RESUMO
Purpose: Metastatic breast cancers continue to elude current therapeutic strategies, including those utilizing PI3K inhibitors. Given the prominent role of PI3Kα,ß in tumor growth and PI3Kγ,δ in immune cell function, we sought to determine whether PI3K inhibition altered antitumor immunity.Experimental Design: The effect of PI3K inhibition on tumor growth, metastasis, and antitumor immune response was characterized in mouse models utilizing orthotopic implants of 4T1 or PyMT mammary tumors into syngeneic or PI3Kγ-null mice, and patient-derived breast cancer xenografts in humanized mice. Tumor-infiltrating leukocytes were characterized by IHC and FACS analysis in BKM120 (30 mg/kg, every day) or vehicle-treated mice and PI3Kγnull versus PI3KγWT mice. On the basis of the finding that PI3K inhibition resulted in a more inflammatory tumor leukocyte infiltrate, the therapeutic efficacy of BKM120 (30 mg/kg, every day) and anti-PD1 (100 µg, twice weekly) was evaluated in PyMT tumor-bearing mice.Results: Our findings show that PI3K activity facilitates tumor growth and surprisingly restrains tumor immune surveillance. These activities could be partially suppressed by BKM120 or by genetic deletion of PI3Kγ in the host. The antitumor effect of PI3Kγ loss in host, but not tumor, was partially reversed by CD8+ T-cell depletion. Treatment with therapeutic doses of both BKM120 and antibody to PD-1 resulted in consistent inhibition of tumor growth compared with either agent alone.Conclusions: PI3K inhibition slows tumor growth, enhances antitumor immunity, and heightens susceptibility to immune checkpoint inhibitors. We propose that combining PI3K inhibition with anti-PD1 may be a viable therapeutic approach for triple-negative breast cancer. Clin Cancer Res; 23(13); 3371-84. ©2016 AACR.
Assuntos
Proliferação de Células/efeitos dos fármacos , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Neoplasias Mamárias Animais/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Aminopiridinas/administração & dosagem , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Imunidade Celular/efeitos dos fármacos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/imunologia , Neoplasias Mamárias Animais/patologia , Camundongos , Morfolinas/administração & dosagem , Metástase Neoplásica , Inibidores de Fosfoinositídeo-3 Quinase , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Inibidores de Proteínas Quinases/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
T cells recognize cancer cells via HLA/peptide complexes, and when disease overtakes these immune mechanisms, immunotherapy can exogenously target these same HLA/peptide surface markers. We previously identified an HLA-A2-presented peptide derived from macrophage migration inhibitory factor (MIF) and generated antibody RL21A against this HLA-A2/MIF complex. The objective of the current study was to assess the potential for targeting the HLA-A2/MIF complex in ovarian cancer. First, MIF peptide FLSELTQQL was eluted from the HLA-A2 of the human cancerous ovarian cell lines SKOV3, A2780, OV90, and FHIOSE118hi and detected by mass spectrometry. By flow cytometry, RL21A was shown to specifically stain these four cell lines in the context of HLA-A2. Next, partially matched HLA-A*02:01+ ovarian cancer (n = 27) and normal fallopian tube (n = 24) tissues were stained with RL21A by immunohistochemistry to assess differential HLA-A2/MIF complex expression. Ovarian tumor tissues revealed significantly increased RL21A staining compared with normal fallopian tube epithelium (P < 0.0001), with minimal staining of normal stroma and blood vessels (P < 0.0001 and P < 0.001 compared with tumor cells) suggesting a therapeutic window. We then demonstrated the anticancer activity of toxin-bound RL21A via the dose-dependent killing of ovarian cancer cells. In summary, MIF-derived peptide FLSELTQQL is HLA-A2-presented and recognized by RL21A on ovarian cancer cell lines and patient tumor tissues, and targeting of this HLA-A2/MIF complex with toxin-bound RL21A can induce ovarian cancer cell death. These results suggest that the HLA-A2/MIF complex should be further explored as a cell-surface target for ovarian cancer immunotherapy.
Assuntos
Biomarcadores Tumorais/metabolismo , Antígeno HLA-A2/metabolismo , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Neoplasias Ovarianas/metabolismo , Peptídeos/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Afinidade de Anticorpos , Especificidade de Anticorpos , Linhagem Celular Tumoral , Feminino , Antígeno HLA-A2/imunologia , Humanos , Neoplasias Ovarianas/patologiaRESUMO
PURPOSE: Preclinical studies show that inhibition of aurora kinases in melanoma tumors induces senescence and reduces tumor growth, but does not cause tumor regression. Additional preclinical models are needed to identify agents that will synergize with aurora kinase inhibitors to induce tumor regression. EXPERIMENTAL DESIGN: We combined treatment with an aurora kinase A inhibitor, MLN8237, with agents that activate death receptors (Apo2L/TRAIL or death receptor 5 agonists) and monitored the ability of this treatment to induce tumor apoptosis and melanoma tumor regression using human cell lines and patient-derived xenograft (PDX) mouse models. RESULTS: We found that this combined treatment led to apoptosis and markedly reduced cell viability. Mechanistic analysis showed that the induction of tumor cell senescence in response to the AURKA inhibitor resulted in a decreased display of Apo2L/TRAIL decoy receptors and increased display of one Apo2L/TRAIL receptor (death receptor 5), resulting in enhanced response to death receptor ligand/agonists. When death receptors were activated in senescent tumor cells, both intrinsic and extrinsic apoptotic pathways were induced independent of BRAF, NRAS, or p53 mutation status. Senescent tumor cells exhibited BID-mediated mitochondrial depolarization in response to Apo2L/TRAIL treatment. In addition, senescent tumor cells had a lower apoptotic threshold due to decreased XIAP and survivin expression. Melanoma tumor xenografts of one human cell line and one PDX displayed total blockage of tumor growth when treated with MLN8237 combined with DR5 agonist antibody. CONCLUSIONS: These findings provide a strong rationale for combining senescence-inducing therapeutics with death receptor agonists for improved cancer treatment.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Aurora Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Receptores de Morte Celular/agonistas , Animais , Apoptose/genética , Azepinas/farmacologia , Caspases/metabolismo , Linhagem Celular Tumoral , Senescência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Pirimidinas/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Membro 10c de Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.
Assuntos
Biomarcadores Tumorais/análise , Técnicas Biossensoriais/métodos , Neoplasias da Mama/química , Antígeno HLA-A2/análise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Antígeno HLA-A2/química , Antígeno HLA-A2/imunologia , Humanos , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/imunologia , Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Therapeutics that induce cancer cell senescence can block cell proliferation and promote immune rejection. However, the risk of tumor relapse due to senescence escape may remain high due to the long lifespan of senescent cells that are not cleared. Here, we show how combining a senescence-inducing inhibitor of the mitotic kinase Aurora A (AURKA) with an MDM2 antagonist activates p53 in senescent tumors harboring wild-type 53. In the model studied, this effect is accompanied by proliferation arrest, mitochondrial depolarization, apoptosis, and immune clearance of cancer cells by antitumor leukocytes in a manner reliant upon Ccl5, Ccl1, and Cxcl9. The AURKA/MDM2 combination therapy shows adequate bioavailability and low toxicity to the host. Moreover, the prominent response of patient-derived melanoma tumors to coadministered MDM2 and AURKA inhibitors offers a sound rationale for clinical evaluation. Taken together, our work provides a preclinical proof of concept for a combination treatment that leverages both senescence and immune surveillance to therapeutic ends.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Aurora Quinase A/antagonistas & inibidores , Melanoma Experimental/tratamento farmacológico , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Aurora Quinase A/metabolismo , Azepinas/administração & dosagem , Azepinas/farmacologia , Proliferação de Células/efeitos dos fármacos , Humanos , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Melanoma/metabolismo , Melanoma/patologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Pirimidinas/administração & dosagem , Pirimidinas/farmacologiaRESUMO
Myeloid cells are capable of promoting or eradicating tumor cells and the nodal functions that contribute to their different roles are still obscure. Here, we show that mice with myeloid-specific genetic loss of the NF-κB pathway regulatory kinase IKKß exhibit more rapid growth of cutaneous and lung melanoma tumors. In a BRAF(V600E/PTEN(-/-)) allograft model, IKKß loss in macrophages reduced recruitment of myeloid cells into the tumor, lowered expression of MHC class II molecules, and enhanced production of the chemokine CCL11, thereby negatively regulating dendritic-cell maturation. Elevated serum and tissue levels of CCL11 mediated suppression of dendritic-cell differentiation/maturation within the tumor microenvironment, skewing it toward a Th2 immune response and impairing CD8(+) T cell-mediated tumor cell lysis. Depleting macrophages or CD8(+) T cells in mice with wild-type IKKß myeloid cells enhanced tumor growth, where the myeloid cell response was used to mediate antitumor immunity against melanoma tumors (with less dependency on a CD8(+) T-cell response). In contrast, myeloid cells deficient in IKKß were compromised in tumor cell lysis, based on their reduced ability to phagocytize and digest tumor cells. Thus, mice with continuous IKKß signaling in myeloid-lineage cells (IKKß(CA)) exhibited enhanced antitumor immunity and reduced melanoma outgrowth. Collectively, our results illuminate new mechanisms through which NF-κB signaling in myeloid cells promotes innate tumor surveillance.
Assuntos
Quinase I-kappa B/genética , Imunidade Inata , Melanoma Experimental/genética , Microambiente Tumoral/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas , Humanos , Quinase I-kappa B/metabolismo , Macrófagos/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Knockout , Células Mieloides/imunologia , Células Mieloides/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais/imunologia , Microambiente Tumoral/genéticaRESUMO
Bone marrow-derived human mesenchymal stem cells (hMSCs) either promote or inhibit cancer progression, depending on factors that heretofore have been undefined. Here we have utilized extreme hypoxia (0.5% O2) and concurrent treatment with metal carcinogen (nickel) to evaluate the passage-dependent response of hMSCs toward cancerous transformation. Effects of hypoxia and nickel treatment on hMSC proliferation, apoptosis, gene and protein expression, replicative senescence, reactive oxygen species (ROS), redox mechanisms, and in vivo tumor growth were analyzed. The behavior of late passage hMSCs in a carcinogenic hypoxia environment follows a profile similar to that of transformed cancer cells (i.e., increased expression of oncogenic proteins, decreased expression of tumor suppressor protein, increased proliferation, decreased apoptosis, and aberrant redox mechanisms), but this effect was not observed in earlier passage control cells. These events resulted in accumulated intracellular ROS in vitro and excessive proliferation in vivo. We suggest a mechanism by which carcinogenic hypoxia modulates the activity of three critical transcription factors (c-MYC, p53, and HIF1), resulting in accumulated ROS and causing hMSCs to undergo cancer-like behavioral changes. This is the first study to utilize carcinogenic hypoxia as an environmentally relevant experimental model for studying the age-dependent cancerous transformation of hMSCs.
Assuntos
Transformação Celular Neoplásica , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Níquel/farmacologia , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Knockout , Camundongos Nus , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transplante Heterólogo , Carga Tumoral/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Oncogene-induced senescence can provide a protective mechanism against tumour progression. However, production of cytokines and growth factors by senescent cells may contribute to tumour development. Thus, it is unclear whether induction of senescence represents a viable therapeutic approach. Here, using a mouse model with orthotopic implantation of metastatic melanoma tumours taken from 19 patients, we observed that targeting aurora kinases with MLN8054/MLN8237 impaired mitosis, induced senescence and markedly blocked proliferation in patient tumour implants. Importantly, when a subset of tumour-bearing mice were monitored for tumour progression after pausing MLN8054 treatment, 50% of the tumours did not progress over a 12-month period. Mechanistic analyses revealed that inhibition of aurora kinases induced polyploidy and the ATM/Chk2 DNA damage response, which mediated senescence and a NF-κB-related, senescence-associated secretory phenotype (SASP). Blockade of IKKß/NF-κB led to reversal of MLN8237-induced senescence and SASP. Results demonstrate that removal of senescent tumour cells by infiltrating myeloid cells is crucial for inhibition of tumour re-growth. Altogether, these data demonstrate that induction of senescence, coupled with immune surveillance, can limit melanoma growth.
Assuntos
Azepinas/farmacologia , Benzazepinas/farmacologia , Melanoma Experimental/tratamento farmacológico , NF-kappa B/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Aurora Quinases , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Quinase do Ponto de Checagem 2 , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Melanoma Experimental/secundário , Camundongos , Camundongos Nus , Poliploidia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although human angiosarcoma has been associated frequently with mutational inactivation of the tumor suppressor gene Ink4a/Arf, the underlying mechanisms have not been delineated. Here we report that malignant angiosarcoma is associated with high levels of RelA/NF-κB and IL-6 in contrast to normal vessels or benign hemagiomas. Studies of Ink4a/Arf deficient mice not only recapitulate genetic traits observed in human angiosarcoma, but also unveil a possible therapeutic link comprised of the NF-kB/IL-6/Stat3 signaling axis. In Ink4a/Arf(-/-) cells, NF-κB controlled Stat3 signaling by transcriptionally controlling the expression of IL-6, gp130, and Jak2. Further, IL-6 mediated Stat3 signaling through the sIL-6R. Inhibition of Ikkß solely in myeloid cells was insufficient to block angiosarcoma development; in contrast, systemic inhibition of Ikkß, IL-6, or Stat3 markedly inhibited angiosarcoma growth. Our findings offer clinical implications for targeting the NF-kB/IL-6/STAT3 pathway as a rational strategy to treat angiosarcoma.
Assuntos
Hemangiossarcoma/metabolismo , Quinase I-kappa B/metabolismo , Interleucina-6/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Animais , Western Blotting , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Análise Serial de Tecidos , Fator de Transcrição RelA/metabolismoRESUMO
The contribution of CXCR7 to the tumor microenvironment has introduced a new level of complexity to CXCL12 signaling in breast cancer. In the previous issue of Breast Cancer Research, Hernandez and colleagues delineate the roles of CXCR4 and CXCR7 in tumor invasion and metastasis. The authors demonstrate that co-expression of CXCR7 and CXCR4 results in inhibition of CXCL12-mediated invasion, reduced intravasation of tumor cells into the vasculature, and fewer lung metastases compared with parental tumors. The results of this study suggest the combination of small molecule inhibitors of CXCR4 and CXCR7 could dramatically reduce invasion, intravasation, and metastasis and could be highly beneficial for the treatment of invasive breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Animais , Feminino , HumanosRESUMO
PURPOSE: The purpose of this preclinical study was to determine the effectiveness of RAF265, a multikinase inhibitor, for treatment of human metastatic melanoma and to characterize traits associated with drug response. EXPERIMENTAL DESIGN: Advanced metastatic melanoma tumors from 34 patients were orthotopically implanted to nude mice. Tumors that grew in mice (17 of 34) were evaluated for response to RAF265 (40 mg/kg, every day) over 30 days. The relation between patient characteristics, gene mutation profile, global gene expression profile, and RAF265 effects on tumor growth, mitogen-activated protein/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation, proliferation, and apoptosis markers was evaluated. RESULTS: Nine of the 17 tumors that successfully implanted (53%) were mutant BRAF (BRAF(V600E/K)), whereas eight of 17 (47%) tumors were BRAF wild type (BRAF(WT)). Tumor implants from 7 of 17 patients (41%) responded to RAF265 treatment with more than 50% reduction in tumor growth. Five of the 7 (71%) responders were BRAF(WT), of which 1 carried c-KIT(L576P) and another N-RAS(Q61R) mutation, while only 2 (29%) of the responding tumors were BRAF(V600E/K). Gene expression microarray data from nonimplanted tumors revealed that responders exhibited enriched expression of genes involved in cell growth, proliferation, development, cell signaling, gene expression, and cancer pathways. Although response to RAF265 did not correlate with pERK1/2 reduction, RAF265 responders did exhibit reduced pMEK1, reduced proliferation based upon reduced Ki-67, cyclin D1 and polo-like kinase1 levels, and induction of the apoptosis mediator BCL2-like 11. CONCLUSIONS: Orthotopic implants of patient tumors in mice may predict prognosis and treatment response for melanoma patients. A subpopulation of human melanoma tumors responds to RAF265 and can be characterized by gene mutation and gene expression profiles.
Assuntos
Imidazóis/farmacologia , Imidazóis/uso terapêutico , Melanoma/tratamento farmacológico , Piridinas/farmacologia , Piridinas/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Proteínas de Ciclo Celular/biossíntese , Proliferação de Células/efeitos dos fármacos , Ciclina D1/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Antígeno Ki-67/biossíntese , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Melanoma/metabolismo , Melanoma/patologia , Melanoma/secundário , Camundongos , Camundongos Nus , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência de DNA , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/biossíntese , Quinase 1 Polo-LikeRESUMO
The identification and validation of new cancer-specific T cell epitopes continues to be a major area of research interest. Nevertheless, challenges remain to develop strategies that can easily discover and validate epitopes expressed in primary cancer cells. Regarded as targets for T cells, peptides presented in the context of the major histocompatibility complex (MHC) are recognized by monoclonal antibodies (mAbs). These mAbs are of special importance as they lend themselves to the detection of epitopes expressed in primary tumor cells. Here, we use an approach that has been successfully utilized in two different infectious disease applications (WNV and influenza). A direct peptide-epitope discovery strategy involving mass spectrometric analysis led to the identification of peptide YLLPAIVHI in the context of MHC A*02 allele (YLL/A2) from human breast carcinoma cell lines. We then generated and characterized an anti-YLL/A2 mAb designated as RL6A TCRm. Subsequently, the TCRm mAb was used to directly validate YLL/A2 epitope expression in human breast cancer tissue, but not in normal control breast tissue. Moreover, mice implanted with human breast cancer cells grew tumors, yet when treated with RL6A TCRm showed a marked reduction in tumor size. These data demonstrate for the first time a coordinated direct discovery and validation strategy that identified a peptide/MHC complex on primary tumor cells for antibody targeting and provide a novel approach to cancer immunotherapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/terapia , Epitopos de Linfócito T/imunologia , Fragmentos de Peptídeos/uso terapêutico , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/imunologia , Western Blotting , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/imunologia , Vacinas Anticâncer/uso terapêutico , RNA Helicases DEAD-box/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Nus , Mimetismo Molecular , Fragmentos de Peptídeos/imunologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ressonância de Plasmônio de SuperfícieRESUMO
HLA-B27- and -B57-positive HIV-infected humans have long been associated with control of HIV replication, implying that CD8(+) T cell responses contribute to control of viral replication. In a similar fashion, 50% of Mamu-B*08-positive Indian rhesus macaques control SIVmac239 replication and become elite controllers with chronic-phase viremia <1000 viral RNA copies/ml. Interestingly, Mamu-B*08-restricted SIV-derived epitopes appeared to match the peptide binding profile for HLA-B*2705 in humans. We therefore defined a detailed peptide-binding motif for Mamu-B*08 and investigated binding similarities between the macaque and human MHC class I molecules. Analysis of a panel of approximately 900 peptides revealed that despite substantial sequence differences between Mamu-B*08 and HLA-B*2705, the peptide-binding repertoires of these two MHC class I molecules share a remarkable degree of overlap. Detailed knowledge of the Mamu-B*08 peptide-binding motif enabled us to identify six additional novel Mamu-B*08-restricted SIV-specific CD8(+) T cell immune responses directed against epitopes in Gag, Vpr, and Env. All 13 Mamu-B*08-restricted epitopes contain an R at the position 2 primary anchor and 10 also possess either R or K at the N terminus. Such dibasic peptides are less prone to cellular degradation. This work highlights the relevance of the Mamu-B*08-positive SIV-infected Indian rhesus macaque as a model to examine elite control of immunodeficiency virus replication. The remarkable similarity of the peptide-binding motifs and repertoires for Mamu-B*08 and HLA-B*2705 suggests that the nature of the peptide bound by the MHC class I molecule may play an important role in control of immunodeficiency virus replication.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/imunologia , Peptídeos/metabolismo , Vírus da Imunodeficiência Símia/imunologia , Replicação Viral , Sequência de Aminoácidos , Animais , DNA Polimerase Dirigida por DNA , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/química , Humanos , Ligantes , Macaca mulatta , Dados de Sequência Molecular , Biblioteca de Peptídeos , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
Cellular immune mechanisms detect and destroy cancerous and infected cells via the human leukocyte antigen (HLA) class I molecules that present peptides of intracellular origin on the surface of all nucleated cells. The identification of novel, tumor-specific epitopes is a critical step in the development of immunotherapeutics for breast cancer. To directly identify peptide epitopes unique to cancerous cells, secreted human class I HLA molecules (sHLA) were constructed by deletion of the transmembrane and cytoplasmic domain of HLA A*0201. The resulting sHLA-A*0201 was transferred and expressed in breast cancer cell lines MCF-7, MDA-MB-231, and BT-20 as well as in the immortal, nontumorigenic cell line MCF10A. Stable transfectants were seeded into bioreactors for production of > 25 mg of sHLA-A*0201. Peptides eluted from affinity purified sHLA were analyzed by mass spectroscopy. Comparative analysis of HLA-A*0201 peptides revealed 5 previously uncharacterized epitopes uniquely presented on breast cancer cells. These peptides were derived from intracellular proteins with either well-defined or putative roles in breast cancer development and progression: Cyclin Dependent Kinase 2 (Cdk2), Ornithine Decarboxylase (ODC1), Kinetochore Associated 2 (KNTC2 or HEC1), Macrophage Migration Inhibitory Factor (MIF), and Exosome Component 6 (EXOSC6). Cellular recognition of the MIF, KNTC2, EXOSC6, and Cdk2 peptides by circulating CD8+ cells was demonstrated by tetramer staining and IFN-gamma ELISPOT. The identification and characterization of peptides unique to the class I of breast cancer cells provide putative targets for the development of immune diagnostic tools and therapeutics.