitumor responses. Here, we generate a ranking algorithm for class I candidate neoepitopes by using next-generation sequencing data and a dataset of 185 neoepitopes that are recognized by HLA class I-restricted TIL from individuals with metastatic cancer. Random forest model analysis showed that the inclusion of multiple factors impacting epitope presentation and recognition increased output sensitivity and specificity compared to the use of predicted HLA binding alone. The ranking score output provides a set of class I candidate neoantigens that may serve as therapeutic targets and provides a tool to facilitate in vitro and in vivo studies aimed at the development of more effective immunotherapies.
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Antígenos de Neoplasias , Neoplasias , Antígenos de Neoplasias/genética , Antígenos HLA/genética , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunoterapia , Linfocitos Infiltrantes de Tumor , Aprendizaje Automático , Neoplasias/genética , Linfocitos TRESUMEN
Immune checkpoint inhibitors and adoptive lymphocyte transfer-based therapies have shown great therapeutic potential in cancers with high tumor mutational burden (TMB), such as melanoma, but not in cancers with low TMB, such as mutant epidermal growth factor receptor (EGFR)-driven lung adenocarcinoma. Precision immunotherapy is an unmet need for most cancers, particularly for cancers that respond inadequately to immune checkpoint inhibitors. Here, we employed large-scale MS-based proteogenomic profiling to identify potential immunogenic human leukocyte antigen (HLA) class I-presented peptides in melanoma and EGFR-mutant lung adenocarcinoma. Similar numbers of peptides were identified from both tumor types. Cell line and patient-specific databases (DBs) were constructed using variants identified from whole-exome sequencing. A de novo search algorithm was used to interrogate the HLA class I immunopeptidome MS data. We identified 12 variant peptides and several classes of tumor-associated antigen-derived peptides. We constructed a cancer germ line (CG) antigen DB with 285 antigens. This allowed us to identify 40 class I-presented CG antigen-derived peptides. The class I immunopeptidome comprised more than 1000 post-translationally modified (PTM) peptides representing 58 different PTMs, underscoring the critical role PTMs may play in HLA binding. Finally, leveraging de novo search algorithm and an annotated long noncoding RNA (lncRNA) DB, we developed a novel lncRNA-encoded peptide discovery pipeline to identify 44 lncRNA-derived peptides that are presented by class I. We validated tandem MS spectra of select variant, CG antigen, and lncRNA-derived peptides using synthetic peptides and performed HLA class I-binding assays to demonstrate binding to class I proteins. In summary, we provide direct evidence of HLA class I presentation of a large number of variant and tumor-associated peptides in both low and high TMB cancer. These results can potentially be useful for precision immunotherapies, such as vaccine or adoptive cell therapies in melanoma and EGFR-mutant lung cancers.
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Adenocarcinoma del Pulmón/metabolismo , Antígenos de Neoplasias/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , Péptidos/metabolismo , Adenocarcinoma del Pulmón/genética , Anciano , Antígenos de Neoplasias/genética , Línea Celular Tumoral , Receptores ErbB/genética , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Neoplasias Pulmonares/genética , Masculino , Melanoma/genética , Mutación , Péptidos/genética , ProteogenómicaRESUMEN
MHC-independent αßTCRs (TCRs) recognize conformational epitopes on native self-proteins and arise in mice lacking both MHC and CD4/CD8 coreceptor proteins. Although naturally generated in the thymus, these TCRs resemble re-engineered therapeutic chimeric antigen receptor (CAR) T cells in their specificity for MHC-independent ligands. Here we identify naturally arising MHC-independent TCRs reactive to three native self-proteins (CD48, CD102, and CD155) involved in cell adhesion. We report that naturally arising MHC-independent TCRs require high affinity TCR-ligand engagements in the thymus to signal positive selection and that high affinity positive selection generates a peripheral TCR repertoire with limited diversity and increased self-reactivity. We conclude that the affinity of TCR-ligand engagements required to signal positive selection in the thymus inversely determines the diversity and self-tolerance of the mature TCR repertoire that is selected.
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Selección Clonal Mediada por Antígenos , Complejo Mayor de Histocompatibilidad/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Autotolerancia/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Timo/fisiología , Animales , Antígenos CD/metabolismo , Antígenos CD8/inmunología , Moléculas de Adhesión Celular/metabolismo , Ligandos , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Complejo Mayor de Histocompatibilidad/genética , Ratones , Ratones Noqueados , Unión Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores Virales/inmunologíaRESUMEN
The adoptive cell transfer (ACT) of T cells targeting mutated neoantigens can cause objective responses in varieties of metastatic cancers, but the development of new T cell-based treatments relies on accurate animal models. To investigate the therapeutic effect of targeting a neoantigen with ACT, we used T cells from pmel-1 T cell receptor-transgenic mice, known to recognize a WT peptide, gp100, and a mutated version of the peptide that has higher avidity. We gene-engineered B16 cells to express the WT or mutated gp100 epitopes and found that pmel-1-specific T cells targeting a neoantigen tumor target augmented recognition as measured by IFN-γ production. Neoantigen expression by B16 also enhanced the capacity of pmel-1 T cells to trigger the complete and durable regression of large, established, vascularized tumor and required less lymphodepleting conditioning. Targeting neoantigen uncovered the possibility of using enforced expression of the IL-2Rα chain (CD25) in mutation-reactive CD8+ T cells to improve their antitumor functionality. These data reveal that targeting of "mutated-self" neoantigens may lead to improved efficacy and reduced toxicities of T cell-based cellular immunotherapies for patients with cancer.
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Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/uso terapéutico , Factores Inmunológicos , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Animales , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer , Quimiocina CCL1 , Modelos Animales de Enfermedad , Epítopos/inmunología , Femenino , Subunidad alfa del Receptor de Interleucina-2/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Antígeno gp100 del Melanoma/genéticaRESUMEN
Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is one of the important molecules that regulate the anti-melanoma T-cell response. Currently, there are some reports showing that CTLA-4 is expressed not only by T cells but also by various kinds of tumor cells, including melanoma cells. However, there is no report that shows the role of CTLA-4 expressed by melanoma cells in melanoma-specific cytotoxic T-lymphocyte (CTL) response. In this report, we confirmed substantial CTLA-4 expression and the localization of CTLA-4 in melanoma cell lines and tissues. Also, we examined its impact on melanoma-specific CTL in vitro, and found that CTLA-4 expressed by melanoma cells does not affect melanoma-specific CTL in the effector phase. Our findings suggest the importance of elucidating the role of CTLA-4 expressed by melanoma cells, particularly in anti-CTLA-4 antibody therapy.
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Antígeno CTLA-4/inmunología , Melanoma/inmunología , Neoplasias Cutáneas/inmunología , Linfocitos T Citotóxicos/inmunología , Antígeno CTLA-4/metabolismo , Línea Celular Tumoral , Humanos , Melanoma/patología , Neoplasias Cutáneas/patología , Linfocitos T Citotóxicos/metabolismoRESUMEN
Purpose: CD70 expression in normal tissues is restricted to activated lymphoid tissues. Targeting CD70 on CD70-expressing tumors could mediate "on-target, off-tumor" toxicity. This study was to evaluate the feasibility and safety of using anti-human CD70 CARs to treat cancer patients whose tumors express CD70.Experimental Design: Seven anti-human CD70 CARs with binding moieties from human CD27 combined with CD3-zeta and different costimulatory domains from CD28 and/or 41BB were constructed. In vitro functionality of these receptors was compared and in vivo treatment efficacy was evaluated in a xenograft mouse model. A homologous, all murine anti-CD70 CAR model was also used to assess treatment-related toxicities.Results: The CAR consisting of the extracellular binding portion of CD27 fused with 41BB and CD3-zeta (trCD27-41BB-zeta) conferred the highest IFNγ production against CD70-expressing tumors in vitro, and NSG mice bearing established CD70-expressing human tumors could be cured by human lymphocytes transduced with this CAR. In the murine CD27-CD3-zeta CAR model, significant reduction of established tumors and prolonged survival were achieved using CAR-transduced splenocytes in a dose-dependent manner. Host preirradiation enhanced treatment efficacy but increased treatment-related toxicities such as transient weight loss and hematopoetic suppression. The treatment did not appear to block adaptive host immune responses.Conclusions: Preclinical testing supports the safety and efficacy of a CD27-containing CAR targeting CD70-expressing tumors. Clin Cancer Res; 23(9); 2267-76. ©2016 AACR.
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Ligando CD27/genética , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/administración & dosificación , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Animales , Ligando CD27/inmunología , Antígenos CD28/genética , Antígenos CD28/inmunología , Complejo CD3/genética , Complejo CD3/inmunología , Línea Celular Tumoral , Humanos , Inmunoterapia Adoptiva , Activación de Linfocitos/efectos de los fármacos , Ratones , Neoplasias/genética , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We used next-generation sequencing to identify somatic alterations in multiple metastatic sites from an "exceptional responder" lung adenocarcinoma patient during his 7-yr course of ERBB2-directed therapies. The degree of heterogeneity was unprecedented, with â¼1% similarity between somatic alterations of the lung and lymph nodes. One novel translocation, PLAG1-ACTA2, present in both sites, up-regulated ACTA2 expression. ERBB2, the predominant driver oncogene, was amplified in both sites, more pronounced in the lung, and harbored an L869R mutation in the lymph node. Functional studies showed increased proliferation, migration, metastasis, and resistance to ERBB2-directed therapy because of L869R mutation and increased migration because of ACTA2 overexpression. Within the lung, a nonfunctional CDK12, due to a novel G879V mutation, correlated with down-regulation of DNA damage response genes, causing genomic instability, and sensitivity to chemotherapy. We propose a model whereby a subclone metastasized early from the primary site and evolved independently in lymph nodes.
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Adenocarcinoma/genética , Neoplasias Pulmonares/genética , Receptor ErbB-2/genética , Adenocarcinoma/metabolismo , Adenocarcinoma del Pulmón , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica/genética , Genes erbB-2/genética , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Persona de Mediana Edad , Mutación , Metástasis de la Neoplasia/genética , Receptor ErbB-2/metabolismo , Resultado del TratamientoRESUMEN
KRAS is one of the most frequently mutated proto-oncogenes in human cancers. The dominant oncogenic mutations of KRAS are single amino acid substitutions at codon 12, in particular G12D and G12V present in 60% to 70% of pancreatic cancers and 20% to 30% of colorectal cancers. The consistency, frequency, and tumor specificity of these "neoantigens" make them attractive therapeutic targets. Recent data associate T cells that target mutated antigens with clinical immunotherapy responses in patients with metastatic melanoma, lung cancer, or cholangiocarcinoma. Using HLA-peptide prediction algorithms, we noted that HLA-A*11:01 could potentially present mutated KRAS variants. By immunizing HLA-A*11:01 transgenic mice, we generated murine T cells and subsequently isolated T-cell receptors (TCR) highly reactive to the mutated KRAS variants G12V and G12D. Peripheral blood lymphocytes (PBL) transduced with these TCRs could recognize multiple HLA-A*11:01(+) tumor lines bearing the appropriate KRAS mutations. In a xenograft model of large established tumor, adoptive transfer of these transduced PBLs reactive with an HLA-A*11:01, G12D-mutated pancreatic cell line could significantly reduce its growth in NSG mice (P = 0.002). The success of adoptive transfer of TCR-engineered T cells against melanoma and other cancers supports clinical trials with these T cells that recognize mutated KRAS in patients with a variety of common cancer types.
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Neoplasias Pancreáticas/terapia , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores de Antígenos de Linfocitos T/fisiología , Traslado Adoptivo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Antígeno HLA-A11/genética , Humanos , Ganglios Linfáticos/patología , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Mutación Missense , Trasplante de Neoplasias , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/inmunología , Bazo/patologíaRESUMEN
Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) results in complete regression of advanced cancer in some patients, but the efficacy of this potentially curative therapy may be limited by poor persistence of TIL after adoptive transfer. Pharmacologic inhibition of the serine/threonine kinase Akt has recently been shown to promote immunologic memory in virus-specific murine models, but whether this approach enhances features of memory (e.g., long-term persistence) in TIL that are characteristically exhausted and senescent is not established. Here, we show that pharmacologic inhibition of Akt enables expansion of TIL with the transcriptional, metabolic, and functional properties characteristic of memory T cells. Consequently, Akt inhibition results in enhanced persistence of TIL after adoptive transfer into an immunodeficient animal model and augments antitumor immunity of CD8 T cells in a mouse model of cell-based immunotherapy. Pharmacologic inhibition of Akt represents a novel immunometabolomic approach to enhance the persistence of antitumor T cells and improve the efficacy of cell-based immunotherapy for metastatic cancer.
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Inmunoterapia Adoptiva/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma Experimental/terapia , Melanoma/terapia , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Animales , Humanos , Memoria Inmunológica , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/patología , Melanoma/inmunología , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-akt/inmunología , Distribución Aleatoria , Células Tumorales CultivadasRESUMEN
Both targeted inhibition of oncogenic driver mutations and immune-based therapies show efficacy in treatment of patients with metastatic cancer, but responses can be either short lived or incompletely effective. Oncogene inhibition can augment the efficacy of immune-based therapy, but mechanisms by which these two interventions might cooperate are incompletely resolved. Using a novel transplantable BRAF(V600E)-mutant murine melanoma model (SB-3123), we explored potential mechanisms of synergy between the selective BRAF(V600E) inhibitor vemurafenib and adoptive cell transfer (ACT)-based immunotherapy. We found that vemurafenib cooperated with ACT to delay melanoma progression without significantly affecting tumor infiltration or effector function of endogenous or adoptively transferred CD8(+) T cells, as previously observed. Instead, we found that the T-cell cytokines IFNγ and TNFα synergized with vemurafenib to induce cell-cycle arrest of tumor cells in vitro. This combinatorial effect was recapitulated in human melanoma-derived cell lines and was restricted to cancers bearing a BRAF(V600E) mutation. Molecular profiling of treated SB-3123 indicated that the provision of vemurafenib promoted the sensitization of SB-3123 to the antiproliferative effects of T-cell effector cytokines. The unexpected finding that immune cytokines synergize with oncogene inhibitors to induce growth arrest has major implications for understanding cancer biology at the intersection of oncogenic and immune signaling and provides a basis for design of combinatorial therapeutic approaches for patients with metastatic cancer.
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Linfocitos T CD8-positivos/inmunología , Citocinas/inmunología , Inmunoterapia Adoptiva , Indoles/uso terapéutico , Melanoma/terapia , Metástasis de la Neoplasia/terapia , Sulfonamidas/uso terapéutico , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de Señal , VemurafenibRESUMEN
Adoptive transfer of tumor-infiltrating lymphocytes (TILs) can mediate regression of metastatic melanoma; however, TILs are a heterogeneous population, and there are no effective markers to specifically identify and select the repertoire of tumor-reactive and mutation-specific CD8⺠lymphocytes. The lack of biomarkers limits the ability to study these cells and develop strategies to enhance clinical efficacy and extend this therapy to other malignancies. Here, we evaluated unique phenotypic traits of CD8⺠TILs and TCR ß chain (TCRß) clonotypic frequency in melanoma tumors to identify patient-specific repertoires of tumor-reactive CD8⺠lymphocytes. In all 6 tumors studied, expression of the inhibitory receptors programmed cell death 1 (PD-1; also known as CD279), lymphocyte-activation gene 3 (LAG-3; also known as CD223), and T cell immunoglobulin and mucin domain 3 (TIM-3) on CD8⺠TILs identified the autologous tumor-reactive repertoire, including mutated neoantigen-specific CD8⺠lymphocytes, whereas only a fraction of the tumor-reactive population expressed the costimulatory receptor 4-1BB (also known as CD137). TCRß deep sequencing revealed oligoclonal expansion of specific TCRß clonotypes in CD8âºPD-1⺠compared with CD8âºPD-1- TIL populations. Furthermore, the most highly expanded TCRß clonotypes in the CD8⺠and the CD8âºPD-1⺠populations recognized the autologous tumor and included clonotypes targeting mutated antigens. Thus, in addition to the well-documented negative regulatory role of PD-1 in T cells, our findings demonstrate that PD-1 expression on CD8⺠TILs also accurately identifies the repertoire of clonally expanded tumor-reactive cells and reveal a dual importance of PD-1 expression in the tumor microenvironment.
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Linfocitos T CD8-positivos/inmunología , Melanoma/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Microambiente Tumoral/inmunología , Traslado Adoptivo , Antígenos CD/genética , Antígenos CD/inmunología , Linfocitos T CD8-positivos/patología , Línea Celular Tumoral , Femenino , Receptor 2 Celular del Virus de la Hepatitis A , Humanos , Masculino , Melanoma/genética , Melanoma/patología , Melanoma/terapia , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Receptor de Muerte Celular Programada 1/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Microambiente Tumoral/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
Despite significant progress in the development of adoptive cell-transfer therapies (ACTs) using gene-engineered T cells, little is known about the fate of cells following infusion. To address that, we performed a comparative analysis of gene expression between T-cell receptor-engineered lymphocytes persisting in the circulation 1 month after administration and the product that was infused. We observed that 156 genes related to immune function were differentially expressed, including underexpression of stimulators of lymphocyte function and overexpression of inhibitory genes in postinfusion cells. Of genes overexpressed postinfusion, the product of programmed cell death 1 (PDCD1), coinhibitory receptor PD-1, was expressed at a higher percentage in postinfusion lymphocytes than in the infusion product. This was associated with a higher sensitivity to inhibition of cytokine production by interaction with its ligand PD-L1. Coinhibitory receptor CD160 was also overexpressed in persisting cells, and its expression was associated with decreased reactivity, which surprisingly was found to be ligand-independent. These results contribute to a deeper understanding of the properties of transgenic lymphocytes used to treat human malignancies and may provide a rationale for the development of combination therapies as a method to improve ACT.
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Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Traslado Adoptivo , Adulto , Animales , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Femenino , Citometría de Flujo , Proteínas Ligadas a GPI/metabolismo , Ingeniería Genética , Humanos , Ligandos , Masculino , Melanoma/sangre , Melanoma/tratamiento farmacológico , Ratones , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores Inmunológicos/metabolismo , Adulto JovenRESUMEN
Clear cell renal cell carcinoma (RCC) is considered an immunogenic tumor, but it has been difficult to identify tumor-infiltrating lymphocytes (TIL) that show in vitro tumor recognition. We compared the characteristics of fresh RCC TIL to peripheral blood lymphocytes (PBL) or melanoma TIL. Our results showed that RCC TIL contained fewer CD27(+) T cells, and fewer naïve and central memory (CM) T cells, but more effector memory (EM) T cells than melanoma TIL or renal PBL. We hypothesized that factors in the RCC microenvironment were skewing TIL phenotype toward EM. One possibility was the expression of CD70 on nearly all human RCCs, but not melanomas. Differentiation of naïve T cells to EM cells only occurred from CD70 costimulation in concert with T-cell receptor (TCR) stimulation (signal one), suggesting that EM TIL responding to CD70 would be enriched for T cells reactive with local antigens, including those associated with RCC. Clonotypic analysis of TCRs in fresh RCCs showed that EM T cells were more clonally expanded than CM or naïve T cells, and the clonal expansion occurred at the tumor site as oligoclonal TCRs were distinct from PBL TCRs from the same patient. In addition, we found that 2 TCRs from the highly represented EM TIL clones, when reexpressed in fresh PBL, recognized an MHC-class II or MHC-class I-restricted antigens shared by multiple RCC lines. Our results suggest that RCC-reactive TIL do exist in situ, but may be difficult to recover and study because of proliferative exhaustion, driven by tumor-expressed CD70.
Asunto(s)
Carcinoma de Células Renales/inmunología , Neoplasias Renales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Ligando CD27/biosíntesis , Ligando CD27/inmunología , Carcinoma de Células Renales/patología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Células Cultivadas , Humanos , Neoplasias Renales/patología , Linfocitos Infiltrantes de Tumor/patología , Melanoma/inmunología , Melanoma/patología , Estadificación de Neoplasias , Fenotipo , Receptores de Antígenos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
Major histocompatibility complex (MHC) restriction is the cardinal feature of T cell antigen recognition and is thought to be intrinsic to αß T cell receptor (TCR) structure because of germline-encoded residues that impose MHC specificity. Here, we analyzed αßTCRs from T cells that had not undergone MHC-specific thymic selection. Instead of recognizing peptide-MHC complexes, the two αßTCRs studied here resembled antibodies in recognizing glycosylation-dependent conformational epitopes on a native self-protein, CD155, and they did so with high affinity independently of MHC molecules. Ligand recognition was via the αßTCR combining site and involved the identical germline-encoded residues that have been thought to uniquely impose MHC specificity, demonstrating that these residues do not only promote MHC binding. This study demonstrates that, without MHC-specific thymic selection, αßTCRs can resemble antibodies in recognizing conformational epitopes on MHC-independent ligands.