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1.
Proc Natl Acad Sci U S A ; 121(27): e2404661121, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38923991

RESUMO

Immune checkpoint therapies (ICT) improve overall survival of patients with cancer but may cause immune-related adverse events (irAEs) such as myocarditis. Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin fusion protein (CTLA-4 Ig), an inhibitor of T cell costimulation through CD28, reverses irAEs in animal models. However, concerns exist about potentially compromising antitumor response of ICT. In mouse tumor models, we administered CTLA-4 Ig 1) concomitantly with ICT or 2) after ICT completion. Concomitant treatment reduced antitumor efficacy, while post-ICT administration improved efficacy without affecting frequency and function of CD8 T cells. The improved response was independent of the ICT used, whether CTLA-4 or PD-1 blockade. The frequency of Tregs was significantly decreased with CTLA-4 Ig. The resulting increased CD8/Treg ratio potentially underlies the enhanced efficacy of ICT followed by CTLA-4 Ig. This paradoxical mechanism shows that a CTLA-4 Ig regimen shown to reduce irAE severity does not compromise antitumor efficacy.


Assuntos
Antígeno CTLA-4 , Imunoterapia , Animais , Camundongos , Imunoterapia/métodos , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/imunologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T Reguladores/imunologia , Linhagem Celular Tumoral , Abatacepte/uso terapêutico , Abatacepte/farmacologia , Feminino , Humanos , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia
2.
N Engl J Med ; 366(3): 207-15, 2012 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-22256804

RESUMO

BACKGROUND: Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors. METHODS: We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed. RESULTS: Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor. CONCLUSIONS: Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).


Assuntos
Carcinoma de Células Escamosas/genética , Genes ras , Indóis/uso terapêutico , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias Cutâneas/genética , Sulfonamidas/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Indóis/administração & dosagem , Masculino , Camundongos , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Sulfonamidas/administração & dosagem , Vemurafenib
3.
BMC Cancer ; 15: 356, 2015 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-25939769

RESUMO

BACKGROUND: Malignant melanoma is an aggressive tumor type that often develops drug resistance to targeted therapeutics. The production of colony stimulating factor 1 (CSF-1) in tumors recruits myeloid cells such as M2-polarized macrophages and myeloid derived suppressor cells (MDSC), leading to an immune suppressive tumor milieu. METHODS: We used the syngeneic mouse model of BRAF (V600E) -driven melanoma SM1, which secretes CSF-1, to evaluate the ability of the CSF-1 receptor (CSF-1R) inhibitor PLX3397 to improve the antitumor efficacy of the oncogenic BRAF inhibitor vemurafenib. RESULTS: Combined BRAF and CSF-1R inhibition resulted in superior antitumor responses compared with either therapy alone. In mice receiving PLX3397 treatment, a dramatic reduction of tumor-infiltrating myeloid cells (TIM) was observed. In this model, we could not detect a direct effect of TIMs or pro-survival cytokines produced by TIMs that could confer resistance to PLX4032 (vemurafenib). However, the macrophage inhibitory effects of PLX3397 treatment in combination with the paradoxical activation of wild type BRAF-expressing immune cells mediated by PLX4032 resulted in more tumor-infiltrating lymphocytes (TIL). Depletion of CD8+ T-cells abrogated the antitumor response to the combination therapy. Furthermore, TILs isolated from SM1 tumors treated with PLX3397 and PLX4032 displayed higher immune potentiating activity. CONCLUSIONS: The combination of BRAF-targeted therapy with CSF-1R blockade resulted in increased CD8 T-cell responses in the SM1 melanoma model, supporting the ongoing evaluation of this therapeutic combination in patients with BRAF (V600) mutant metastatic melanoma.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Melanoma Experimental/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Aminopiridinas/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Indóis/administração & dosagem , Ativação Linfocitária , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Melanoma Experimental/imunologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Pirróis/administração & dosagem , Sulfonamidas/administração & dosagem , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Vemurafenib
4.
Proc Natl Acad Sci U S A ; 107(32): 14286-91, 2010 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-20624956

RESUMO

A key issue in advancing the use of adoptive cell transfer (ACT) of T cell receptor (TCR) engineered lymphocytes for cancer therapy is demonstrating how TCR transgenic cells repopulate lymphopenic hosts and target tumors in an antigen-specific fashion. ACT of splenocytes from fully immunocompetent HLA-A2.1/K(b) mice transduced with a chimeric murine/human TCR specific for tyrosinase, together with lymphodepletion conditioning, dendritic cell (DC)-based vaccination, and high-dose interleukin-2 (IL-2), had profound antitumor activity against large established MHC- and antigen-matched tumors. Genetic labeling with bioluminescence imaging (BLI) and positron emitting tomography (PET) reporter genes allowed visualization of the distribution and antigen-specific tumor homing of TCR transgenic T cells, with trafficking correlated with antitumor efficacy. After an initial brief stage of systemic distribution, TCR-redirected and genetically labeled T cells demonstrated an early pattern of specific distribution to antigen-matched tumors and locoregional lymph nodes, followed by a more promiscuous distribution 1 wk later with additional accumulation in antigen-mismatched tumors. This approach of TCR engineering and molecular imaging reporter gene labeling is directly translatable to humans and provides useful information on how to clinically develop this mode of therapy.


Assuntos
Imunoterapia Adotiva/métodos , Neoplasias/imunologia , Engenharia de Proteínas/métodos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Animais , Quimiotaxia de Leucócito , Genes Reporter , Humanos , Cinética , Camundongos , Monofenol Mono-Oxigenase/imunologia , Neoplasias/terapia , Linfócitos T/transplante , Distribuição Tecidual
5.
Mol Cancer ; 11: 22, 2012 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-22515704

RESUMO

BACKGROUND: TAK733 is a novel allosteric, non-ATP-binding, inhibitor of the BRAF substrates MEK-1/2. METHODS: The growth inhibitory effects of TAK733 were assessed in a panel of 27 cutaneous and five uveal melanoma cell lines genotyped for driver oncogenic mutations. Flow cytometry, Western blots and metabolic tracer uptake assays were used to characterize the changes induced by exposure to TAK733. RESULTS: Fourteen cutaneous melanoma cell lines with different driver mutations were sensitive to the antiproliferative effects of TAK733, with a higher proportion of BRAFV600E mutant cell lines being highly sensitive with IC50s below 1 nM. The five uveal melanoma cell lines had GNAQ or GNA11 mutations and were either moderately or highly sensitive to TAK733. The tested cell lines wild type for NRAS, BRAF, GNAQ and GNA11 driver mutations were moderately to highly resistant to TAK733. TAK733 led to a decrease in pERK and G1 arrest in most of these melanoma cell lines regardless of their origin, driver oncogenic mutations and in vitro sensitivity to TAK733. MEK inhibition resulted in increase in pMEK more prominently in NRASQ61L mutant and GNAQ mutant cell lines than in BRAFV600E mutant cell lines. Uptake of the metabolic tracers FDG and FLT was inhibited by TAK733 in a manner that closely paralleled the in vitro sensitivity assays. CONCLUSIONS: The MEK inhibitor TAK733 has antitumor properties in melanoma cell lines with different oncogenic mutations and these effects could be detectable by differential metabolic tracer uptake.


Assuntos
Antineoplásicos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologia , Neoplasias Cutâneas/metabolismo , Neoplasias Uveais/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Concentração Inibidora 50 , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Traçadores Radioativos , Transdução de Sinais/efeitos dos fármacos
6.
J Transl Med ; 8: 39, 2010 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-20406486

RESUMO

Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation. Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 muM. Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 muM, and three were moderately sensitive with IC50 values between 1 and 10 muM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity.


Assuntos
Indóis/farmacologia , Melanoma/genética , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Substituição de Aminoácidos/genética , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Diagnóstico por Imagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Genoma Humano/genética , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/enzimologia , Melanoma/patologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vemurafenib
7.
Cancer Immunol Immunother ; 58(5): 699-708, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-18807035

RESUMO

Several tumor immunotherapy approaches result in a low percentage of durable responses in selected cancers. We hypothesized that the insensitivity of cancer cells to immunotherapy may be related to an anti-apoptotic cancer cell milieu, which could be pharmacologically reverted through the inhibition of antiapoptotic Bcl-2 family proteins in cancer cells. ABT-737, a small molecule inhibitor of the antiapoptotic proteins Bcl-2, Bcl-w and Bcl-x(L), was tested for the ability to increase antitumor immune responses in two tumor immunotherapy animal models. The addition of systemic therapy with ABT-737 to the immunization of BALB/c mice with tumor antigen peptide-pulsed dendritic cells (DC) resulted in a significant delay in CT26 murine colon carcinoma tumor growth and improvement in survival. However, the addition of ABT-737 to either a vaccine strategy involving priming with TRP-2 melanoma antigen peptide-pulsed DC and boosting with recombinant Listeria monocytogenes expressing the same melanoma antigen, or the adoptive transfer of TCR transgenic cells, did not result in superior antitumor activity against B16 murine melanoma. In vitro studies failed to demonstrate increased cytotoxic lytic activity when testing the combination of ABT-737 with lymphokine activated killer (LAK) cells, or the death receptor agonists Fas, TRAIL-ligand or TNF-alpha against the CT26 and B16 cell lines. In conclusion, the Bcl-2 inhibitor ABT-737 sensitized cancer cells to the antitumor effect of antigen-specific immunotherapy in a vaccine model for the CT26 colon carcinoma in vivo but not in two immunotherapy strategies against B16 melanoma.


Assuntos
Compostos de Bifenilo/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Neoplasias do Colo/terapia , Imunoterapia/métodos , Melanoma Experimental/terapia , Proteínas de Neoplasias/antagonistas & inibidores , Nitrofenóis/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/uso terapêutico , Animais , Antígenos de Neoplasias/imunologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/imunologia , Neoplasias do Colo/imunologia , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imunoterapia Adotiva , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/imunologia , Células Matadoras Ativadas por Linfocina/transplante , Listeria monocytogenes/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Piperazinas/uso terapêutico , Receptores de Morte Celular/agonistas , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Receptor fas/farmacologia
9.
Oncoimmunology ; 5(7): e1052212, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27622011

RESUMO

The combination of targeted therapy with BRAF and MEK inhibitors has become the standard of care in patients with BRAF (V600E) mutant melanoma, but responses are not durable. In addition, the impressive clinical benefits with anti-PD-1 and anti-PD-L1 antibodies (Ab) in patients with heavily pretreated metastatic melanoma and the synergistic effect of dabrafenib, trametinib and anti-PD-1 compared with single therapy alone groups support the idea that combining dabrafenib, trametinib and immunotherapy based on PD-1 blockade could be an interesting approach in the treatment of metastatic melanoma. With our mouse model of syngeneic BRAF (V600E) driven melanoma (SM1), we tested whether the addition of an immunostimulatory Ab targeting CD137 (4-1BB) and/or CD134 (OX40) would enhance the antitumor effect of dabrafenib, trametinib and anti-PD-1 or anti-PD-L1 therapy. In vitro studies showed that the combination group of dabrafenib, trametinib and anti-PD-1 increases CD8(+) tumor infiltrating lymphocytes (TILs), as well as CD4(+) T cells and tumor-associated macrophages (TAMs). An upregulation of PD-L1 was observed in the combination of dabrafenib, trametinib and anti-PD-1 therapy. Combination of dabrafenib, trametinib and anti-PD-1, with either anti-CD137 or anti-CD134, showed a superior antitumor effect, but the five-agent combination was not superior to the four-agent combinations. In conclusion, the combination of dabrafenib, trametinib, anti-PD1 or anti-PD-L1 therapy results in robust antitumor activity, which is further improved by adding the immune-stimulating Ab anti-CD137 or anti-CD134. Our findings support the testing of these combinations in patients with BRAF (V600E) mutant metastatic melanoma.

10.
Cancer Res ; 76(1): 73-82, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26573799

RESUMO

The rapidly advancing field of cancer immunotherapy is currently limited by the scarcity of noninvasive and quantitative technologies capable of monitoring the presence and abundance of CD8(+) T cells and other immune cell subsets. In this study, we describe the generation of (89)Zr-desferrioxamine-labeled anti-CD8 cys-diabody ((89)Zr-malDFO-169 cDb) for noninvasive immuno-PET tracking of endogenous CD8(+) T cells. We demonstrate that anti-CD8 immuno-PET is a sensitive tool for detecting changes in systemic and tumor-infiltrating CD8 expression in preclinical syngeneic tumor immunotherapy models including antigen-specific adoptive T-cell transfer, agonistic antibody therapy (anti-CD137/4-1BB), and checkpoint blockade antibody therapy (anti-PD-L1). The ability of anti-CD8 immuno-PET to provide whole body information regarding therapy-induced alterations of this dynamic T-cell population provides new opportunities to evaluate antitumor immune responses of immunotherapies currently being evaluated in the clinic.


Assuntos
Linfócitos T CD8-Positivos/diagnóstico por imagem , Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/terapia , Imunoterapia Adotiva/métodos , Tomografia por Emissão de Pósitrons/métodos , Radioisótopos/administração & dosagem , Compostos Radiofarmacêuticos/administração & dosagem , Zircônio/administração & dosagem , Animais , Anticorpos Biespecíficos , Antígenos CD8 , Neoplasias do Colo/imunologia , Desferroxamina/administração & dosagem , Desferroxamina/química , Desferroxamina/imunologia , Modelos Animais de Doenças , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/química , Imunoconjugados/imunologia , Linfócitos do Interstício Tumoral/diagnóstico por imagem , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Radioisótopos/química , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/imunologia , Zircônio/química
11.
Sci Transl Med ; 7(279): 279ra41, 2015 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-25787767

RESUMO

Combining immunotherapy and BRAF targeted therapy may result in improved antitumor activity with the high response rates of targeted therapy and the durability of responses with immunotherapy. However, the first clinical trial testing the combination of the BRAF inhibitor vemurafenib and the CTLA4 antibody ipilimumab was terminated early because of substantial liver toxicities. MEK [MAPK (mitogen-activated protein kinase) kinase] inhibitors can potentiate the MAPK inhibition in BRAF mutant cells while potentially alleviating the unwanted paradoxical MAPK activation in BRAF wild-type cells that lead to side effects when using BRAF inhibitors alone. However, there is the concern of MEK inhibitors being detrimental to T cell functionality. Using a mouse model of syngeneic BRAF(V600E)-driven melanoma, SM1, we tested whether addition of the MEK inhibitor trametinib would enhance the antitumor activity of combined immunotherapy with the BRAF inhibitor dabrafenib. Combination of dabrafenib and trametinib with pmel-1 adoptive cell transfer (ACT) showed complete tumor regression, increased T cell infiltration into tumors, and improved in vivo cytotoxicity. Single-agent dabrafenib increased tumor-associated macrophages and T regulatory cells (Tregs) in tumors, which decreased with the addition of trametinib. The triple combination therapy resulted in increased melanosomal antigen and major histocompatibility complex (MHC) expression and global immune-related gene up-regulation. Given the up-regulation of PD-L1 seen with dabrafenib and/or trametinib combined with antigen-specific ACT, we tested the combination of dabrafenib, trametinib, and anti-PD1 therapy in SM1 tumors, and observed superior antitumor effect. Our findings support the testing of triple combination therapy of BRAF and MEK inhibitors with immunotherapy in patients with BRAF(V600E) mutant metastatic melanoma.


Assuntos
Antineoplásicos/uso terapêutico , Imunoterapia/métodos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Animais , Antígenos de Neoplasias/metabolismo , Antineoplásicos/química , Antígeno CTLA-4/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Imidazóis/química , MAP Quinase Quinase Quinases/imunologia , Complexo Principal de Histocompatibilidade , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oximas/química , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/imunologia , Piridonas/química , Pirimidinonas/química , Neoplasias Cutâneas/imunologia
12.
Cancer Res ; 74(1): 153-161, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24247719

RESUMO

Colony stimulating factor 1 (CSF-1) recruits tumor-infiltrating myeloid cells (TIM) that suppress tumor immunity, including M2 macrophages and myeloid-derived suppressor cells (MDSC). The CSF-1 receptor (CSF-1R) is a tyrosine kinase that is targetable by small molecule inhibitors such as PLX3397. In this study, we used a syngeneic mouse model of BRAF(V600E)-driven melanoma to evaluate the ability of PLX3397 to improve the efficacy of adoptive cell therapy (ACT). In this model, we found that combined treatment produced superior antitumor responses compared with single treatments. In mice receiving the combined treatment, a dramatic reduction of TIMs and a skewing of MHCII(low) to MHCII(hi) macrophages were observed. Furthermore, mice receiving the combined treatment exhibited an increase in tumor-infiltrating lymphocytes (TIL) and T cells, as revealed by real-time imaging in vivo. In support of these observations, TILs from these mice released higher levels of IFN-γ. In conclusion, CSF-1R blockade with PLX3397 improved the efficacy of ACT immunotherapy by inhibiting the intratumoral accumulation of immunosuppressive macrophages.


Assuntos
Imunoterapia Adotiva/métodos , Melanoma/imunologia , Melanoma/terapia , Inibidores de Proteínas Quinases/farmacologia , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/imunologia , Melanoma/enzimologia , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor de Fator Estimulador de Colônias de Macrófagos/imunologia
13.
Oncoimmunology ; 3: e29244, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25083336

RESUMO

Targeting immune inhibitory receptors has brought excitement, innovation and hope to cancer patients. Our recent work revealed the immunological effects of blocking the CTLA4 and PD-1 immune checkpoints on T cell receptor usage among peripheral blood cells, and further uncovers how the expansion of the T cell repertoire matches the immunotoxicity profile of the therapy.

14.
Clin Cancer Res ; 20(9): 2424-32, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24583799

RESUMO

PURPOSE: To evaluate the immunomodulatory effects of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) blockade with tremelimumab in peripheral blood mononuclear cells (PBMC). EXPERIMENTAL DESIGN: We used next-generation sequencing to study the complementarity-determining region 3 (CDR3) from the rearranged T-cell receptor (TCR) variable beta (V-beta) in PBMCs of 21 patients, at baseline and 30 to 60 days after receiving tremelimumab. RESULTS: After receiving tremelimumab, there was a median of 30% increase in unique productive sequences of TCR V-beta CDR3 in 19 out of 21 patients, and a median decrease of 30% in only 2 out of 21 patients. These changes were significant for richness (P = 0.01) and for Shannon index diversity (P = 0.04). In comparison, serially collected PBMCs from four healthy donors did not show a significant change in TCR V-beta CDR3 diversity over 1 year. There was a significant difference in the total unique productive TCR V-beta CDR3 sequences between patients experiencing toxicity with tremelimumab compared with patients without toxicity (P = 0.05). No relevant differences were noted between clinical responders and nonresponders. CONCLUSIONS: CTLA4 blockade with tremelimumab diversifies the peripheral T-cell pool, representing a pharmacodynamic effect of how this class of antibodies modulates the human immune system.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Antígeno CTLA-4/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/metabolismo , Adulto , Idoso , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antineoplásicos/uso terapêutico , Análise por Conglomerados , Regiões Determinantes de Complementaridade/genética , Biologia Computacional , Feminino , Variação Genética , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Melanoma/diagnóstico , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/mortalidade , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Análise de Sequência de DNA , Resultado do Tratamento
15.
Cancer Res ; 73(9): 2782-94, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23418320

RESUMO

Radiotherapy is used to treat many types of cancer, but many treated patients relapse with local tumor recurrence. Tumor-infiltrating myeloid cells (TIM), including CD11b (ITGAM)(+)F4/80 (EMR1)+ tumor-associated macrophages (TAM), and CD11b(+)Gr-1 (LY6G)+ myeloid-derived suppressor cells (MDSC), respond to cancer-related stresses and play critical roles in promoting tumor angiogenesis, tissue remodeling, and immunosuppression. In this report, we used a prostate cancer model to investigate the effects of irradiation on TAMs and MDSCs in tumor-bearing animals. Unexpectedly, when primary tumor sites were irradiated, we observed a systemic increase of MDSCs in spleen, lung, lymph nodes, and peripheral blood. Cytokine analysis showed that the macrophage colony-stimulating factor CSF1 increased by two-fold in irradiated tumors. Enhanced macrophage migration induced by conditioned media from irradiated tumor cells was completely blocked by a selective inhibitor of CSF1R. These findings were confirmed in patients with prostate cancer, where serum levels of CSF1 increased after radiotherapy. Mechanistic investigations revealed the recruitment of the DNA damage-induced kinase ABL1 into cell nuclei where it bound the CSF1 gene promoter and enhanced CSF1 gene transcription. When added to radiotherapy, a selective inhibitor of CSF1R suppressed tumor growth more effectively than irradiation alone. Our results highlight the importance of CSF1/CSF1R signaling in the recruitment of TIMs that can limit the efficacy of radiotherapy. Furthermore, they suggest that CSF1 inhibitors should be evaluated in clinical trials in combination with radiotherapy as a strategy to improve outcomes.


Assuntos
Células Mieloides/citologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/radioterapia , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Cromatina/metabolismo , Dano ao DNA , Humanos , Imuno-Histoquímica/métodos , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/métodos , Transplante de Neoplasias , Radioterapia/métodos
16.
J Clin Invest ; 123(3): 1371-81, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23454771

RESUMO

The BRAF mutant, BRAF(V600E), is expressed in nearly half of melanomas, and oral BRAF inhibitors induce substantial tumor regression in patients with BRAF(V600E) metastatic melanoma. The inhibitors are believed to work primarily by inhibiting BRAF(V600E)-induced oncogenic MAPK signaling; however, some patients treated with BRAF inhibitors exhibit increased tumor immune infiltration, suggesting that a combination of BRAF inhibitors and immunotherapy may be beneficial. We used two relatively resistant variants of Braf(V600E)-driven mouse melanoma (SM1 and SM1WT1) and melanoma-prone mice to determine the role of host immunity in type I BRAF inhibitor PLX4720 antitumor activity. We found that PLX4720 treatment downregulated tumor Ccl2 gene expression and decreased tumor CCL2 expression in both Braf(V600E) mouse melanoma transplants and in de novo melanomas in a manner that was coincident with reduced tumor growth. While PLX4720 did not directly increase tumor immunogenicity, analysis of SM1 tumor-infiltrating leukocytes in PLX4720-treated mice demonstrated a robust increase in CD8(+) T/FoxP3(+)CD4(+) T cell ratio and NK cells. Combination therapy with PLX4720 and anti-CCL2 or agonistic anti-CD137 antibodies demonstrated significant antitumor activity in mouse transplant and de novo tumorigenesis models. These data elucidate a role for host CCR2 in the mechanism of action of type I BRAF inhibitors and support the therapeutic potential of combining BRAF inhibitors with immunotherapy.


Assuntos
Antineoplásicos/farmacologia , Indóis/farmacologia , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sulfonamidas/farmacologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Antineoplásicos/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CCL2/metabolismo , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Imunidade Celular , Imunoterapia , Indóis/administração & dosagem , Masculino , Melanoma/imunologia , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas B-raf/genética , Receptores CCR2/metabolismo , Sulfonamidas/administração & dosagem , Linfócitos T Reguladores/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Cancer Res ; 72(16): 3928-37, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22693252

RESUMO

Combining immunotherapy with targeted therapy blocking oncogenic BRAFV600 may result in improved treatments for advanced melanoma. In this study, we developed a BRAFV600E-driven murine model of melanoma, SM1, which is syngeneic to fully immunocompetent mice. SM1 cells exposed to the BRAF inhibitor vemurafenib (PLX4032) showed partial in vitro and in vivo sensitivity resulting from the inhibition of MAPK pathway signaling. Combined treatment of vemurafenib plus adoptive cell transfer therapy with lymphocytes genetically modified with a T-cell receptor (TCR) recognizing chicken ovalbumin (OVA) expressed by SM1-OVA tumors or pmel-1 TCR transgenic lymphocytes recognizing gp100 endogenously expressed by SM1 resulted in superior antitumor responses compared with either therapy alone. T-cell analysis showed that vemurafenib did not significantly alter the expansion, distribution, or tumor accumulation of the adoptively transferred cells. However, vemurafenib paradoxically increased mitogen-activated protein kinase (MAPK) signaling, in vivo cytotoxic activity, and intratumoral cytokine secretion by adoptively transferred cells. Taken together, our findings, derived from 2 independent models combining BRAF-targeted therapy with immunotherapy, support the testing of this therapeutic combination in patients with BRAFV600 mutant metastatic melanoma.


Assuntos
Imunoterapia Adotiva/métodos , Indóis/farmacologia , Melanoma/terapia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sulfonamidas/farmacologia , Animais , Terapia Combinada , Feminino , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Melanoma/enzimologia , Melanoma/imunologia , Melanoma Experimental/enzimologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Linfócitos T/imunologia , Linfócitos T/patologia , Células Tumorais Cultivadas , Vemurafenib
19.
Clin Cancer Res ; 17(12): 4101-9, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21558401

RESUMO

BACKGROUND: CTLA4 blocking monoclonal antibodies provide durable clinical benefit in a subset of patients with advanced melanoma mediated by intratumoral lymphocytic infiltrates. A key question is defining whether the intratumoral infiltration (ITI) is a differentiating factor between patients with and without tumor responses. METHODS: Paired baseline and postdosing tumor biopsy specimens were prospectively collected from 19 patients with metastatic melanoma, including 3 patients with an objective tumor response, receiving the anti-CTLA4 antibody tremelimumab within a clinical trial with primary endpoint of quantitating CD8(+) cytotoxic T-lymphocyte (CTL) infiltration in tumors. Samples were analyzed for cell density by automated imaging capture and further characterized for functional lymphocyte properties by assessing the cell activation markers HLA-DR and CD45RO, the cell proliferation marker Ki67, and the regulatory T-cell marker FOXP3. RESULTS: There was a highly significant increase in ITI by CD8(+) cells in biopsy samples taken after tremelimumab treatment. This included increases between 1-fold and 100-fold changes in 14 of 18 evaluable cases regardless of clinical tumor response or progression. There was no difference between the absolute number, location, or cell density of infiltrating cells between clinical responders and patients with nonresponding lesions that showed acquired intratumoral infiltrates. There were similar levels of expression of T-cell activation markers (CD45RO, HLA-DR) in both groups and no difference in markers for cell replication (Ki67) or the suppressor cell marker FOXP3. CONCLUSION: CTLA4 blockade induces frequent increases in ITI by T cells despite which only a minority of patients have objective tumor responses.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígenos CD/imunologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Anticorpos Bloqueadores/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Biópsia , Antígeno CTLA-4 , Feminino , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Melanoma/imunologia , Melanoma/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fenótipo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Resultado do Tratamento
20.
Cancer Res ; 69(22): 8693-9, 2009 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19861533

RESUMO

Tumors grow in the presence of antigen-specific T cells, suggesting the existence of intrinsic cancer cell escape mechanisms. We hypothesized that a histone deacetylase (HDAC) inhibitor could sensitize tumor cells to immunotherapy because this class of agents has been reported to increase tumor antigen expression and shift gene expression to a proapoptotic milieu in cancer cells. To test this question, we treated B16 murine melanoma with the combination of the HDAC inhibitor LAQ824 and the adoptive transfer of gp100 melanoma antigen-specific pmel-1 T cells. The combined therapy significantly improved antitumor activity through several mechanisms: (a) increase in MHC and tumor-associated antigen expression by tumor cells; (b) decrease in competing endogenous lymphocytes in recipient mice, resulting in a proliferative advantage for the adoptively transferred cells; and (c) improvement in the functional activity of the adoptively transferred lymphocytes. We confirmed the beneficial effects of this HDAC inhibitor as a sensitizer to immunotherapy in a different model of prophylactic prime-boost vaccination with the melanoma antigen tyrosinase-related protein 2, which also showed a significant improvement in antitumor activity against B16 melanoma. In conclusion, the HDAC inhibitor LAQ824 significantly enhances tumor immunotherapy through effects on target tumor cells as well as improving the antitumor activity of tumor antigen-specific lymphocytes.


Assuntos
Transferência Adotiva , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Melanoma Experimental/terapia , Linfócitos T/transplante , Transferência Adotiva/métodos , Animais , Linhagem Celular Tumoral , Terapia Combinada , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica , Imunoterapia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL
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