RESUMO
ER+ breast cancers (BC) are characterized by the elevated expression and signaling of estrogen receptor alpha (ESR1), which renders them sensitive to anti-endocrine therapy. While these therapies are clinically effective, prolonged treatment inevitably results in therapeutic resistance, which can occur through the emergence of gain-of-function mutations in ESR1. The central importance of ESR1 and development of mutated forms of ESR1 suggest that vaccines targeting these proteins could potentially be effective in preventing or treating endocrine resistance. To explore the potential of this approach, we developed several recombinant vaccines encoding different mutant forms of ESR1 (ESR1mut) and validated their ability to elicit ESR1-specific T cell responses. We then developed novel ESR1mut-expressing murine mammary cancer models to test the anti-tumor potential of ESR1mut vaccines. We found that these vaccines could suppress tumor growth, ESR1mut expression and estrogen signaling in vivo. To illustrate the applicability of these findings, we utilize HPLC to demonstrate the presentation of ESR1 and ESR1mut peptides on human ER+ BC cell MHC complexes. We then show the presence of human T cells reactive to ESR1mut epitopes in an ER+ BC patient. These findings support the development of ESR1mut vaccines, which we are testing in a Phase I clinical trial.
Assuntos
Neoplasias da Mama , Vacinas , Humanos , Animais , Camundongos , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Mutação , Estrogênios/uso terapêutico , Transdução de Sinais , Vacinas/uso terapêuticoRESUMO
Therapeutic cancer vaccines, designed to activate immune effectors against tumor antigens, utilize a number of different platforms for antigen delivery. Among these are messenger RNAs (mRNA), successfully deployed in some prophylactic SARS-CoV2 vaccines. To enhance the immunogenicity of mRNA-delivered epitopes, self-replicating RNAs (srRNA) that markedly increase epitope expression have been developed. These vectors are derived from positive-strand RNA viruses in which the structural protein genes have been replaced with heterologous genes of interest, and the structural proteins are provided in trans to create single cycle viral replicon particles (VRPs). Clinical stage srRNA vectors have been derived from alphaviruses, including Venezuelan Equine Encephalitis (VEE), Sindbis, and Semliki Forest virus (SFV) and have encoded the tumor antigens carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER2), prostate specific membrane antigen (PSMA), and human papilloma virus (HPV) antigens E6 and E7. Adverse events have mainly been grade 1 toxicities and minimal injection site reactions. We review here the clinical experience with these vaccines and our recent safety data from a study combining a VRP encoding HER2 plus an anti-PD1 monoclonal antibody (pembrolizumab). This experience with VRP-based srRNA supports recent development of fully synthetic srRNA technologies, where the viral structural proteins are replaced with protective lipid nanoparticles (LNP), cationic nanoemulsions or polymers.
Assuntos
COVID-19 , Vacinas Anticâncer , Vírus da Encefalite Equina Venezuelana , Neoplasias , Humanos , RNA Viral/genética , Vacinas Anticâncer/genética , Vírus da Encefalite Equina Venezuelana/genética , COVID-19/genética , SARS-CoV-2/genética , RNA Mensageiro , Replicon , Vetores Genéticos , Neoplasias/genética , Neoplasias/terapiaRESUMO
BACKGROUND: There remains a significant need to eliminate the risk of recurrence of resected cancers. Cancer vaccines are well tolerated and activate tumor-specific immune effectors and lead to long-term survival in some patients. We hypothesized that vaccination with alphaviral replicon particles encoding tumor associated antigens would generate clinically significant antitumor immunity to enable prolonged overall survival (OS) in patients with both metastatic and resected cancer. METHODS: OS was monitored for patients with stage IV cancer treated in a phase I study of virus-like replicon particle (VRP)-carcinoembryonic antigen (CEA), an alphaviral replicon particle encoding a modified CEA. An expansion cohort of patients (n=12) with resected stage III colorectal cancer who had completed their standard postoperative adjuvant chemotherapy was administered VRP-CEA every 3 weeks for a total of 4 immunizations. OS and relapse-free survival (RFS) were determined, as well as preimmunization and postimmunization cellular and humoral immunity. RESULTS: Among the patients with stage IV cancer, median follow-up was 10.9 years and 5-year survival was 17%, (95% CI 6% to 33%). Among the patients with stage III cancer, the 5-year RFS was 75%, (95%CI 40% to 91%); no deaths were observed. At a median follow-up of 5.8 years (range: 3.9-7.0 years) all patients were still alive. All patients demonstrated CEA-specific humoral immunity. Patients with stage III cancer had an increase in CD8 +TEM (in 10/12) and decrease in FOXP3 +Tregs (in 10/12) following vaccination. Further, CEA-specific, IFNγ-producing CD8+granzyme B+TCM cells were increased. CONCLUSIONS: VRP-CEA induces antigen-specific effector T cells while decreasing Tregs, suggesting favorable immune modulation. Long-term survivors were identified in both cohorts, suggesting the OS may be prolonged.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/tratamento farmacológico , Memória Imunológica/fisiologia , Linfócitos T Reguladores/imunologia , Neoplasias do Colo/mortalidade , Feminino , Humanos , Masculino , Estadiamento de Neoplasias , Análise de SobrevidaRESUMO
PURPOSE: Immune-based therapy for metastatic breast cancer has had limited success, particularly in molecular subtypes with low somatic mutations rates. Strategies to augment T-cell infiltration of tumors include vaccines targeting established oncogenic drivers such as the genomic amplification of HER2. We constructed a vaccine based on a novel alphaviral vector encoding a portion of HER2 (VRP-HER2). PATIENTS AND METHODS: In preclinical studies, mice were immunized with VRP-HER2 before or after implantation of hHER2+ tumor cells and HER2-specific immune responses and antitumor function were evaluated. We tested VRP-HER2 in a phase I clinical trial where subjects with advanced HER2-overexpressing malignancies in cohort 1 received VRP-HER2 every 2 weeks for a total of 3 doses. In cohort 2, subjects received the same schedule concurrently with a HER2-targeted therapy. RESULTS: Vaccination in preclinical models with VRP-HER2 induced HER2-specific T cells and antibodies while inhibiting tumor growth. VRP-HER2 was well tolerated in patients and vaccination induced HER2-specific T cells and antibodies. Although a phase I study, there was 1 partial response and 2 patients with continued stable disease. Median OS was 50.2 months in cohort 1 (n = 4) and 32.7 months in cohort 2 (n = 18). Perforin expression by memory CD8 T cells post-vaccination significantly correlated with improved PFS. CONCLUSIONS: VRP-HER2 increased HER2-specific memory CD8 T cells and had antitumor effects in preclinical and clinical studies. The expansion of HER2-specific memory CD8 T cells in vaccinated patients was significantly correlated with increased PFS. Subsequent studies will seek to enhance T-cell activity by combining with anti-PD-1.
Assuntos
Neoplasias da Mama/terapia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/administração & dosagem , Imunidade Humoral/imunologia , Memória Imunológica/imunologia , Receptor ErbB-2/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Adulto , Idoso , Alphavirus/genética , Animais , Apoptose , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Vacinas Anticâncer/imunologia , Proliferação de Células , Células Dendríticas/imunologia , Feminino , Seguimentos , Vetores Genéticos/administração & dosagem , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Receptor ErbB-2/metabolismo , Taxa de Sobrevida , Células Tumorais Cultivadas , Vacinas de Subunidades Antigênicas/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Upregulation of human epidermal growth factor receptor 3 (HER3) is a major mechanism of acquired resistance to therapies targeting its heterodimerization partners epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2), but also exposes HER3 as a target for immune attack. We generated an adenovirus encoding full length human HER3 (Ad-HER3) to serve as a cancer vaccine. Previously we reported the anti-tumor efficacy and function of the T cell response to this vaccine. We now provide a detailed assessment of the antitumor efficacy and functional mechanisms of the HER3 vaccine-induced antibodies (HER3-VIAs) in serum from mice immunized with Ad-HER3. METHODS: Serum containing HER3-VIA was tested in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) assays and for its effect on HER3 internalization and degradation, downstream signaling of HER3 heterodimers and growth of metastatic HER2+ (BT474M1), HER2 therapy-resistant (rBT474), and triple negative (MDA-MB-468) breast cancers. RESULTS: HER3-VIAs mediated CDC and ADCC, HER3 internalization, interruption of HER3 heterodimer-driven tumor signaling pathways, and anti-proliferative effects against HER2+ tumor cells in vitro and significant antitumor effects against metastatic HER2+ BT474M1, treatment refractory HER2+ rBT474 and triple negative MDA-MB-468 in vivo. CONCLUSIONS: In addition to the T cell anti-tumor response induced by Ad-HER3, the HER3-VIAs provide additional functions to eliminate tumors in which HER3 signaling mediates aggressive behavior or acquired resistance to HER2-targeted therapy. These data support clinical studies of vaccination against HER3 prior to or concomitantly with other therapies to prevent outgrowth of therapy-resistant HER2+ and triple negative clones.
Assuntos
Anticorpos/imunologia , Antineoplásicos/farmacologia , Vacinas Anticâncer/imunologia , Receptor ErbB-3/imunologia , Neoplasias de Mama Triplo Negativas/terapia , Adenoviridae/genética , Animais , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos/uso terapêutico , Mama/patologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Mapeamento de Epitopos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Imunização Passiva/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Receptor ErbB-3/genética , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To determine whether 1 of 2 vaccines based on dendritic cells (DCs) and poxvectors encoding CEA (carcinoembryonic antigen) and MUC1 (PANVAC) would lengthen survival in patients with resected metastases of colorectal cancer (CRC). BACKGROUND: Recurrences after complete resections of metastatic CRC remain frequent. Immune responses to CRC are associated with fewer recurrences, suggesting a role for cancer vaccines as adjuvant therapy. Both DCs and poxvectors are potent stimulators of immune responses against cancer antigens. METHODS: Patients, disease-free after CRC metastasectomy and perioperative chemotherapy (n = 74), were randomized to injections of autologous DCs modified with PANVAC (DC/PANVAC) or PANVAC with per injection GM-CSF (granulocyte-macrophage colony-stimulating factor). Endpoints were recurrence-free survival overall survival, and rate of CEA-specific immune responses. Clinical outcome was compared with that of an unvaccinated, contemporary group of patients who had undergone CRC metastasectomy, received similar perioperative therapy, and would have otherwise been eligible for the study. RESULTS: Recurrence-free survival at 2 years was similar (47% and 55% for DC/PANVAC and PANVAC/GM-CSF, respectively) (χ P = 0.48). At a median follow-up of 35.7 months, there were 2 of 37 deaths in the DC/PANVAC arm and 5 of 37 deaths in the PANVAC/GM-CSF arm. The rate and magnitude of T-cell responses against CEA was statistically similar between study arms. As a group, vaccinated patients had superior survival compared with the contemporary unvaccinated group. CONCLUSIONS: Both DC and poxvector vaccines have similar activity. Survival was longer for vaccinated patients than for a contemporary unvaccinated group, suggesting that a randomized trial of poxvector vaccinations compared with standard follow-up after metastasectomy is warranted. (NCT00103142).
Assuntos
Vacinas Anticâncer , Antígeno Carcinoembrionário , Neoplasias Colorretais/prevenção & controle , Células Dendríticas , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Imunização/métodos , Glicoproteínas de Membrana , Mucina-1 , Recidiva Local de Neoplasia/prevenção & controle , Adulto , Idoso , Antígeno Carcinoembrionário/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mucina-1/genética , Metástase Neoplásica , Poxviridae/genéticaRESUMO
First-generation, E1-deleted adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen carcinoembryonic antigen (CEA), induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of preexisting or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of preexisting Ad5 immunity in a majority (61.3 %) of patients. Importantly, there was minimal toxicity, and overall patient survival (48 % at 12 months) was similar regardless of preexisting Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.
Assuntos
Vacinas Anticâncer/imunologia , Antígeno Carcinoembrionário/imunologia , Neoplasias Colorretais/imunologia , Vetores Genéticos/imunologia , Linfócitos T/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Adulto , Idoso , Anticorpos Neutralizantes/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Antígeno Carcinoembrionário/genética , Estudos de Coortes , Neoplasias Colorretais/terapia , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos/genética , Humanos , Imunização/métodos , Interferon gama/imunologia , Interferon gama/metabolismo , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Linfócitos T/metabolismo , Fatores de Tempo , Resultado do TratamentoRESUMO
INTRODUCTION: Sustained HER2 signaling at the cell surface is an oncogenic mechanism in a significant proportion of breast cancers. While clinically effective therapies targeting HER2 such as mAbs and tyrosine kinase inhibitors exist, tumors overexpressing HER2 eventually progress despite treatment. Thus, abrogation of persistent HER2 expression at the plasma membrane to synergize with current approaches may represent a novel therapeutic strategy. METHODS: We generated polyclonal anti-HER2 antibodies (HER2-VIA) by vaccinating mice with an adenovirus expressing human HER2, and assessed their signaling effects in vitro and anti-tumor effects in a xenograft model. In addition, we studied the signaling effects of human HER2-specific antibodies induced by vaccinating breast cancer patients with a HER2 protein vaccine. RESULTS: HER2-VIA bound HER2 at the plasma membrane, initially activating the downstream kinases extracellular signal-regulated protein kinase 1/2 and Akt, but subsequently inducing receptor internalization in clathrin-coated pits in a HER2 kinase-independent manner, followed by ubiquitination and degradation of HER2 into a 130 kDa fragment phosphorylated at tyrosine residues 1,221/1,222 and 1,248. Following vaccination of breast cancer patients with the HER2 protein vaccine, HER2-specific antibodies were detectable and these antibodies bound to cell surface-expressed HER2 and inhibited HER2 signaling through blocking tyrosine 877 phosphorylation of HER2. In contrast to the murine antibodies, human anti-HER2 antibodies induced by protein vaccination did not mediate receptor internalization and degradation. CONCLUSION: These data provide new insight into HER2 trafficking at the plasma membrane and the changes induced by polyclonal HER2-specific antibodies. The reduction of HER2 membrane expression and HER2 signaling by polyclonal antibodies induced by adenoviral HER2 vaccines supports human clinical trials with this strategy for those breast cancer patients with HER2 therapy-resistant disease.
Assuntos
Anticorpos/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Receptor ErbB-2/imunologia , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células , Vesículas Revestidas por Clatrina/metabolismo , Endocitose/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Transplante de Neoplasias , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Transplante Heterólogo , Ubiquitinação , VacinaçãoRESUMO
BACKGROUND: Patients with HER2-overexpressing metastatic breast cancer, despite initially benefiting from the monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib, will eventually have progressive disease. HER2-based vaccines induce polyclonal antibody responses against HER2 that demonstrate enhanced anti-tumor activity when combined with lapatinib in murine models. We wished to test the clinical safety, immunogenicity, and activity of a HER2-based cancer vaccine, when combined with lapatinib. METHODS: We immunized women (n = 12) with metastatic, trastuzumab-refractory, HER2-overexpressing breast cancer with dHER2, a recombinant protein consisting of extracellular domain (ECD) and a portion of the intracellular domain (ICD) of HER2 combined with the adjuvant AS15, containing MPL, QS21, CpG and liposome. Lapatinib (1250 mg/day) was administered concurrently. Peripheral blood antibody and T cell responses were measured. RESULTS: This regimen was well tolerated, with no cardiotoxicity. Anti-HER2-specific antibody was induced in all patients whereas HER2-specific T cells were detected in one patient. Preliminary analyses of patient serum demonstrated downstream signaling inhibition in HER2 expressing tumor cells. The median time to progression was 55 days, with the majority of patients progressing prior to induction of peak anti-HER2 immune responses; however, 300-day overall survival was 92% (95% CI: 77-100%). CONCLUSIONS: dHER2 combined with lapatinib was safe and immunogenic with promising long term survival in those with HER2-overexpressing breast cancers refractory to trastuzumab. Further studies to define the anticancer activity of the antibodies induced by HER2 vaccines along with lapatinib are underway. TRIAL REGISTRY: ClinicalTrials.gov NCT00952692.
Assuntos
Imunoterapia , Quinazolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Idoso , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/imunologia , Demografia , Epitopos/imunologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Lapatinib , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Resultado do Tratamento , VacinaçãoRESUMO
PURPOSE: The purpose of this study is to test whether peptide epitopes chosen from among those naturally processed and overpresented within MHC molecules by malignant, but not normal cells, when formulated into cancer vaccines, could activate antitumor T-cell responses in humans. EXPERIMENTAL DESIGN: Mixtures of human leukocyte antigen A2 (HLA-A2)-binding ovarian cancer-associated peptides were used to activate naive T cells to generate antigen-specific T cells that could recognize ovarian and breast cancers in vitro. Combinations of these peptides (0.3 mg of each peptide or 1 mg of each peptide) were formulated into vaccines in conjunction with Montanide ISA-51 and granulocyte monocyte colony stimulating factor which were used to vaccinate patients with ovarian and breast cancer without evidence of clinical disease in parallel pilot clinical trials. RESULTS: T cells specific for individual peptides could be generated in vitro by using mixtures of peptides, and these T cells recognized ovarian and breast cancers but not nonmalignant cells. Patient vaccinations were well tolerated with the exception of local erythema and induration at the injection site. Nine of the 14 vaccinated patients responded immunologically to their vaccine by inducing peptide-specific T-cell responses that were capable of recognizing HLA-matched breast and ovarian cancer cells. CONCLUSION: Mixtures of specific peptides identified as naturally presented on cancer cells and capable of activating tumor-specific T cells in vitro also initiate or augment immune responses toward solid tumors in cancer patients.
Assuntos
Antígenos de Neoplasias/isolamento & purificação , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Neoplasias Ovarianas/metabolismo , Proteômica/métodos , Linfócitos T Citotóxicos/imunologia , Adulto , Idoso , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Neoplasias da Mama/imunologia , Vacinas Anticâncer/imunologia , Carcinoma/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunoprecipitação/métodos , Ativação Linfocitária/imunologia , Pessoa de Meia-Idade , Neoplasias Ovarianas/imunologia , Linfócitos T Citotóxicos/metabolismoRESUMO
Regulatory T cells (Treg) have become increasingly relevant in the study of human disease including cancer. Treg cells have been shown to inhibit anti-tumor immune responses, and elevated Treg levels have been associated with certain types of cancer. Similarly, depletion of Tregs by various methods can also enhance anti-tumor immune responses. We have found a prevalence of Treg in cancer patients when compared to normal volunteers. In addition, we have shown that the depletion of Treg using the IL-2 fusion protein denileukin diftitox decreased Treg function and increased antigen-specific T cell response to a cancer vaccine. These results indicate the potential for combining Treg depletion with anti-cancer vaccines to enhance tumor antigen-specific immune responses and the need to explore the dose and schedule of Treg depletion strategies in optimizing vaccine efforts.
Assuntos
Toxina Diftérica/farmacologia , Interleucina-2/farmacologia , Depleção Linfocítica/métodos , Linfócitos T Reguladores/efeitos dos fármacos , Antígenos de Neoplasias/imunologia , Humanos , Neoplasias/imunologia , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Therapeutic anticancer vaccines are designed to boost patients' immune responses to tumors. One approach is to use a viral vector to deliver antigen to in situ DCs, which then activate tumor-specific T cell and antibody responses. However, vector-specific neutralizing antibodies and suppressive cell populations such as Tregs remain great challenges to the efficacy of this approach. We report here that an alphavirus vector, packaged in virus-like replicon particles (VRP) and capable of efficiently infecting DCs, could be repeatedly administered to patients with metastatic cancer expressing the tumor antigen carcinoembryonic antigen (CEA) and that it overcame high titers of neutralizing antibodies and elevated Treg levels to induce clinically relevant CEA-specific T cell and antibody responses. The CEA-specific antibodies mediated antibody-dependent cellular cytotoxicity against tumor cells from human colorectal cancer metastases. In addition, patients with CEA-specific T cell responses exhibited longer overall survival. These data suggest that VRP-based vectors can overcome the presence of neutralizing antibodies to break tolerance to self antigen and may be clinically useful for immunotherapy in the setting of tumor-induced immunosuppression.
Assuntos
Alphavirus/genética , Anticorpos Neutralizantes , Vetores Genéticos , Neoplasias/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Alphavirus/imunologia , Citotoxicidade Celular Dependente de Anticorpos/genética , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/imunologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , RepliconRESUMO
The monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib improve the clinical outcome of patients with HER2-overexpressing breast cancer. However, the majority of metastatic cancers will eventually progress, suggesting the need for other therapies. Because HER2 overexpression persists, we hypothesized that the anti-HER2 immune response induced by cancer vaccines would be an effective strategy for treating trastuzumab- and lapatinib-refractory tumors. Furthermore, we hypothesized that the antibody response could synergize with lapatinib to enhance tumor inhibition. We developed a recombinant adenoviral vector expressing a kinase-inactive HER2 (Ad-HER2-ki) to use as a cancer vaccine. Vaccine-induced polyclonal HER2-specific antiserum was analyzed for receptor internalization and signaling effects alone and in combination with lapatinib. Ad-HER2-ki vaccine-induced potent T cell and antibody responses in mice and the vaccine-induced polyclonal HER2-specific antiserum mediated receptor internalization and degradation much more effectively than trastuzumab. Our in vitro studies demonstrated that HER2 vaccine-induced antibodies effectively caused a decrease in HER2 expression, but when combined with lapatinib caused significant inhibition of HER2 signaling, decreased pERK and pAKT levels and reduced breast tumor cell proliferation. In addition, a known mechanism of resistance to lapatinib, induction of survivin, was inhibited. The combination of Ad-HER2-ki plus lapatinib also showed superior antitumor efficacy in vivo. Based on these results, we feel clinical studies using this approach to target HER2-overexpressing breast cancer, including trastuzumab- and lapatinib-resistant tumors is warranted.
Assuntos
Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Vacinas Anticâncer/uso terapêutico , Terapia Genética , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Receptor ErbB-2/genética , Adenoviridae/genética , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Proliferação de Células , Terapia Combinada , Sinergismo Farmacológico , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Lapatinib , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Infantile Pompe disease progresses to a lethal cardiomyopathy in absence of effective treatment. Enzyme-replacement therapy (ERT) with recombinant human acid alpha-glucosidase (rhGAA) has been effective in most patients with Pompe disease, but efficacy was reduced by high-titer antibody responses. Immunomodulatory gene therapy with a low dose adeno-associated virus (AAV) vector (2 x 10(10) particles) containing a liver-specific regulatory cassette significantly lowered immunoglobin G (IgG), IgG1, and IgE antibodies to GAA in Pompe disease mice, when compared with mock-treated mice (P < 0.05). AAV-LSPhGAApA had the same effect on GAA-antibody production whether it was given prior to, following, or simultaneously with the initial GAA injection. Mice given AAV-LSPhGAApA had significantly less decrease in body temperature (P < 0.001) and lower anaphylactic scores (P < 0.01) following the GAA challenge. Mouse mast cell protease-1 (MMCP-1) followed the pattern associated with hypersensitivity reactions (P < 0.05). Regulatory T cells (Treg) were demonstrated to play a role in the tolerance induced by gene therapy as depletion of Treg led to an increase in GAA-specific IgG (P < 0.001). Treg depleted mice were challenged with GAA and had significantly stronger allergic reactions than mice given gene therapy without subsequent Treg depletion (temperature: P < 0.01; symptoms: P < 0.05). Ubiquitous GAA expression failed to prevent antibody formation. Thus, immunomodulatory gene therapy could provide adjunctive therapy in lysosomal storage disorders treated by enzyme replacement.
Assuntos
Formação de Anticorpos/imunologia , Dependovirus/fisiologia , Terapia Genética/métodos , Doença de Depósito de Glicogênio Tipo II/imunologia , Doença de Depósito de Glicogênio Tipo II/terapia , Animais , Formação de Anticorpos/genética , Linhagem Celular , Dependovirus/genética , Terapia de Reposição de Enzimas/métodos , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos Endogâmicos C57BL , alfa-Glucosidases/genética , alfa-Glucosidases/fisiologiaRESUMO
CD4(+)CD25(high)FoxP3(+) regulatory T (Treg) cells limit antigen-specific immune responses and are a cause of suppressed anticancer immunity. In preclinical and clinical studies, we assessed the immune consequences of FoxP3(+) Treg-cell depletion in patients with advanced malignancies. We demonstrated that a CD25(high) targeting immunotoxin (denileukin diftitox) depleted FoxP3(+) Treg cells, decreased Treg-cell function, and enhanced antigen-specific T-cell responses in vitro. We then attempted to enhance antitumor immune responses in patients with carcinoembryonic antigen (CEA)-expressing malignancies by Treg-cell depletion. In a pilot study (n = 15), denileukin diftitox, given as a single dose or repeated dosing, was followed by immunizations with dendritic cells modified with the fowlpox vector rF-CEA(6D)-TRICOM. By flow cytometric analysis, we report the first direct evidence that circulating CD4(+)CD25(high)FoxP3(+) Treg cells are depleted after multiple doses of denileukin diftitox. Earlier induction of, and overall greater exposure to, the T-cell response to CEA was observed in the multiple-dose group, but not the single-dose group. These results indicate the potential for combining Treg-cell depletion with anticancer vaccines to enhance tumor antigen-specific immune responses and the need to explore dose and schedule of Treg depletion strategies in optimizing vaccine efforts.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Toxina Diftérica/administração & dosagem , Interleucina-2/administração & dosagem , Depleção Linfocítica/métodos , Linfócitos T Reguladores , Antígeno Carcinoembrionário/imunologia , Células Dendríticas/transplante , Toxina Diftérica/farmacologia , Humanos , Imunidade , Imunoterapia Adotiva/métodos , Interleucina-2/farmacologia , Projetos Piloto , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Resultado do TratamentoRESUMO
Two decades of research into the role of immunosuppression and angiogenesis in tumor biology have revealed multiple links between the two. Vascular endothelial growth factor, originally thought to be solely involved in vascular growth and permeability, has emerged as a significant agent of immune tolerance in the tumor microenvironment. This review examines two major elements of this field: the research behind the role of vascular endothelial growth factor in immunosuppression, especially as pertains to dendritic cell function; and the subsequent research into the potential for using antiangiogenic therapy to both starve tumors by hypoxia and enhance the response of tumors to immunotherapy. Several strategies tested so far have yielded incomplete, yet promising, results.
Assuntos
Células Dendríticas/fisiologia , Terapia de Imunossupressão , Neoplasias/terapia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Inibidores da Angiogênese/uso terapêutico , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Bevacizumab , Ensaios Clínicos como Assunto , Células Dendríticas/metabolismo , Humanos , Imunoterapia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
PURPOSE: To determine the safety and immunologic and clinical efficacy of a dendritic cell vaccine modified to hyperexpress costimulatory molecules and tumor antigen. EXPERIMENTAL DESIGN: In this phase I study, we administered one or two cycles of four triweekly s.c./intradermal injections of ex vivo generated dendritic cells modified with a recombinant fowlpox vector encoding carcinoembryonic antigen (CEA) and a triad of costimulatory molecules [rF-CEA(6D)-TRICOM]. Controls consisted of immature dendritic cells loaded with tetanus toxoid and a HLA A2-restricted peptide derived from cytomegalovirus pp65 protein. RESULTS: Fourteen patients (11 with colorectal cancer and 3 with non-small cell lung cancer) were enrolled and 12 completed at least one cycle of immunization. There were no grade 3/4 toxicities directly referable to the immunizations. One patient had a decrease in the CEA level from 46 to 6.8 and a minor regression in adenopathy that occurred several months after completion of the immunizations. Five other patients were stable through at least one cycle of immunization (3 months). Direct analysis of peripheral blood mononuclear cells using the ELISpot assay showed an increase in the frequency of CEA-specific T cells in 10 patients (range, 10-541 CEA-specific cells/10(5) peripheral blood mononuclear cells). There was a trend for a greater peak frequency of CEA-specific T cells among those with either a minor response or a stable disease following at least one cycle of therapy. A second cycle was not associated with higher T-cell frequencies. Cytokine flow cytometry showed CEA-specific immune response among both CD4(+) and CD8(+) T cells in all immune responders. CONCLUSION: This immunization strategy is safe and activates potent CEA-specific immune responses.
Assuntos
Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Neoplasias Colorretais/imunologia , Células Dendríticas/imunologia , Neoplasias Pulmonares/imunologia , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígenos CD58/genética , Antígenos CD58/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/uso terapêutico , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/imunologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Citocinas/metabolismo , Células Dendríticas/metabolismo , Feminino , Citometria de Fluxo , Vírus da Varíola das Aves Domésticas/genética , Antígeno HLA-A2/imunologia , Humanos , Imunoterapia Adotiva/métodos , Injeções Intradérmicas , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Toxoide Tetânico/imunologia , Resultado do TratamentoRESUMO
Detection of the circulating antigen-specific T-cell response to immunization is an important biologic end point in clinical trials of cancer vaccines. Typically employed assays are peptide MHC tetramer, ELISpot, and intracellular cytokine analysis. Although there is no agreement on the definition of a positive response in these assays, many groups have chosen a number of T cells greater than 2 standard deviations above the mean of the negative controls. The authors wished to determine how well this cutoff performed for each of these assays in detecting positive and negative T-cell responses to a model antigen, the immunodominant HLA-A*0201-restricted epitope of cytomegalovirus (CMV) pp65. For each assay, the mean + 2 standard deviations of the response for CMV seronegatives was the point that best separated the two groups. Using this value, each assay had a sensitivity of 87.5% and specificity of 95% to 100% and exhibited a high degree of concordance (kappa 0.76-0.9) with the other two. The authors conclude that currently available immunologic assays perform well in detecting biologically relevant levels of antigen-specific T cells. These assays will better define the quantity and quality of protective immune responses to viral disease and offer insight into the requirements for protective anti-cancer immunity.
Assuntos
Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Fragmentos de Peptídeos/imunologia , Fosfoproteínas/imunologia , Linfócitos T/imunologia , Proteínas da Matriz Viral/imunologia , Antígenos/imunologia , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Linfócitos T CD8-Positivos/imunologia , Citomegalovirus/imunologia , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Citometria de Fluxo , Genes MHC Classe I/imunologia , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , Humanos , Testes Imunológicos/métodos , Testes Imunológicos/estatística & dados numéricos , Interferon gama/metabolismo , Lectinas Tipo C , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Fosfoproteínas/sangue , Reprodutibilidade dos Testes , Testes Sorológicos/métodos , Linfócitos T/citologia , Linfócitos T/metabolismo , Proteínas da Matriz Viral/sangueRESUMO
Immunizations with dendritic cells (DC) transfected with RNA encoding tumor antigens induce potent tumor antigen-specific immune responses in vitro and in murine models. We performed a phase I study of patients with advanced carcinoembryonic antigen (CEA)-expressing malignancies followed by a phase II study of patients with resected hepatic metastases of colon cancer to assess safety and feasibility of administering autologous DC loaded with CEA mRNA. The immunizations were well tolerated. Of the 24 evaluable patients in the dose-escalation phase, there was 1 complete response (by tumor marker), 2 minor responses, 3 with stable disease, and 18 with progressive disease. In the phase II study, 9 of 13 patients have relapsed at a median of 122 days. Evidence of an immunologic response was demonstrated in biopsies of DC injection sites and peripheral blood of selected patients. We conclude that it is feasible and safe to administer mRNA-loaded DC to patients with advanced malignancies.
Assuntos
Antígeno Carcinoembrionário/genética , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Células Dendríticas/transplante , Imunoterapia/métodos , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/terapia , Transplante Autólogo , Adulto , Idoso , Antígeno Carcinoembrionário/efeitos adversos , Antígeno Carcinoembrionário/uso terapêutico , Células Dendríticas/fisiologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Leucaférese , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Proteínas Recombinantes/uso terapêutico , Análise de Sobrevida , Transfecção/métodos , Transplante Autólogo/efeitos adversos , Transplante Autólogo/mortalidadeRESUMO
Standardized biomarkers for the detection of clinically significant immunological responses would be extremely valuable in immunotherapy trials. Most available assays measure either the frequency or function of antigen-specific T-cells, or the titers of antibodies or immune complexes. These assays have generally exhibited either inadequate sensitivity or too high a signal-to-noise ratio to reliably detect the low-frequency T-cell responses induced by cancer vaccines. In addition, such assays reflect only one aspect of the immune response rather than the complete picture. Proteomics, the study of proteins within a cell or biological sample, may offer a novel approach to immunological monitoring that complements existing immunological assays. By studying the protein content of T-cells responding to a vaccine or in the serum of vaccinated individuals, it may be possible to develop a metric for quantitating the magnitude of immunological responses. Proteomics could also provide a tool for establishing the quality of the immune response and for obtaining valuable information regarding the underlying regulatory mechanisms and pathways. Advances in miniaturization and automation may also permit characterization of the immune response more rapidly and from smaller amounts of biological material than is possible with existing assay systems.