RESUMO
Immunotherapy is widely accepted as a powerful new treatment modality for the treatment of cancer. The most successful form of immunotherapy to date has been the blockade of the immune checkpoints PD-1 and CTLA-4. Combining inhibitors of both PD-1 and CTLA-4 increases the proportion of patients who respond to immunotherapy. However, most patients still do not respond to checkpoint inhibitors, and prognostic biomarkers are currently lacking. Therefore, a better understanding of the mechanism by which these checkpoint inhibitors enhance antitumor immune responses is required to more accurately predict which patients are likely to respond and further enhance this treatment modality. Our current study of two mouse tumor models revealed that CD4+Foxp3- cells activated by dual PD-1/CTLA-4 blockade modulated the myeloid compartment, including activation of conventional CD103+ dendritic cells (DC) and expansion of a myeloid subset that produces TNFα and iNOS (TIP-DCs). CD4+Foxp3- T cell-mediated activation of CD103+ DCs resulted in enhanced IL12 production by these cells and IL12 enhanced the therapeutic effect of dual PD-1/CTLA-4 blockade. Given the importance of these myeloid subsets in the antitumor immune response, our data point to a previously underappreciated role of CD4+Foxp3- cells in modulating this arm of the antitumor immune response. Cancer Immunol Res; 6(9); 1069-81. ©2018 AACR.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígeno CTLA-4/antagonistas & inibidores , Células Dendríticas/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Antígenos CD/genética , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/imunologia , Linhagem Celular Tumoral , Fator 3-gama Nuclear de Hepatócito/genética , Imunoterapia , Cadeias alfa de Integrinas/genética , Interleucina-12/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/imunologiaRESUMO
Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs). Herein, we have observed that CAR activation resulted in increased A2AR expression and suppression of both murine and human CAR T cells. This was reversible using either A2AR antagonists or genetic targeting of A2AR using shRNA. In 2 syngeneic HER2+ self-antigen tumor models, we found that either genetic or pharmacological targeting of the A2AR profoundly increased CAR T cell efficacy, particularly when combined with PD-1 blockade. Mechanistically, this was associated with increased cytokine production of CD8+ CAR T cells and increased activation of both CD8+ and CD4+ CAR T cells. Given the known clinical relevance of the CD73/adenosine pathway in several solid tumor types, and the initiation of phase I trials for A2AR antagonists in oncology, this approach has high translational potential to enhance CAR T cell efficacy in several cancer types.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/terapia , Receptor A2A de Adenosina/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Feminino , Humanos , Neoplasias Mamárias Experimentais/genética , Camundongos , Receptor A2A de Adenosina/genética , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
Adoptive immunotherapy utilizing chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematologic cancers, but results against solid malignancies have been limited to date, due in part to the immunosuppressive tumor microenvironment. Activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong costimulatory signals for augmenting and diversifying T-cell responses. We therefore hypothesized that a combination of α-4-1BB and CAR T-cell therapy would result in improved antitumor responses. Using a human-Her2 self-antigen mouse model, we report here that α-4-1BB significantly enhanced CAR T-cell efficacy directed against the Her2 antigen in two different established solid tumor settings. Treatment also increased the expression of IFNγ and the proliferation marker Ki67 in tumor-infiltrating CAR T cells when combined with α-4-1BB. Strikingly, α-4-1BB significantly reduced host immunosuppressive cells at the tumor site, including regulatory T cells and myeloid-derived suppressor cells, correlating with an increased therapeutic response. We conclude that α-4-1BB has a multifunctional role for enhancing CAR T-cell responses and that this combination therapy has high translational potential, given current phase I/II clinical trials with α-4-1BB against various types of cancer. Cancer Res; 77(6); 1296-309. ©2017 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias do Colo/terapia , Imunoterapia Adotiva , Neoplasias Mamárias Experimentais/tratamento farmacológico , Receptores de Antígenos de Linfócitos T/imunologia , Sarcoma Experimental/tratamento farmacológico , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Citotoxicidade Imunológica/imunologia , Feminino , Humanos , Ativação Linfocitária , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Sarcoma Experimental/imunologia , Sarcoma Experimental/patologia , Células Tumorais Cultivadas , Microambiente Tumoral/imunologiaRESUMO
While adoptive immunotherapy using chimeric antigen receptor (CAR)-modified T cells can induce remission of some tumors, the role of other CAR-modified leukocytes is not well characterized. In this study, we characterize the function of leukocytes including natural killer (NK) cells, macrophages and CAR T cells from transgenic mice expressing a CAR under the control of the pan-hematopoietic promoter, vav, and determine the ability of these mice to respond to ERB expressing tumors. We demonstrate the anti-tumor functions of leukocytes, including antigen specific cytotoxicity and cytokine secretion. The adoptive transfer of CAR T cells provided a greater survival advantage in the E0771ERB tumor model than their wildtype (WT) counterparts. In addition, CAR NK cells and CAR T cells also mediated increased survival in the RMAERB tumor model. When challenged with Her2 expressing tumors, F38 mice were shown to mount an effective immune response, resulting in tumor rejection and long-term survival. This was shown to be predominantly dependent on both CD8+ T cells and NK cells. However, macrophages and CD4+ T cells were also shown to contribute to this response. Overall, this study highlights the use of the vav-CAR mouse model as a unique tool to determine the anti-tumor function of various immune subsets, either alone or when acting alongside CAR T cells in adoptive immunotherapy.
Assuntos
Transferência Adotiva/métodos , Citotoxicidade Imunológica/imunologia , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/transplante , Linhagem Celular Tumoral , Citocinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/mortalidade , Proteínas Proto-Oncogênicas c-vav/genética , Receptor ErbB-2/metabolismoRESUMO
Advanced stages of cancer often involve multiple tumors in different locations in the body. These tumors are associated with a microenvironment that can influence tumor responses to immunotherapy. Whether tumors and their disparate microenvironment can interact together at distance in a multiple tumor setting, through a form of cross-talk, and affect their responses to immunotherapy has never been described. Our study investigated the cross-talk between two tumors with disparate microenvironments in a mouse model. We demonstrated that immunosuppressive visceral tumors could influence distant subcutaneous (SC) tumors to render them resistant to immunotherapy. We observed distinct modifications in the SC tumor microenvironment following cross-talk with kidney tumors that exhibit a type-2 macrophage-related immunosuppressive microenvironment. Indeed, when a concomitant kidney tumor was present in the mouse, the SC tumors were highly infiltrated with M2 macrophages and had a reduced T cell and NK cell effector immune profile. Finally, blocking the M2-associated chemokine CCL2 or depleting macrophages, significantly improved the effect of immunotherapy on growth of SC tumors in the presence of concomitant kidney tumors. This work emphasizes the potential negative influence that a tumor, with a strong immunosuppressive microenvironment, can exert on distant tumors that would normally be treatment-responsive. This report may lead to a new vision of the prioritization in the treatment of advanced metastatic cancer.
RESUMO
Immunotherapy is rapidly emerging as a cancer treatment with high potential. Recent clinical trials with anti-CTLA-4 and anti-PD-1/PD-L1 antibodies (mAbs) suggest that targeting multiple immunosuppressive pathways may significantly improve patient survival. The generation of adenosine by CD73 also suppresses antitumor immune responses through the activation of A2A receptors on T cells and natural killer (NK) cells. We sought to determine whether blockade of A2A receptors could enhance the efficacy of anti-PD-1 mAb. The expression of CD73 by tumor cells limited the efficacy of anti-PD-1 mAb in two tumor models, and this was alleviated with concomitant treatment with an A2A adenosine receptor antagonist. The blockade of PD-1 enhanced A2A receptor expression on tumor-infiltrating CD8(+) T cells, making them more susceptible to A2A-mediated suppression. Thus, dual blockade of PD-1 and A2A significantly enhanced the expression of IFNγ and Granzyme B by tumor-infiltrating CD8(+) T cells and, accordingly, increased growth inhibition of CD73(+) tumors and survival of mice. The results of our study indicate that CD73 expression may constitute a potential biomarker for the efficacy of anti-PD-1 mAb in patients with cancer and that the efficacy of anti-PD-1 mAb can be significantly enhanced by A2A antagonists. We have therefore revealed a potentially novel biomarker for the efficacy of anti-PD-1 that warrants further investigation in patients. Because our studies used SYN-115, a drug that has already undergone phase IIb testing in Parkinson disease, our findings have immediate translational relevance for patients with cancer.
Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Anticorpos Monoclonais/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Receptor de Morte Celular Programada 1/imunologia , Pirimidinas/farmacologia , Triazóis/farmacologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Humanos , Interferon gama/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The transcription factor Foxp3 represents the most specific functional marker of CD4+ regulatory T cells (TRegs). However, previous reports have described Foxp3 expression in other cell types including some subsets of macrophages, although there are conflicting reports and Foxp3 expression in cells other than Treg is not well characterized. We performed detailed investigations into Foxp3 expression in macrophages in the normal tissue and tumor settings. We detected Foxp3 protein in macrophages infiltrating mouse renal cancer tumors injected subcutaneously or in the kidney. Expression was demonstrated using flow cytometry and Western blot with two individual monoclonal antibodies. Further analyses confirmed Foxp3 expression in macrophages by RT PCR, and studies using ribonucleic acid-sequencing (RNAseq) demonstrated a previously unknown Foxp3 messenger (m)RNA transcript in tumor-associated macrophages. In addition, depletion of Foxp3+ cells using diphtheria toxin in Foxp3DTR mice reduced the frequency of type-2 macrophages (M2) in kidney tumors. Collectively, these results indicate that tumor-associated macrophages could express Foxp3.
Assuntos
Fatores de Transcrição Forkhead/biossíntese , Neoplasias Renais/imunologia , Macrófagos/imunologia , Linfócitos T Reguladores/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Fatores de Transcrição Forkhead/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , RNA Mensageiro/biossínteseRESUMO
Genetic modification of primary mouse T cells with chimeric antigen receptors (CAR) has emerged as an important tool for optimizing adoptive T cell immunotherapy strategies. However, limitations in current protocols for generating highly pure and sufficient numbers of enriched effector and helper CAR(+) T cell subsets remain problematic. Here, we describe a new retroviral transduction protocol for successfully generating transduced CD8(+) and CD4(+) T lymphocytes for in vitro and in vivo characterization.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Engenharia Genética/métodos , Receptores de Antígenos/genética , Proteínas Recombinantes de Fusão/genética , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Separação Celular , Sobrevivência Celular , Citocinas/metabolismo , Expressão Gênica , Imunoterapia Adotiva , Camundongos , Receptores de Antígenos/imunologia , Receptores de Antígenos/metabolismo , Retroviridae/genética , Baço/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Transdução GenéticaRESUMO
The tumor microenvironment can promote tumor growth and reduce treatment efficacy. Tumors can occur in many sites in the body, but how surrounding normal tissues at different anatomical sites affect tumor microenvironments and their subsequent response to therapy is not known.We demonstrated that tumors from renal, colon, or prostate cell lines in orthotopic locations responded to immunotherapy consisting of three agonist antibodies, termed Tri-mAb, to a much lesser extent than the same tumor type located subcutaneously. A tissue-specific response to Tri-mAb was confirmed by ex vivo separation of subcutaneous (SC) or orthotopic tumor cells from stromal cells, followed by reinjection of tumor cells into the opposite site. Compared with SC tumors, orthotopic tumors had a microenvironment associated with a type 2 immune response, related to immunosuppression, and an involvement of alternatively activated macrophages in the kidney model. Orthotopic kidney tumors were more highly vascularized than SC tumors. Neutralizing the macrophage- and Th2-associated molecules chemokine (C-C motif) ligand 2 or interleukin-13 led to a significantly improved therapeutic effect. This study highlights the importance of the tissue of implantation in sculpting the tumor microenvironment. These are important fundamental issues in tumor biology and crucial factors to consider in the design of experimental models and treatment strategies.
Assuntos
Imunoterapia , Neoplasias/imunologia , Neoplasias/patologia , Microambiente Tumoral/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos CD40/antagonistas & inibidores , Antígenos CD40/imunologia , Linhagem Celular Tumoral , Quimiocina CCL2/imunologia , Neoplasias do Colo/imunologia , Modelos Animais de Doenças , Expressão Gênica , Interleucina-13/imunologia , Neoplasias Renais/imunologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Neoplasias/mortalidade , Neoplasias/terapia , Neovascularização Patológica/imunologia , Especificidade de Órgãos/imunologia , Próstata/imunologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Resultado do Tratamento , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
In many cancers, regulatory T cells (Treg) play a crucial role in suppressing the effector immune response thereby permitting tumor development. Indeed, in mouse models, their depletion can promote the regression of tumors of various origins, including renal cell carcinoma when located subcutaneous (SC). In the present study, we aimed to assess the importance of Treg immunosuppression in the physiologic context of metastatic renal carcinoma (Renca) disease. To that purpose we inoculated renal tumors orthotopically, intra-kidney (IK), in mice. Treg depletions were performed using anti-CD4 antibody in wild type mice or diphtheria toxin (DT) in Foxp3DTR transgenic mice. Our main observation was that Treg were not the key immunosuppressive component of the IK tumoral microenvironment, compared to the same tumors located SC. We demonstrated that the CD8+ effector immune response was still suppressed in IK tumors when compared to SC tumors, following Treg depletion. Furthermore, the level of program cell death protein (PD)-1 was increased on the surface of CD4+ T cells infiltrating IK tumors compared to SC tumors. Finally, the Treg-independent immunosuppression, occurring in IK tumors, was potent enough to inhibit regression of concomitant SC tumors, normally responsive to Treg depletion. Our findings provide further insight into the immunosuppressive nature of the immune response generated in the kidney microenvironment, suggesting that it can have additional mechanisms in addition to Treg. These observations might help to identify better targets from the kidney tumor microenvironment for future cancer therapies.
RESUMO
The presence of an immunosuppressive microenvironment can limit the full potential of adoptive T cell immunotherapy. However, specific blockade of the PD-1 immunosuppressive pathway can significantly enhance the function of gene-modified T cells expressing a chimeric antigen receptor (CAR) leading to enhanced tumor eradication.
RESUMO
The tumor microenvironment is a complex assortment of cells that includes a variety of leukocytes. The overall effect of the microenvironment is to support the growth of tumors and suppress immune responses. Immunotherapy is a highly promising form of cancer treatment, but its efficacy can be severely compromised by an immunosuppressive tumor microenvironment. Chemotherapy and radiation treatment can mediate tumor reduction through cytotoxic effects, but it is becoming increasingly clear that these forms of treatment can be used to modify the tumor microenvironment to liberate tumor antigens and decrease immunosuppression. Chemotherapy and radiotherapy can be used to modulate the tumor microenvironment to enhance immunotherapy.
RESUMO
There is much promise in the use of immunotherapy for the treatment of cancer. Approaches such as those using antibodies or adoptive cell transfer can mediate complete tumor regression in a proportion of patients. However, the tumor microenvironment can inhibit immune responses leading to ineffective or suboptimal responses of tumors to immunotherapy in the majority of cases. As our knowledge of the tumor microenvironment increases, many strategies are emerging for changing the immunosuppressive nature of the tumor toward a microenvironment able to support immunity. These strategies aim to enhance the ability of immunotherapies to initiate effective immune responses able to destroy tumors. In this article, we review approaches that use immunomodulators specifically to modify the tumor microenvironment, and their use in combination with other immune-based strategies for cancer therapy.
RESUMO
CD73 inhibits antitumor immunity through the activation of adenosine receptors expressed on multiple immune subsets. CD73 also enhances tumor metastasis, although the nature of the immune subsets and adenosine receptor subtypes involved in this process are largely unknown. In this study, we revealed that A2A/A2B receptor antagonists were effective in reducing the metastasis of tumors expressing CD73 endogenously (4T1.2 breast tumors) and when CD73 was ectopically expressed (B16F10 melanoma). A2A(-/-) mice were strongly protected against tumor metastasis, indicating that host A2A receptors enhanced tumor metastasis. A2A blockade enhanced natural killer (NK) cell maturation and cytotoxic function in vitro, reduced metastasis in a perforin-dependent manner, and enhanced NK cell expression of granzyme B in vivo, strongly suggesting that the antimetastatic effect of A2A blockade was due to enhanced NK cell function. Interestingly, A2B blockade had no effect on NK cell cytotoxicity, indicating that an NK cell-independent mechanism also contributed to the increased metastasis of CD73(+) tumors. Our results thus revealed that CD73 promotes tumor metastasis through multiple mechanisms, including suppression of NK cell function. Furthermore, our data strongly suggest that A2A or A2B antagonists may be useful for the treatment of metastatic disease. Overall, our study has potential therapeutic implications given that A2A/A2B receptor antagonists have already entered clinical trials in other therapeutic settings.
Assuntos
5'-Nucleotidase/imunologia , Células Matadoras Naturais/imunologia , Neoplasias Experimentais/imunologia , Receptor A2A de Adenosina/imunologia , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Antagonistas do Receptor A2 de Adenosina/farmacologia , Animais , Linhagem Celular Tumoral , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/imunologia , Citometria de Fluxo , Granzimas/imunologia , Granzimas/metabolismo , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Metástase Neoplásica , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Pirimidinas/farmacologia , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/imunologia , Receptor A2B de Adenosina/metabolismo , Triazóis/farmacologia , Xantinas/farmacologiaRESUMO
PURPOSE: To determine the antitumor efficacy and toxicity of a novel combination approach involving adoptive T-cell immunotherapy using chimeric antigen receptor (CAR) T cells with an immunomodulatory reagent for blocking immunosuppression. EXPERIMENTAL DESIGN: We examined whether administration of a PD-1 blocking antibody could increase the therapeutic activity of CAR T cells against two different Her-2(+) tumors. The use of a self-antigen mouse model enabled investigation into the efficacy, mechanism, and toxicity of this combination approach. RESULTS: In this study, we first showed a significant increase in the level of PD-1 expressed on transduced anti-Her-2 CD8(+) T cells following antigen-specific stimulation with PD-L1(+) tumor cells and that markers of activation and proliferation were increased in anti-Her-2 T cells in the presence of anti-PD-1 antibody. In adoptive transfer studies in Her-2 transgenic recipient mice, we showed a significant improvement in growth inhibition of two different Her-2(+) tumors treated with anti-Her-2 T cells in combination with anti-PD-1 antibody. The therapeutic effects observed correlated with increased function of anti-Her-2 T cells following PD-1 blockade. Strikingly, a significant decrease in the percentage of Gr1(+) CD11b(+) myeloid-derived suppressor cells (MDSC) was observed in the tumor microenvironment of mice treated with the combination therapy. Importantly, increased antitumor effects were not associated with any autoimmune pathology in normal tissue expressing Her-2 antigen. CONCLUSION: This study shows that specifically blocking PD-1 immunosuppression can potently enhance CAR T-cell therapy that has significant implications for potentially improving therapeutic outcomes of this approach in patients with cancer.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Neoplasias/genética , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transferência Adotiva , Animais , Anticorpos Monoclonais/administração & dosagem , Antígenos/imunologia , Antineoplásicos/administração & dosagem , Autoimunidade , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Células Mieloides/imunologia , Neoplasias/patologia , Neoplasias/terapia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologiaRESUMO
AIM: The aim of the current study was to fully elucidate the functions of T cells genetically modified with an erbB2-specific chimeric antigen receptor (CAR). MATERIAL & METHODS: In this study, key functional parameters of CAR T cells were examined following antigen-specific stimulation of the chimeric anti-erbB2 receptor. RESULTS: Gene-modified T cells produced the cytokines IFN-γ, IL-2, IL-4, IL-10, TNF-α and IL-17, and the chemokine RANTES upon CAR ligation. A multifunctional capacity of these CAR T cells was also demonstrated, where 13.7% of cells were found to simultaneously express IFN-γ and CD107a, indicative of cytolytic granule release. In addition, the CAR T cells were able to respond to a greater degree on the second ligation of CAR, which has not been previously shown. IFN-γ secretion levels were significantly higher on second ligation than those secreted following initial ligation. CAR-expressing T cells were also demonstrated to lyze erbB2-expressing tumor cells in the absence of activity against bystander cells not expressing the erbB2 antigen, thereby demonstrating exquisite specificity. CONCLUSION: This study demonstrates the specificity of CAR gene-engineered T cells and their capacity to deliver a wide range of functions against tumor cells with an enhanced response capability after initial receptor engagement.
Assuntos
Antígenos CD28/metabolismo , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptor ErbB-2/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/transplante , Animais , Antígenos CD28/genética , Citocinas/metabolismo , Citotoxicidade Imunológica/genética , Células HCT116 , Humanos , Memória Imunológica , Ativação Linfocitária/genética , Camundongos , Engenharia de Proteínas , Receptor ErbB-2/genética , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologiaRESUMO
Genetic engineering of cellular function holds much promise for the treatment of a variety of diseases including gene deficiencies and cancer. However, engineering the full complement of cellular functions can be a daunting genetic exercise since many molecular triggers need to be activated to achieve complete function. In the case of T cells, genes encoding chimeric antigen receptors (CARs) covalently linking antibodies to cytoplasmic signaling domains can trigger some, but not all, cellular functions against cancer cells. To date, relatively few CAR formats have been investigated using a candidate molecule approach, in which rationally chosen molecules were trialed one by one. Therefore, to expedite this arduous process we developed an innovative screening method to screen many thousands of CAR formats to identify genes able to enhance the anticancer ability of T cells. We used a directional in-frame library of randomly assembled signaling domains in a CAR specific for the tumor associated antigen erbB2. Several new and original CARs were identified, one of which had an enhanced ability to lyse cancer cells and inhibit tumor growth in mice. This study highlights novel technology that could be used to screen a variety of molecules for their capacity to induce diverse functions in cells.
Assuntos
Engenharia Celular/métodos , Biblioteca Gênica , Receptores de Antígenos/genética , Proteínas Recombinantes de Fusão/genética , Linfócitos T/citologia , Animais , Linhagem Celular Tumoral , Genes erbB-2/genética , Vetores Genéticos/genética , Humanos , Camundongos , Receptores Imunológicos/genética , Reprodutibilidade dos Testes , Retroviridae/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genéticaRESUMO
Members of the Ikaros family of zinc-finger transcription factors have been shown to be critical for immune and blood cell development. However, the role of the most divergent family member, Pegasus, has remained elusive, although it shows conservation to invertebrate Hunchback proteins that influence embryonic patterning through regulation of homeodomain genes. Zebrafish was employed as a relevant model to investigate the function of Pegasus since it possesses a single pegasus orthologue with high homology to its mammalian counterparts. During zebrafish embryogenesis pegasus transcripts were initially maternally-derived and later replaced by zygotic expression in the diencephalon, tectum, hindbrain, thymus, eye, and ultimately the exocrine pancreas and intestine. Morpholino-mediated knockdown of the zebrafish pegasus gene resulted in disrupted left-right asymmetry of the gut and pancreas. Molecular analysis indicated that zebrafish Pegasus localised to the nucleus in discrete non-nucleolar structures and bound the 'atypical' DNA sequence GN3GN2G, confirming its presumed role as a transcriptional regulator. In vivo transcriptome analysis identified candidate target genes, several of which encoded homeodomain transcription factors. One of these, pitx2, implicated in left-right asymmetry, possessed appropriate 'atypical' Pegasus binding sites in its promoter. Knockdown of Pegasus affected both the level and asymmetry of pitx2 expression, as well as disrupting the asymmetry of the lefty2 and spaw genes, explaining the perturbed left-right patterning in pegasus morphants. Collectively these results provide the first definitive insights into the in vivo role of Pegasus, supporting the notion that it acts as a broader regulator of development, with potential parallels to the related invertebrate Hunchback proteins.
Assuntos
Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Fator de Transcrição Ikaros/metabolismo , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Padronização Corporal/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Marcação de Genes , Células HEK293 , Humanos , Fator de Transcrição Ikaros/química , Fator de Transcrição Ikaros/genética , Dados de Sequência Molecular , Morfolinos/farmacologia , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
Malignant cells are susceptible to viral infection and consequent cell death. Virus-induced cell death is endowed with features that are known to stimulate innate and adaptive immune responses. Thus danger signals emitted by cells succumbing to viral infection as well as viral nucleic acids are detected by specific receptors, and tumor cell antigens can be routed to professional antigen-presenting cells. The anticancer immune response triggered by viral infection is frequently insufficient to eradicate malignancy but may be further amplified. For this purpose, transgenes encoding cytokines as co-stimulatory molecules can be genetically engineered into viral vectors. Alternatively, or in addition, it is possible to use monoclonal antibodies that either block inhibitory receptors of immune effector cells, or act as agonists for co-stimulatory receptors. Combined strategies are based on the ignition of a local immune response at the malignant site plus systemic immune boosting. We have recently reported examples of this approach involving the Vaccinia virus or Semliki Forest virus, interleukin-12 and anti-CD137 monoclonal antibodies.