RESUMO
Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1126-134 (TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (ß1i), which is required for presentation of WT1126-134. An analysis of a second patient treated with TTCR-C4 demonstrated specific loss of AML cells coexpressing ß1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT137-45 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37-45) killed the first patients' relapsed AML resistant to WT1126-134 targeting, as well as other primary AML, in vitro. TTCR37-45 controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.
Assuntos
Leucemia Mieloide Aguda , Complexo de Endopeptidases do Proteassoma , Proteínas WT1 , Animais , Antígenos de Neoplasias , Epitopos , Antígeno HLA-A2 , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Camundongos , Peptídeos , Complexo de Endopeptidases do Proteassoma/imunologia , Complexo de Endopeptidases do Proteassoma/uso terapêutico , Receptores de Antígenos de Linfócitos T , Proteínas WT1/uso terapêuticoRESUMO
Adoptively transferred T cell receptor (TCR)-transgenic T cells (TCR-T cells) are not restricted by cell surface expression of their targets and are therefore poised to become a main pillar of cellular cancer immunotherapies. Addressing clinical and laboratory data, we discuss emerging features for the efficient deployment of novel TCR-T therapies, such as selection of ideal TCRs targeting validated epitopes with well-characterized cancer cell expression and processing, enhancing TCR-T effector function, trafficking, expansion, persistence, and memory formation by strategic selection of substrate cells, and gene-engineering with synthetic co-stimulatory circuits. Overall, a better understanding of the relevant mechanisms of action and resistance will help prioritize the vast array of potential TCR-T optimizations for future clinical products.
Assuntos
Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T Citotóxicos/transplante , Linfócitos T Auxiliares-Indutores/transplante , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Autoantígenos/genética , Autoantígenos/imunologia , Autoantígenos/metabolismo , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Humanos , Camundongos , Mutação , Neoplasias/genética , Neoplasias/imunologia , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Resultado do TratamentoRESUMO
Identifying engineered T cells in situ is important to understand the location, persistence, and phenotype of these cells in patients after adoptive T cell therapy. While engineered cells are routinely characterized in fresh tissue or blood from patients by flow cytometry, it is difficult to distinguish them from endogenous cells in formalin-fixed, paraffin-embedded (FFPE) tissue biopsies. To overcome this limitation, we have developed a method for characterizing engineered T cells in fixed tissue using in situ hybridization (ISH) to the woodchuck hepatitis post-transcriptional regulatory element (WPRE) common in many lentiviral vectors used to transduce chimeric antigen receptor T (CAR-T) and T cell receptor T (TCR-T) cells, coupled with alternative permeabilization conditions that allows subsequent multiplex immunohistochemical (mIHC) staining within the same image. This new method provides the ability to mark the cells by ISH, and simultaneously stain for cell-associated proteins to immunophenotype CAR/TCR modified T cells within tumors, as well as assess potential roles of these cells in on-target/off-tumor toxicity in other tissue.
Assuntos
Imuno-Histoquímica/métodos , Imunofenotipagem/métodos , Receptores de Antígenos Quiméricos/análise , Linfócitos T/imunologia , Animais , Biópsia , Engenharia Celular , Técnicas de Cocultura , Vetores Genéticos/genética , Vírus da Hepatite B da Marmota/genética , Humanos , Hibridização in Situ Fluorescente , Lentivirus/genética , Linfonodos/patologia , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Inclusão em Parafina , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Pele/citologia , Pele/imunologia , Pele/patologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Fixação de Tecidos , Transdução Genética , Quimeras de TransplanteRESUMO
Relapse after allogeneic hematopoietic cell transplantation (HCT) is the leading cause of death in patients with acute myeloid leukemia (AML) entering HCT with poor-risk features1-3. When HCT does produce prolonged relapse-free survival, it commonly reflects graft-versus-leukemia effects mediated by donor T cells reactive with antigens on leukemic cells4. As graft T cells have not been selected for leukemia specificity and frequently recognize proteins expressed by many normal host tissues, graft-versus-leukemia effects are often accompanied by morbidity and mortality from graft-versus-host disease5. Thus, AML relapse risk might be more effectively reduced with T cells expressing receptors (TCRs) that target selected AML antigens6. We therefore isolated a high-affinity Wilms' Tumor Antigen 1-specific TCR (TCRC4) from HLA-A2+ normal donor repertoires, inserted TCRC4 into Epstein-Bar virus-specific donor CD8+ T cells (TTCR-C4) to minimize graft-versus-host disease risk and enhance transferred T cell survival7,8, and infused these cells prophylactically post-HCT into 12 patients ( NCT01640301 ). Relapse-free survival was 100% at a median of 44 months following infusion, while a concurrent comparative group of 88 patients with similar risk AML had 54% relapse-free survival (P = 0.002). TTCR-C4 maintained TCRC4 expression, persisted long-term and were polyfunctional. This strategy appears promising for preventing AML recurrence in individuals at increased risk of post-HCT relapse.
Assuntos
Genes Codificadores dos Receptores de Linfócitos T , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Leucemia Mieloide Aguda/terapia , Proteínas WT1/genética , Adulto , Idoso , Feminino , Humanos , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Recidiva , Transplante HomólogoRESUMO
Osteosarcoma is the most common bone cancer in children and adolescents. Although 70% of patients with localized disease are cured with chemotherapy and surgical resection, patients with metastatic osteosarcoma are typically refractory to treatment. Numerous lines of evidence suggest that cytotoxic T lymphocytes (CTLs) limit the development of metastatic osteosarcoma. We have investigated the role of PD-1, an inhibitory TNFR family protein expressed on CTLs, in limiting the efficacy of immune-mediated control of metastatic osteosarcoma. We show that human metastatic, but not primary, osteosarcoma tumors express a ligand for PD-1 (PD-L1) and that tumor-infiltrating CTLs express PD-1, suggesting this pathway may limit CTLs control of metastatic osteosarcoma in patients. PD-L1 is also expressed on the K7M2 osteosarcoma tumor cell line that establishes metastases in mice, and PD-1 is expressed on tumor-infiltrating CTLs during disease progression. Blockade of PD-1/PD-L1 interactions dramatically improves the function of osteosarcoma-reactive CTLs in vitro and in vivo, and results in decreased tumor burden and increased survival in the K7M2 mouse model of metastatic osteosarcoma. Our results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma should be pursued as a therapeutic strategy.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Neoplasias Ósseas/imunologia , Imunomodulação/efeitos dos fármacos , Osteossarcoma/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T/imunologia , Adolescente , Adulto , Animais , Anticorpos Monoclonais/administração & dosagem , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Criança , Citocinas/metabolismo , Citotoxicidade Imunológica , Modelos Animais de Doenças , Progressão da Doença , Expressão Gênica , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Metástase Neoplásica , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/mortalidade , Osteossarcoma/patologia , Prognóstico , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
T cell expression of inhibitory proteins can be a critical component for the regulation of immunopathology owing to self-reactivity or potentially exuberant responses to pathogens, but it may also limit T cell responses to some malignancies, particularly if the tumor Ag being targeted is a self-protein. We found that the abrogation of Src homology region 2 domain-containing phosphatase-1 (SHP-1) in tumor-reactive CD8(+) T cells improves the therapeutic outcome of adoptive immunotherapy in a mouse model of disseminated leukemia, with benefit observed in therapy employing transfer of CD8(+) T cells alone or in the context of also providing supplemental IL-2. SHP-1(-/-) and SHP-1(+/+) effector T cells were expanded in vitro for immunotherapy. Following transfer in vivo, the SHP-1(-/-) effector T cells exhibited enhanced short-term accumulation, followed by greater contraction, and they ultimately formed similar numbers of long-lived, functional memory cells. The increased therapeutic effectiveness of SHP-1(-/-) effector cells was also observed in recipients that expressed the tumor Ag as a self-antigen in the liver, without evidence of inducing autoimmune toxicity. SHP-1(-/-) effector CD8(+) T cells expressed higher levels of eomesodermin, which correlated with enhanced lysis of tumor cells. Furthermore, reduction of SHP-1 expression in tumor-reactive effector T cells by retroviral transduction with vectors that express SHP-1-specific small interfering RNA, a translatable strategy, also exhibited enhanced antitumor activity in vivo. These studies suggest that abrogating SHP-1 in effector T cells may improve the efficacy of tumor elimination by T cell therapy without affecting the ability of the effector cells to persist and provide a long-term response.