RESUMO
Adoptive cell transfer therapies (ACTs) with cytotoxic T cells that target melanocytic antigens can achieve remissions in patients with metastatic melanomas, but tumours frequently relapse. Hypotheses explaining the acquired resistance to ACTs include the selection of antigen-deficient tumour cell variants and the induction of T-cell tolerance. However, the lack of appropriate experimental melanoma models has so far impeded clear insights into the underlying mechanisms. Here we establish an effective ACT protocol in a genetically engineered mouse melanoma model that recapitulates tumour regression, remission and relapse as seen in patients. We report the unexpected observation that melanomas acquire ACT resistance through an inflammation-induced reversible loss of melanocytic antigens. In serial transplantation experiments, melanoma cells switch between a differentiated and a dedifferentiated phenotype in response to T-cell-driven inflammatory stimuli. We identified the proinflammatory cytokine tumour necrosis factor (TNF)-α as a crucial factor that directly caused reversible dedifferentiation of mouse and human melanoma cells. Tumour cells exposed to TNF-α were poorly recognized by T cells specific for melanocytic antigens, whereas recognition by T cells specific for non-melanocytic antigens was unaffected or even increased. Our results demonstrate that the phenotypic plasticity of melanoma cells in an inflammatory microenvironment contributes to tumour relapse after initially successful T-cell immunotherapy. On the basis of our work, we propose that future ACT protocols should simultaneously target melanocytic and non-melanocytic antigens to ensure broad recognition of both differentiated and dedifferentiated melanoma cells, and include strategies to sustain T-cell effector functions by blocking immune-inhibitory mechanisms in the tumour microenvironment.
Assuntos
Desdiferenciação Celular , Imunoterapia , Inflamação/patologia , Melanoma/patologia , Melanoma/terapia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/transplante , Transferência Adotiva , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Inflamação/imunologia , Melanoma/imunologia , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Microambiente Tumoral/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Antígeno gp100 de Melanoma/metabolismoRESUMO
Intorduction: Chondroitin sulfate proteoglycan 4 (CSPG4), also known as high molecular weight-melanoma associated antigen, is expressed in melanoma but also other tumor entities and constitutes an attractive target for immunotherapeutic approaches. While recent preclinical reports focused on anti-CSPG4 chimeric antigen receptors (CAR), we here explore T-cell receptor (TCR)-based approaches targeting CSPG4. Methods: The TCRs of two CSPG4-reactive T-cell clones (11C/73 and 2C/165) restricted by the highly prevalent HLA-C*07:01 allele were isolated and the respective αßTCR pairs were retrovirally expressed in CRISPR/Cas9-edited TCR-knockout T cells for functional testing. We also combined alpha and beta TCR chains derived from 11C/73 and 2C/165 in a cross-over fashion to assess for hemichain dominance. CSPG4+ melanoma, glioblastoma and lung cancer cell lines were identified and, if negative, retrovirally transduced with HLA-C*07:01. Results: Functional tests confirmed specific recognition of CSPG4+HLA-C*07:01+ target cells by the αßTCR retrieved from the parental T-cell clones and in part also by the cross-over TCR construct 2Cα-11Cß. Despite high surface expression, the 11Cα-2Cß combination, however, was not functional. Discussion: Collectively, 11C/73- and 2C/165-expressing T cells specifically and efficiently recognized CSPG4+HLA-C*07:01+ cancer cells which warrants further preclinical and clinical evaluation of these TCRs.
Assuntos
Antígenos HLA-C , Melanoma , Humanos , Antígenos HLA-C/genética , Receptores de Antígenos de Linfócitos T , Linfócitos T , Proteínas de Membrana , Proteoglicanas de Sulfatos de CondroitinaRESUMO
To facilitate preclinical testing of T-cell receptors (TCRs) derived from tumor-reactive T-cell clones it is necessary to develop convenient and rapid cloning strategies for the generation of TCR expression constructs. Herein, we describe a pDONR™221 vector backbone allowing to generate Gateway™ compatible entry clones encoding optimized bicistronic αßTCR constructs. It harbors P2A-linked TCR constant regions and head-to-head-oriented recognition sites of the Type IIS restriction enzymes BsmBI and BsaI for seamless cloning of the TCRα and TCRß V(D)J regions, respectively. Additional well-established TCR optimizations were incorporated to enhance TCR functionality. This included replacing of the human αßTCR constant regions with their codon-optimized murine counterparts for chimerization, addition of a second interchain disulfide bond and arrangement of the TCR chains in the order ß-P2A-α. We exemplified the utility of our vector backbone by cloning and functional testing of three melanoma-reactive TCRs in primary human T cells.
Assuntos
Clonagem Molecular/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Retroviridae/genética , Linfócitos T/citologia , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Técnicas de Cocultura , Vetores Genéticos/genética , Células HEK293 , Humanos , Linfócitos T/imunologia , Linfócitos T/virologia , Transdução Genética , Recombinação V(D)JRESUMO
T lymphocytes against tumor-specific mutated neoantigens can induce tumor regression. Also, the size of the immunogenic cancer mutanome is supposed to correlate with the clinical efficacy of checkpoint inhibition. Herein, we studied the susceptibility of tumor cell lines from lymph node metastases occurring in a melanoma patient over several years towards blood-derived, neoantigen-specific CD8+ T cells. In contrast to a cell line established during early stage III disease, all cell lines generated at later time points from stage IV metastases exhibited partial or complete loss of HLA class I expression. Whole exome and transcriptome sequencing of the four tumor lines and a germline control were applied to identify expressed somatic single nucleotide substitutions (SNS), insertions and deletions (indels). Candidate peptides encoded by these variants and predicted to bind to the patient's HLA class I alleles were synthesized and tested for recognition by autologous mixed lymphocyte-tumor cell cultures (MLTCs). Peptides from four mutated proteins, HERPUD1G161S, INSIG1S238F, MMS22LS437F and PRDM10S1050F, were recognized by MLTC responders and MLTC-derived T cell clones restricted by HLA-A*24:02 or HLA-B*15:01. Intracellular peptide processing was verified with transfectants. All four neoantigens could only be targeted on the cell line generated during early stage III disease. HLA loss variants of any kind were uniformly resistant. These findings corroborate that, although neoantigens represent attractive therapeutic targets, they also contribute to the process of cancer immunoediting as a serious limitation to specific T cell immunotherapy.
Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Genes MHC Classe I , Melanoma/genética , Melanoma/imunologia , Mutação , Linfócitos T/imunologia , Linfócitos T/metabolismo , Alelos , Animais , Apresentação de Antígeno , Linhagem Celular Tumoral , Biologia Computacional/métodos , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL , Melanoma/patologia , Camundongos , Metástase Neoplásica , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , TranscriptomaRESUMO
The ability to use circulating peripheral blood cells and matched tumor sequencing data as a basis for neoantigen prediction has exciting possibilities for application in the personalized treatment of cancer patients. We have used a high-throughput screening approach, combining whole-exome sequence data, mRNA microarrays, and publicly available epitope prediction algorithm output to identify mutated proteins processed and displayed by patient tumors and recognized by circulating immune cells. Matched autologous melanoma cell lines and peripheral blood mononuclear cells were used to create mixed lymphocyte tumor cell cultures, resulting in an expansion of tumor-reactive T cells to use for mutated peptide screening. Five patients were investigated, three of whom had a durable complete response (CR; 15+ years) in an autologous melanoma-pulsed dendritic cell clinical trial. We identified seven mutated antigens in total that stimulated T-effector memory cells in two of the five patients. While the procedure did not result in clinically applicable neoantigens for all patients, those identified were likely important in tumor clearance, leading to durable CR. The nature of the screening process allows results to be obtained rapidly and is easily applicable to a wide variety of different tumor types.
Assuntos
Antígenos de Neoplasias/genética , Exoma/imunologia , Melanoma/genética , Melanoma/imunologia , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Exoma/genética , Humanos , Interferon gama/biossíntese , Linfócitos do Interstício Tumoral/imunologia , MutaçãoRESUMO
Our understanding of pathways leading to antitumor immunity may depend on an undistorted knowledge of the primary antigenic targets of patients' autologous T cell responses. In the melanoma model derived from patient DT, we applied cryopreserved short-term autologous mixed lymphocyte-tumor cell cultures (MLTCs) in combination with an IFN-gamma enzyme-linked immunospot (ELISPOT) assay to cDNA expression screening. We identified three previously unknown peptides processed from melanosomal proteins tyrosinase (presented by HLA-A(*)2601 and -B(*)3801) and gp100 (presented by HLA-B(*)07021) and five neoantigens generated by somatic point mutations in the patient's melanoma. The mutations were found in the genes SIRT2, GPNMB, SNRP116, SNRPD1, and RBAF600. Peptides containing the mutated residues were presented by HLA-A(*)03011, -B(*)07021, and -B(*)3801. Mutation-induced functional impairment was so far demonstrated for SIRT2. Within MLTC responder populations that were independently expanded from the patient's peripheral blood lymphocytes of different years, T cells against mutated epitopes clearly predominated. These results document a high degree of individuality for the cellular antitumor response and support the need for individualizing the monitoring and therapeutic approaches to the primary targets of the autologous T cell response, which may finally lead to a more effective cancer immunotherapy.