RESUMEN
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.
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Anticuerpos/inmunología , Antineoplásicos/farmacología , Vacunas contra el Cáncer/inmunología , Receptor ErbB-3/inmunología , Neoplasias de la Mama Triple Negativas/terapia , Adenoviridae/genética , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Antineoplásicos/uso terapéutico , Mama/patología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/genética , Línea Celular Tumoral , Proliferación Celular , Resistencia a Antineoplásicos , Mapeo Epitopo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Inmunización Pasiva/métodos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/metabolismo , Receptor ErbB-3/genética , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The ability to temporally regulate gene expression and track labeled cells makes animal models powerful biomedical tools. However, sudden expression of xenobiotic genes [e.g., GFP, luciferase (Luc), or rtTA3] can trigger inadvertent immunity that suppresses foreign protein expression or results in complete rejection of transplanted cells. Germline exposure to foreign antigens somewhat addresses these challenges; however, native fluorescence and bioluminescence abrogates the utility of reporter proteins and highly spatiotemporally restricted expression can lead to suboptimal xenoantigen tolerance. To overcome these unwanted immune responses and enable reliable cell tracking/gene regulation, we developed a novel mouse model that selectively expresses antigen-intact but nonfunctional forms of GFP and Luc, as well as rtTA3, after CRE-mediated recombination. Using tissue-specific CREs, we observed model and sex-based differences in immune tolerance to the encoded xenoantigens, illustrating the obstacles of tolerizing animals to foreign genes and validating the utility of these "NoGlow" mice to dissect mechanisms of central and peripheral tolerance. Critically, tissue unrestricted NoGlow mice possess no detectable background fluorescence or luminescence and exhibit limited adaptive immunity against encoded transgenic xenoantigens after vaccination. Moreover, we demonstrate that NoGlow mice allow tracking and tetracycline-inducible gene regulation of triple-transgenic cells expressing GFP/Luc/rtTA3, in contrast to transgene-negative immune-competent mice that eliminate these cells or prohibit metastatic seeding. Notably, this model enables de novo metastasis from orthotopically implanted, triple-transgenic tumor cells, despite high xenoantigen expression. Altogether, the NoGlow model provides a critical resource for in vivo studies across disciplines, including oncology, developmental biology, infectious disease, autoimmunity, and transplantation. SIGNIFICANCE: Multitolerant NoGlow mice enable tracking and gene manipulation of transplanted tumor cells without immune-mediated rejection, thus providing a platform to investigate novel mechanisms of adaptive immunity related to metastasis, immunotherapy, and tolerance.
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Antígenos Heterófilos , Rastreo Celular , Animales , Ratones , Regulación de la Expresión Génica , Ratones Transgénicos , Modelos Animales de EnfermedadRESUMEN
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.
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Neoplasias de la Mama , Vacunas , Humanos , Animales , Ratones , Femenino , Neoplasias de la Mama/genética , Neoplasias de la Mama/terapia , Mutación , Estrógenos/uso terapéutico , Transducción de Señal , Vacunas/uso terapéuticoRESUMEN
Approximately 30% of breast cancer survivors deemed free of disease will experience locoregional or metastatic recurrence even up to 30 years after initial diagnosis, yet how residual/dormant tumor cells escape immunity elicited by the primary tumor remains unclear. We demonstrate that intrinsically dormant tumor cells are indeed recognized and lysed by antigen-specific T cells in vitro and elicit robust immune responses in vivo. However, despite close proximity to CD8+ killer T cells, dormant tumor cells themselves support early accumulation of protective FoxP3+ T regulatory cells (Tregs), which can be targeted to reduce tumor burden. These intrinsically dormant tumor cells maintain a hybrid epithelial/mesenchymal state that is associated with immune dysfunction, and we find that the tumor-derived, stem cell/basal cell protein Dickkopf WNT signaling pathway inhibitor 3 (DKK3) is critical for Treg inhibition of CD8+ T cells. We also demonstrate that DKK3 promotes immune-mediated progression of proliferative tumors and is significantly associated with poor survival and immunosuppression in human breast cancers. Together, these findings reveal that latent tumors can use fundamental mechanisms of tolerance to alter the T cell microenvironment and subvert immune detection. Thus, targeting these pathways, such as DKK3, may help render dormant tumors susceptible to immunotherapies.
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Neoplasias de la Mama , Linfocitos T Reguladores , Humanos , Femenino , Linfocitos T Citotóxicos , Neoplasias de la Mama/patología , Terapia de Inmunosupresión , Inmunidad Adaptativa , Microambiente Tumoral , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
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.
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Anticuerpos/inmunología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/terapia , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Receptor ErbB-2/inmunología , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Proliferación Celular , Vesículas Cubiertas por Clatrina/metabolismo , Endocitosis/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Trasplante de Neoplasias , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Trasplante Heterólogo , Ubiquitinación , VacunaciónRESUMEN
BACKGROUND: The majority of colorectal carcinomas (CRCs) are insensitive to programmed death protein-1/programmed death-ligand 1 (anti-PD-1/PD-L1) immune checkpoint inhibitor (ICI) antibodies. While there are many causes for ICI insensitivity, recent studies suggest that suppression of innate immune gene expression in tumor cells could be a root cause of this insensitivity and an important factor in the evolution of tumor immunosuppression. METHODS: We first assessed the reduction of mitochondrial antiviral signaling gene (MAVS) and related RIG-I pathway gene expression in several patient RNA expression datasets. We then engineered MAVS expressing tumor cells and tested their ability to elicit innate and adaptive anti-tumor immunity using both in vitro and in vivo approaches, which we then confirmed using MAVS expressing viral vectors. Finally, we observed that MAVS stimulated PD-L1 expression in multiple cell types and then assessed the combination of PD-L1 ICI antibodies with MAVS tumor expression in vivo. RESULTS: MAVS was significantly downregulated in CRCs, but its re-expression could stimulate broad cellular interferon-related responses, in both murine and patient-derived CRCs. In vivo, local MAVS expression elicited significant anti-tumor responses in both immune-sensitive and insensitive CRC models, through the stimulation of an interferon responsive axis that provoked tumor antigen-specific adaptive immunity. Critically, we found that tumor-intrinsic MAVS expression triggered systemic adaptive immune responses that enabled abscopal CD8 +T cell cytotoxicity against distant CRCs. As MAVS also induced PD-L1 expression, we further found synergistic anti-tumor responses in combination with anti-PD-L1 ICIs. CONCLUSION: These data demonstrate that intratumoral MAVS expression results in local and systemic tumor antigen-specific T cell responses, which could be combined with PD-L1 ICI to permit effective anti-tumor immunotherapy in ICI resistant cancers.
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Neoplasias Colorrectales , Inhibidores de Puntos de Control Inmunológico , Animales , Antivirales , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia/métodos , Ratones , Transducción de SeñalRESUMEN
Two HER2-specific mAbs, trastuzumab and pertuzumab (T+P), combined with chemotherapy comprise standard-of-care treatment for advanced HER2+ breast cancers (BC). While this antibody combination is highly effective, its synergistic mechanism-of-action (MOA) remains incompletely understood. Past studies have suggested that the synergy underlying this combination occurs through the different mechanisms elicited by these antibodies, with pertuzumab suppressing HER2 heterodimerization and trastuzumab inducing antitumor immunity. However, in vivo evidence for this synergy is lacking. In this study, we found that the therapeutic efficacy elicited by their combination occurs through their joint ability to activate the classical complement pathway, resulting in both complement-dependent cytotoxicity and complement-dependent cellular phagocytosis of HER2+ tumors. We also demonstrate that tumor C1q expression is positively associated with survival outcome in HER2+ BC patients and that complement regulators CD55 and CD59 were inversely correlated with outcome, suggesting the clinical importance of complement activity. Accordingly, inhibition of C1q in mice abolished the synergistic therapeutic activity of T+P therapy, whereas knockdown of CD55 and CD59 expression enhanced T+P efficacy. In summary, our study identifies classical complement activation as a significant antitumor MOA for T+P therapy that may be functionally enhanced to potentially augment clinical therapeutic efficacy.
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Neoplasias de la Mama , Receptor ErbB-2 , Animales , Anticuerpos Monoclonales Humanizados , Neoplasias de la Mama/patología , Línea Celular Tumoral , Complemento C1q , Femenino , Humanos , Ratones , Fagocitosis , Receptor ErbB-2/metabolismo , Trastuzumab/farmacología , Trastuzumab/uso terapéuticoRESUMEN
BACKGROUND: Clinical studies have linked usage of progestins (synthetic progesterone [P4]) to breast cancer risk. However, little is understood regarding the role of native P4, signaling through the progesterone receptor (PR), in breast tumor formation. Recently, we reported a link between PR and immune signaling pathways, showing that P4/PR can repress type I interferon signaling pathways. Given these findings, we sought to investigate whether P4/PR drive immunomodulation in the mammary gland and promote tumor formation. METHODS: To determine the effect of P4 on immune cell populations in the murine mammary gland, mice were treated with P4 or placebo pellets for 21 days. Immune cell populations in the mammary gland, spleen, and inguinal lymph nodes were subsequently analyzed by flow cytometry. To assess the effect of PR overexpression on mammary gland tumor development as well as immune cell populations in the mammary gland, a transgenic mouse model was used in which PR was overexpressed throughout the entire mouse. Immune cell populations were assessed in the mammary glands, spleens, and inguinal lymph nodes of 6-month-old transgenic and control mice by flow cytometry. Transgenic mice were also monitored for mammary gland tumor development over a 2-year time span. Following development of mammary gland tumors, immune cell populations in the tumors and spleens of transgenic and control mice were analyzed by flow cytometry. RESULTS: We found that mice treated with P4 exhibited changes in the mammary gland indicative of an inhibited immune response compared with placebo-treated mice. Furthermore, transgenic mice with PR overexpression demonstrated decreased numbers of immune cell populations in their mammary glands, lymph nodes, and spleens. On long-term monitoring, we determined that multiparous PR-overexpressing mice developed significantly more mammary gland tumors than control mice. Additionally, tumors from PR-overexpressing mice contained fewer infiltrating immune cells. Finally, RNA sequencing analysis of tumor samples revealed that immune-related gene signatures were lower in tumors from PR-overexpressing mice as compared with control mice. CONCLUSION: Together, these findings offer a novel mechanism of P4-driven mammary gland tumor development and provide rationale in investigating the usage of antiprogestin therapies to promote immune-mediated elimination of mammary gland tumors.
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Neoplasias de la Mama/inducido químicamente , Transformación Celular Neoplásica/inducido químicamente , Glándulas Mamarias Animales/efectos de los fármacos , Progesterona/administración & dosificación , Receptores de Progesterona/agonistas , Escape del Tumor/efectos de los fármacos , Microambiente Tumoral/inmunología , Inmunidad Adaptativa/efectos de los fármacos , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Implantes de Medicamentos , Femenino , Galectina 4/genética , Galectina 4/metabolismo , Inmunidad Innata/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Glándulas Mamarias Animales/inmunología , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Ratones Transgénicos , Ovariectomía , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Transducción de Señal , Factores de Tiempo , Carga Tumoral/efectos de los fármacosRESUMEN
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.
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Neoplasias de la Mama/inmunología , Neoplasias de la Mama/terapia , Vacunas contra el Cáncer/uso terapéutico , Terapia Genética , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Receptor ErbB-2/genética , Adenoviridae/genética , Animales , Western Blotting , Neoplasias de la Mama/metabolismo , Proliferación Celular , Terapia Combinada , Sinergismo Farmacológico , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Lapatinib , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor ErbB-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Wilms' tumor protein (WT1) is overexpressed in most leukemias and many solid tumors and is a promising target for tumor immunotherapy. WT1 peptide-based cancer vaccines have been reported but have limited application due to HLA restriction of the peptides. We sought to vaccinate using adenoviral (Ad) vectors encoding tumor-associated antigens such as WT1 that can stimulate tumor-associated antigen-specific immunity across a broad array of HLA types and multiple class I and class II epitopes. EXPERIMENTAL DESIGN: We developed a novel Ad vector encoding a truncated version of WT1 (Ad-tWT1) lacking the highly conserved COOH terminus zinc finger domains and tested its ability to stimulate WT1-specific immune responses and antitumor immunity in two murine models of WT1-expressing tumors. RESULTS: Despite encoding a transcription factor, we found that Ad-tWT1-transduced murine and human dendritic cells showed cytoplasmic expression of the truncated WT1 protein. In addition, vaccination of C57BL/6 mice with Ad-tWT1 generated WT1-specific cell-mediated and humoral immune responses and conferred protection against challenge with the leukemia cell line, mWT1-C1498. Moreover, in a tumor therapy model, Ad-tWT1 vaccination of TRAMP-C2 tumor-bearing mice significantly suppressed tumor growth. CONCLUSIONS: This is the first report of a WT1-encoding Ad vector that is capable of inducing effective immunity against WT1-expressing malignancies. Based on these findings, Ad-tWT1 warrants investigation in human clinical trials to evaluate its applications as a vaccine for patients with WT1-expressing cancers.
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Vacunas contra el Cáncer/uso terapéutico , Leucemia/terapia , Proteínas WT1/inmunología , Animales , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Células Dendríticas/inmunología , Vectores Genéticos , Humanos , Inmunoterapia , Leucemia/inmunología , Ratones , Ratones Endogámicos C57BL , Linfocitos T Citotóxicos/inmunología , Proteínas WT1/genéticaRESUMEN
BACKGROUND: The advent of immune checkpoint blockade antibodies has demonstrated that effective mobilization of T cell responses can cause tumor regression of metastatic cancers, although these responses are heterogeneous and restricted to certain histologic types of cancer. To enhance these responses, there has been renewed emphasis in developing effective cancer-specific vaccines to stimulate and direct T cell immunity to important oncologic targets, such as the oncogene human epidermal growth factor receptor 2 (HER2), expressed in ~20% of breast cancers (BCs). METHODS: In our study, we explored the use of alternative antigen trafficking through use of a lysosome-associated membrane protein 1 (LAMP) domain to enhance vaccine efficacy against HER2 and other model antigens in both in vitro and in vivo studies. RESULTS: We found that inclusion of this domain in plasmid vaccines effectively trafficked antigens to endolysosomal compartments, resulting in enhanced major histocompatibility complex (MHC) class I and II presentation. Additionally, this augmented the expansion/activation of antigen-specific CD4+ and CD8+ T cells and also led to elevated levels of antigen-specific polyfunctional CD8+ T cells. Significantly, vaccination with HER2-LAMP produced tumor regression in ~30% of vaccinated mice with established tumors in an endogenous model of metastatic HER2+ BC, compared with 0% of HER2-WT vaccinated mice. This therapeutic benefit is associated with enhanced tumor infiltration of activated CD4+ and CD8+ T cells. CONCLUSIONS: These data demonstrate the potential of using LAMP-based endolysosomal trafficking as a means to augment the generation of polyfunctional, antigen-specific T cells in order to improve antitumor therapeutic responses using cancer antigen vaccines.
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Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Linfocitos T/metabolismo , Animales , Femenino , Humanos , Ratones , TransfecciónRESUMEN
IL26 is a unique amphipathic member of the IL10 family of cytokines that participates in inflammatory signaling through a canonical receptor pathway. It also directly binds DNA to facilitate cellular transduction and intracellular inflammatory signaling. Although IL26 has almost no described role in cancer, our in vivo screen of inflammatory and cytokine pathway genes revealed IL26 to be one of the most significant inflammatory mediators of mammary engraftment and lung metastatic growth in triple-negative breast cancer (TNBC). Examination of human breast cancers demonstrated elevated IL26 transcripts in TNBC specimens, specifically in tumor cells as well as in Th17 CD4+ T cells within clinical TNBC specimens. IL26 did not have an autocrine effect on human TNBC cells, but rather its effect on engraftment and growth in vivo required neutrophils. IL26 enhanced mouse-derived DNA induction of inflammatory cytokines, which were collectively important for mammary and metastatic lung engraftment. To neutralize this effect, we developed a novel IL26 vaccine to stimulate antibody production and suppress IL26-enhanced engraftment in vivo, suggesting that targeting this inflammatory amplifier could be a unique means to control cancer-promoting inflammation in TNBC and other autoimmune diseases. Thus, we identified IL26 as a novel key modulator of TNBC metastasis and a potential therapeutic target in TNBC as well as other diseases reliant upon IL26-mediated inflammatory stimulation. SIGNIFICANCE: These findings identify IL26 as a unique, clinically relevant, inflammatory amplifier that enhances TNBC engraftment and dissemination in association with neutrophils, which has potential as a therapeutic target. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/15/3088/F1.large.jpg.
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Adhesión Celular , Interleucinas/fisiología , Trasplante de Neoplasias , Neutrófilos/fisiología , Neoplasias de la Mama Triple Negativas/patología , Animales , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Células Cultivadas , ADN de Neoplasias/efectos de los fármacos , ADN de Neoplasias/metabolismo , Progresión de la Enfermedad , Trampas Extracelulares/efectos de los fármacos , Trampas Extracelulares/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Mediadores de Inflamación/farmacología , Mediadores de Inflamación/fisiología , Interleucinas/genética , Interleucinas/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Trasplante de Neoplasias/inmunología , Trasplante de Neoplasias/patología , Neutrófilos/patología , Neoplasias de la Mama Triple Negativas/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Despite promising advances in breast cancer immunotherapy, augmenting T-cell infiltration has remained a significant challenge. Although neither individual vaccines nor immune checkpoint blockade (ICB) have had broad success as monotherapies, we hypothesized that targeted vaccination against an oncogenic driver in combination with ICB could direct and enable antitumor immunity in advanced cancers. EXPERIMENTAL DESIGN: Our models of HER2+ breast cancer exhibit molecular signatures that are reflective of advanced human HER2+ breast cancer, with a small numbers of neoepitopes and elevated immunosuppressive markers. Using these, we vaccinated against the oncogenic HER2Δ16 isoform, a nondriver tumor-associated gene (GFP), and specific neoepitopes. We further tested the effect of vaccination or anti-PD-1, alone and in combination. RESULTS: We found that only vaccination targeting HER2Δ16, a driver of oncogenicity and HER2-therapeutic resistance, could elicit significant antitumor responses, while vaccines targeting a nondriver tumor-specific antigen or tumor neoepitopes did not. Vaccine-induced HER2-specific CD8+ T cells were essential for responses, which were more effective early in tumor development. Long-term tumor control of advanced cancers occurred only when HER2Δ16 vaccination was combined with αPD-1. Single-cell RNA sequencing of tumor-infiltrating T cells revealed that while vaccination expanded CD8 T cells, only the combination of vaccine with αPD-1 induced functional gene expression signatures in those CD8 T cells. Furthermore, we show that expanded clones are HER2-reactive, conclusively demonstrating the efficacy of this vaccination strategy in targeting HER2. CONCLUSIONS: Combining oncogenic driver targeted vaccines with selective ICB offers a rational paradigm for precision immunotherapy, which we are clinically evaluating in a phase II trial (NCT03632941).
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Neoplasias de la Mama/terapia , Vacunas contra el Cáncer/administración & dosificación , Inhibidores de Puntos de Control Inmunológico/administración & dosificación , Neoplasias Mamarias Experimentales/terapia , Receptor ErbB-2/inmunología , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Inmunoterapia/métodos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Vacunas Combinadas/administración & dosificaciónRESUMEN
The HER2-specific monoclonal antibody (mAb), trastuzumab, has been the mainstay of therapy for HER2+ breast cancer (BC) for approximately 20 years. However, its therapeutic mechanism of action (MOA) remains unclear, with antitumor responses to trastuzumab remaining heterogeneous and metastatic HER2+ BC remaining incurable. Consequently, understanding its MOA could enable rational strategies to enhance its efficacy. Using both murine and human versions of trastuzumab, we found its antitumor activity dependent on Fcγ receptor stimulation of tumor-associated macrophages (TAMs) and antibody-dependent cellular phagocytosis (ADCP), but not cellular cytotoxicity (ADCC). Trastuzumab also stimulated TAM activation and expansion, but did not require adaptive immunity, natural killer cells, and/or neutrophils. Moreover, inhibition of the innate immune ADCP checkpoint, CD47, significantly enhanced trastuzumab-mediated ADCP and TAM expansion and activation, resulting in the emergence of a unique hyperphagocytic macrophage population, improved antitumor responses, and prolonged survival. In addition, we found that tumor-associated CD47 expression was inversely associated with survival in HER2+ BC patients and that human HER2+ BC xenografts treated with trastuzumab plus CD47 inhibition underwent complete tumor regression. Collectively, our study identifies trastuzumab-mediated ADCP as an important antitumor MOA that may be clinically enabled by CD47 blockade to augment therapeutic efficacy.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Antígeno CD47/antagonistas & inhibidores , Fagocitosis/efectos de los fármacos , Trastuzumab/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Mama/patología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Antígeno CD47/inmunología , Antígeno CD47/metabolismo , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Humanos , Inmunidad Innata/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Fagocitosis/inmunología , Pronóstico , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/metabolismo , Trastuzumab/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Triple-negative breast cancer (TNBC) is an aggressive and molecularly diverse breast cancer subtype typified by the presence of p53 mutations (â¼80%), elevated immune gene signatures and neoantigen expression, as well as the presence of tumor infiltrating lymphocytes (TILs). As these factors are hypothesized to be strong immunologic prerequisites for the use of immune checkpoint blockade (ICB) antibodies, multiple clinical trials testing single ICBs have advanced to Phase III, with early indications of heterogeneous response rates of <20% to anti-PD1 and anti-PDL1 ICB. While promising, these modest response rates highlight the need for mechanistic studies to understand how different ICBs function, how their combination impacts functionality and efficacy, as well as what immunologic parameters predict efficacy to different ICBs regimens in TNBC. To address these issues, we tested anti-PD1 and anti-CTLA4 in multiple models of TNBC and found that their combination profoundly enhanced the efficacy of either treatment alone. We demonstrate that this efficacy is due to anti-CTLA4-driven expansion of an individually unique T-cell receptor (TCR) repertoire whose functionality is enhanced by both intratumoral Treg suppression and anti-PD1 blockade of tumor expressed PDL1. Notably, the individuality of the TCR repertoire was observed regardless of whether the tumor cells expressed a nonself antigen (ovalbumin) or if tumor-specific transgenic T-cells were transferred prior to sequencing. However, responsiveness was strongly correlated with systemic measures of tumor-specific T-cell and B-cell responses, which along with systemic assessment of TCR expansion, may serve as the most useful predictors for clinical responsiveness in future clinical trials of TNBC utilizing anti-PD1/anti-CTLA4 ICB.
RESUMEN
HER2 overexpression occurs in approximately 25% of breast cancers, where it correlates with poor prognosis. Likewise, systemic inflammation in breast cancer correlates with poor prognosis, although the process is not understood. In this study, we explored the relationship between HER2 and inflammation, comparing the effects of overexpressing wild-type or mutated inactive forms of HER2 in primary human breast cells. Wild-type HER2 elicited a profound transcriptional inflammatory profile, including marked elevation of interleukin-6 (IL-6) expression, which we established to be a critical determinant of HER2 oncogenesis. Mechanistic investigations revealed that IL-6 secretion induced by HER2 overexpression activated Stat3 and altered gene expression, enforcing an autocrine loop of IL-6/Stat3 expression. Both mouse and human in vivo models of HER2-amplified breast carcinoma relied critically on this HER2-IL-6-Stat3 signaling pathway. Our studies offer the first direct evidence linking HER2 to a systemic inflammatory mechanism that orchestrates HER2-mediated tumor growth. We suggest that the HER2-IL-6-STAT3 signaling axis we have defined in breast cancer could prompt new therapeutic or prevention strategies for treatment of HER2-amplified cancers.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Interleucina-6/metabolismo , Receptor ErbB-2/biosíntesis , Células 3T3 , Animales , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación/metabolismo , Interleucina-6/biosíntesis , Interleucina-6/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Fosforilación , Receptor ErbB-2/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
While many tumor associated antigens (TAAs) have been identified in human cancers, efforts to develop efficient TAA "cancer vaccines" using classical vaccine approaches have been largely ineffective. Recently, a process to specifically target proteins to exosomes has been established which takes advantage of the ability of the factor V like C1C2 domain of lactadherin to specifically address proteins to exosomes. Using this approach, we hypothesized that TAAs could be targeted to exosomes to potentially increase their immunogenicity, as exosomes have been demonstrated to traffic to antigen presenting cells (APC). To investigate this possibility, we created adenoviral vectors expressing the extracellular domain (ECD) of two non-mutated TAAs often found in tumors of cancer patients, carcinoembryonic antigen (CEA) and HER2, and coupled them to the C1C2 domain of lactadherin. We found that these C1C2 fusion proteins had enhanced expression in exosomes in vitro. We saw significant improvement in antigen specific immune responses to each of these antigens in naïve and tolerant transgenic animal models and could further demonstrate significantly enhanced therapeutic anti-tumor effects in a human HER2+ transgenic animal model. These findings demonstrate that the mode of secretion and trafficking can influence the immunogenicity of different human TAAs, and may explain the lack of immunogenicity of non-mutated TAAs found in cancer patients. They suggest that exosomal targeting could enhance future anti-tumor vaccination protocols. This targeting exosome process could also be adapted for the development of more potent vaccines in some viral and parasitic diseases where the classical vaccine approach has demonstrated limitations.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Vacunas contra el Cáncer/inmunología , Antígeno Carcinoembrionario/inmunología , Exosomas/inmunología , Receptor ErbB-2/inmunología , Adenoviridae/inmunología , Animales , Antígenos de Superficie/inmunología , Línea Celular Tumoral , Vectores Genéticos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de la Leche/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Although critical for initiating and regulating immune responses, the therapeutic use of individual cytokines as anticancer immunotherapeutic agents has achieved only modest clinical success. Consequently, many current strategies have focused on the use of specific immunotherapeutic agonists that engage individual receptors of innate immune networks, such as the Toll-like receptor (TLR) system, each resulting in specific patterns of gene expression, cytokine production, and inflammatory outcome. However, these immunotherapeutics are constrained by variable cellular TLR expression and responsiveness to particular TLR agonists, as well as the specific cellular context of different tumors. We hypothesized that overexpression of MyD88, a pivotal regulator of multiple TLR signaling pathways, could circumvent these constraints and mimic coordinated TLR signaling across all cell types in a ligand-independent fashion. To explore this hypothesis, we generated an adenoviral vector expressing MyD88 and show that Ad-MyD88 infection elicits extensive Th1-specific transcriptional and secreted cytokine signatures in all murine and human cell types tested in vitro and in vivo. Importantly, in vivo intratumoral injection of Ad-MyD88 into established tumor masses enhanced adaptive immune responses and inhibited local tumor immunosuppression, resulting in significantly inhibited local and systemic growth of multiple tumor types. Finally, Ad-MyD88 infection of primary human dendritic cells, tumor-associated fibroblasts, and colorectal carcinoma cells elicited significant Th1-type cytokine responses, resulting in enhanced tumor cell lysis and expansion of human tumor antigen-specific T cells. Thus, Ad-MyD88 initiated robust antitumor activity in established murine tumor microenvironments and in human contexts, suggesting its potential effectiveness as a clinical immunotherapeutic strategy.
Asunto(s)
Factor 88 de Diferenciación Mieloide/inmunología , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Receptores Toll-Like/inmunología , Adenoviridae/genética , Animales , Línea Celular Tumoral , Células Dendríticas/inmunología , Epítopos de Linfocito T/inmunología , Terapia Genética/métodos , Vectores Genéticos/genética , Humanos , Inmunoterapia/métodos , Células Asesinas Naturales/inmunología , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Factor 88 de Diferenciación Mieloide/biosíntesis , Factor 88 de Diferenciación Mieloide/genética , Neoplasias Experimentales/genética , Transducción de Señal , Linfocitos T/inmunología , Receptores Toll-Like/metabolismoRESUMEN
PURPOSE: Overexpression of the breast cancer oncogene HER2 correlates with poor survival. Current HER2-directed therapies confer limited clinical benefits and most patients experience progressive disease. Because refractory tumors remain strongly HER2+, vaccine approaches targeting HER2 have therapeutic potential, but wild type (wt) HER2 cannot safely be delivered in immunogenic viral vectors because it is a potent oncogene. We designed and tested several HER2 vaccines devoid of oncogenic activity to develop a safe vaccine for clinical use. EXPERIMENTAL DESIGN: We created recombinant adenoviral vectors expressing the extracellular domain of HER2 (Ad-HER2-ECD), ECD plus the transmembrane domain (Ad-HER2-ECD-TM), and full-length HER2 inactivated for kinase function (Ad-HER2-ki), and determined their immunogenicity and antitumor effect in wild type (WT) and HER2-tolerant mice. To assess their safety, we compared their effect on the cellular transcriptome, cell proliferation, anchorage-dependent growth, and transformation potential in vivo. RESULTS: Ad-HER2-ki was the most immunogenic vector in WT animals, retained immunogenicity in HER2-transgenic tolerant animals, and showed strong therapeutic efficacy in treatment models. Despite being highly expressed, HER2-ki protein was not phosphorylated and did not produce an oncogenic gene signature in primary human cells. Moreover, in contrast to HER2-wt, cells overexpressing HER2-ki were less proliferative, displayed less anchorage-independent growth, and were not transformed in vivo. CONCLUSIONS: Vaccination with mutationally inactivated, nononcogenic Ad-HER2-ki results in robust polyclonal immune responses to HER2 in tolerant models, which translates into strong and effective antitumor responses in vivo. Ad-HER2-ki is thus a safe and promising vaccine for evaluation in clinical trials.