RESUMEN
Clinical studies have demonstrated that local expression of the cytokine IL-12 drives interferon-gamma expression and recruits T cells to the tumor microenvironment, ultimately yielding durable systemic T cell responses. Interrogation of longitudinal biomarker data from our late-stage melanoma trials identified a significant on-treatment increase of intratumoral CXCR3 transcripts that was restricted to responding patients, underscoring the clinical relevance of tumor-infiltrating CXCR3+ immune cells. In this study, we sought to understand if the addition of DNA-encodable CXCL9 could augment the anti-tumor immune responses driven by intratumoral IL-12. We show that localized IL-12 and CXCL9 treatment reshapes the tumor microenvironment to promote dendritic cell licensing and CD8+ T cell activation. Additionally, this combination treatment results in a significant abscopal anti-tumor response and provides a concomitant benefit to anti-PD-1 therapies. Collectively, these data demonstrate that a functional tumoral CXCR3/CXCL9 axis is critical for IL-12 anti-tumor efficacy. Furthermore, restoring or amplifying the CXCL9 gradient in the tumors via intratumoral electroporation of plasmid CXCL9 can not only result in efficient trafficking of cytotoxic CD8+ T cells into the tumor but can also reshape the microenvironment to promote systemic immune response.
RESUMEN
Intratumoral delivery of plasmid IL12 via electroporation (IT-tavo-EP) induces localized expression of IL12 leading to regression of treated and distant tumors with durable responses and minimal toxicity. A key driver in amplifying this local therapy into a systemic response is the magnitude and composition of immune infiltrate in the treated tumor. While intratumoral IL12 typically increases the density of CD3+ tumor-infiltrating lymphocytes (TIL), this infiltrate is composed of a broad range of T-cell subsets, including activated tumor-specific T cells, less functional bystander T cells, as well as suppressive T regulatory cells. To encourage a more favorable on-treatment tumor microenvironment (TME), we explored combining this IL12 therapy with an intratumoral polyclonal T-cell stimulator membrane-anchored anti-CD3 to productively engage a diverse subset of lymphocytes including the nonreactive and suppressive T cells. This study highlighted that combined intratumoral electroporation of IL12 and membrane-anchored anti-CD3 plasmids can enhance cytokine production, T-cell cytotoxicity, and proliferation while limiting the suppressive capacity within the TME. These collective antitumor effects not only improve regression of treated tumors but drive systemic immunity with control of nontreated contralateral tumors in vivo. Moreover, combination of IL12 and anti-CD3 restored the function of TIL isolated from a patient with melanoma actively progressing on programmed cell death protein 1 (PD-1) checkpoint inhibitor therapy. IMPLICATIONS: This DNA-encodable polyclonal T-cell stimulator (membrane-anchored anti-CD3 plasmid) may represent a key addition to intratumoral IL12 therapies in the clinic.
Asunto(s)
Interleucina-12 , Melanoma , Electroporación , Humanos , Inmunoterapia , Interleucina-12/genética , Interleucina-12/metabolismo , Melanoma/patología , Plásmidos/genética , Microambiente TumoralRESUMEN
PURPOSE: Triple-negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Antibodies targeting programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) have entered the therapeutic landscape in TNBC, but only a minority of patients benefit. A way to reliably enhance immunogenicity, T-cell infiltration, and predict responsiveness is critically needed. PATIENTS AND METHODS: Using mouse models of TNBC, we evaluate immune activation and tumor targeting of intratumoral IL12 plasmid followed by electroporation (tavokinogene telseplasmid; Tavo). We further present a single-arm, prospective clinical trial of Tavo monotherapy in patients with treatment refractory, advanced TNBC (OMS-I140). Finally, we expand these findings using publicly available breast cancer and melanoma datasets. RESULTS: Single-cell RNA sequencing of murine tumors identified a CXCR3 gene signature (CXCR3-GS) following Tavo treatment associated with enhanced antigen presentation, T-cell infiltration and expansion, and PD-1/PD-L1 expression. Assessment of pretreatment and posttreatment tissue from patients confirms enrichment of this CXCR3-GS in tumors from patients that exhibited an enhancement of CD8+ T-cell infiltration following treatment. One patient, previously unresponsive to anti-PD-L1 therapy, but who exhibited an increased CXCR3-GS after Tavo treatment, went on to receive additional anti-PD-1 therapy as their immediate next treatment after OMS-I140, and demonstrated a significant clinical response. CONCLUSIONS: These data show a safe, effective intratumoral therapy that can enhance antigen presentation and recruit CD8 T cells, which are required for the antitumor efficacy. We identify a Tavo treatment-related gene signature associated with improved outcomes and conversion of nonresponsive tumors, potentially even beyond TNBC.
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Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Resistencia a Antineoplásicos/genética , Interleucina-12/genética , Plásmidos/administración & dosificación , Receptores CXCR3/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/terapia , Animales , Línea Celular Tumoral , Manejo de la Enfermedad , Modelos Animales de Enfermedad , Electroporación , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunofenotipificación , Inyecciones Intralesiones , Compuestos de Hierro , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Melanoma/terapia , Ratones , Plásmidos/genética , Resultado del Tratamiento , Neoplasias de la Mama Triple Negativas/etiología , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
PURPOSE: Tumors with low frequencies of checkpoint positive tumor-infiltrating lymphocytes (cpTIL) have a low likelihood of response to PD-1 blockade. We conducted a prospective multicenter phase II trial of intratumoral plasmid IL-12 (tavokinogene telseplasmid; "tavo") electroporation combined with pembrolizumab in patients with advanced melanoma with low frequencies of checkpoint positive cytotoxic lymphocytes (cpCTL). PATIENTS AND METHODS: Tavo was administered intratumorally days 1, 5, and 8 every 6 weeks while pembrolizumab (200 mg, i.v.) was administered every 3 weeks. The primary endpoint was objective response rate (ORR) by RECIST, secondary endpoints included duration of response, overall survival and progression-free survival. Toxicity was evaluated by the CTCAE v4. Extensive correlative analysis was done. RESULTS: The combination of tavo and pembrolizumab was well tolerated with adverse events similar to those previously reported with pembrolizumab alone. Patients had a 41% ORR (n = 22, RECIST 1.1) with 36% complete responses. Correlative analysis showed that the combination enhanced immune infiltration and sustained the IL-12/IFNγ feed-forward cycle, driving intratumoral cross-presenting dendritic cell subsets with increased TILs, emerging T cell receptor clones and, ultimately, systemic cellular immune responses. CONCLUSIONS: The combination of tavo and pembrolizumab was associated with a higher than expected response rate in this poorly immunogenic population. No new or unexpected toxicities were observed. Correlative analysis showed T cell infiltration with enhanced immunity paralleling the clinical activity in low cpCTL tumors.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Melanoma/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Adulto , Anciano , Anticuerpos Monoclonales Humanizados/administración & dosificación , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Humanos , Interleucina-12/administración & dosificación , Masculino , Melanoma/patología , Persona de Mediana Edad , Pronóstico , Estudios ProspectivosRESUMEN
PURPOSE OF REVIEW: Recent developments in immunotherapy have transformed the landscape of melanoma therapy. Here, we review markers for response to immunotherapy. RECENT FINDINGS: Current immunotherapies disable immune checkpoints on T cells and other immune cells and allow immune rejection of tumor. This process depends crucially on a preexisting response to the development of the melanoma. Here we describe the complexity of the anti-tumor immune response and the links to the development of markers that are currently used or under investigation in the clinic. We describe immune response biomarkers along with new developments that could translate into advances.
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Biomarcadores de Tumor/análisis , Inhibidores de Puntos de Control Inmunológico , Melanoma/inmunología , Melanoma/terapia , Antineoplásicos/efectos adversos , Antineoplásicos/inmunología , Antineoplásicos/uso terapéutico , Antígeno B7-H1/biosíntesis , Antígeno B7-H1/inmunología , Biomarcadores de Tumor/sangre , Antígeno CTLA-4/antagonistas & inhibidores , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/inmunología , Humanos , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Inhibidores de Puntos de Control Inmunológico/inmunología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia/métodos , Melanoma/genética , Pronóstico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Linfocitos T/inmunología , Escape del Tumor/efectos de los fármacos , Escape del Tumor/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
Whereas systemic IL12 is associated with potentially life-threatening toxicity, intratumoral delivery of IL12 through tavokinogene telseplasmid electroporation (tavo) is safe and can induce tumor regression at distant sites. The mechanism by which these responses are mediated is unknown but is presumed to result from a cellular immune response. In a phase II clinical trial of tavo (NCT01502293), samples from 29 patients with cutaneous melanoma with in-transit disease were assessed for immune responses induced with this treatment. Within the blood circulating immune cell population, we found that the frequencies of circulating PD-1+ CD4+ and CD8+ T cells declined with treatment. Circulating immune responses to gp100 were also detected following treatment as measured by IFNγ ELISpot. Patients with a greater antigen-specific circulating immune response also had higher numbers of CD8+ T cells within the tumor. Clinical response was also associated with increased intratumoral CD3+ T cells. Finally, intratumoral T-cell clonality and convergence were increased after treatment, indicating a focusing of the T-cell receptor repertoire. These results indicated that local treatment with tavo can induce a systemic T-cell response and recruit T cells to the tumor microenvironment.
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Linfocitos T CD8-positivos/inmunología , Electroporación/métodos , Interleucina-12/uso terapéutico , Melanoma/inmunología , Melanoma/terapia , Plásmidos/administración & dosificación , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/terapia , Microambiente Tumoral/inmunología , Adyuvantes Inmunológicos/uso terapéutico , Antígenos de Neoplasias/inmunología , Biomarcadores/sangre , Biomarcadores/metabolismo , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunoterapia/métodos , Interferón gamma/inmunología , Melanoma/metabolismo , Estadificación de Neoplasias , Seguridad del Paciente , Neoplasias Cutáneas/metabolismo , Resultado del Tratamiento , Melanoma Cutáneo MalignoRESUMEN
PURPOSE: IL12 promotes adaptive type I immunity and has demonstrated antitumor efficacy, but systemic administration leads to severe adverse events (AE), including death. This pilot trial investigated safety, efficacy, and immunologic activity of intratumoral delivery of IL12 plasmid DNA (tavo) via in vivo electroporation (i.t.-tavo-EP) in patients with Merkel cell carcinoma (MCC), an aggressive virus-associated skin cancer. PATIENTS AND METHODS: Fifteen patients with MCC with superficial injectable tumor(s) received i.t.-tavo-EP on days 1, 5, and 8 of each cycle. Patients with locoregional MCC (cohort A, N = 3) received one cycle before definitive surgery in week 4. Patients with metastatic MCC (cohort B, N = 12) received up to four cycles total, administered at least 6 weeks apart. Serial tumor and blood samples were collected. RESULTS: All patients successfully completed at least one cycle with transient, mild (grades 1 and 2) AEs and without significant systemic toxicity. Sustained (day 22) intratumoral expression of IL12 protein was observed along with local inflammation and increased tumor-specific CD8+ T-cell infiltration, which led to systemic immunologic and clinical responses. The overall response rate was 25% (3/12) in cohort B, with 2 patients experiencing durable clinical benefit (16 and 55+ months, respectively). Two cohort A patients (1 with pathologic complete remission) were recurrence-free at 44+ and 75+ months, respectively. CONCLUSIONS: I.t.-tavo-EP was safe and feasible without systemic toxicity. Sustained local expression of IL12 protein and local inflammation led to systemic immune responses and clinically meaningful benefit in some patients. Gene electrotransfer, specifically i.t.-tavo-EP, warrants further investigation for immunotherapy of cancer.
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Carcinoma de Células de Merkel/tratamiento farmacológico , Electroporación/métodos , Técnicas de Transferencia de Gen , Inmunoterapia/métodos , Interleucina-12/administración & dosificación , Plásmidos/administración & dosificación , Neoplasias Cutáneas/tratamiento farmacológico , Anciano , Anciano de 80 o más Años , Linfocitos T CD8-positivos/inmunología , Carcinoma de Células de Merkel/inmunología , Carcinoma de Células de Merkel/patología , Estudios de Cohortes , Femenino , Humanos , Interleucina-12/genética , Interleucina-12/metabolismo , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Seguridad del Paciente , Proyectos Piloto , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Resultado del TratamientoRESUMEN
Anti-PD-1 immune checkpoint blockers can induce sustained clinical responses in cancer but how they function in vivo remains incompletely understood. Here, we combined intravital real-time imaging with single-cell RNA sequencing analysis and mouse models to uncover anti-PD-1 pharmacodynamics directly within tumors. We showed that effective antitumor responses required a subset of tumor-infiltrating dendritic cells (DCs), which produced interleukin 12 (IL-12). These DCs did not bind anti-PD-1 but produced IL-12 upon sensing interferon γ (IFN-γ) that was released from neighboring T cells. In turn, DC-derived IL-12 stimulated antitumor T cell immunity. These findings suggest that full-fledged activation of antitumor T cells by anti-PD-1 is not direct, but rather involves T cell:DC crosstalk and is licensed by IFN-γ and IL-12. Furthermore, we found that activating the non-canonical NF-κB transcription factor pathway amplified IL-12-producing DCs and sensitized tumors to anti-PD-1 treatment, suggesting a therapeutic strategy to improve responses to checkpoint blockade.
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Células Dendríticas/inmunología , Interferón gamma/inmunología , Interleucina-12/inmunología , Neoplasias/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Células Dendríticas/metabolismo , Femenino , Humanos , Inmunoterapia/métodos , Interferón gamma/metabolismo , Interleucina-12/administración & dosificación , Interleucina-12/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , FN-kappa B/inmunología , FN-kappa B/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Tumor cells express a number of immunosuppressive molecules that can suppress anti-tumor immune responses. Efficient delivery of small interfering RNAs to treat a wide range of diseases including cancers remains a challenge. Retroviral replicating vectors (RRV) can be used to stably and selectively introduce genetic material into cancer cells. Here, we designed RRV to express shRNA (RRV-shPDL1) or microRNA30-derived shRNA (RRV-miRPDL1) using Pol II or Pol III promoters to downregulate PDL1 in human cancer cells. We also designed RRV expressing cytosine deaminase (yCD2) and miRPDL1 for potential combinatorial therapy. Among various configurations tested, we showed that RRV-miRPDL1 vectors with Pol II or Pol III promoter replicated efficiently and exhibited sustained downregulation of PDL1 protein expression by more than 75% in human cancer cell lines with high expression of PDL1. Immunologic effects of RRV-miRPDL1 were assessed by a trans-suppression lymphocyte assay. In vitro data showed downregulation of PDL1+ tumor cells restored activation of CD8+ T cells and bio-equivalency compared to anti-PDL1 antibody treatment. These results suggest RRV-miRPDL1 may be an alternative therapeutic approach to enhance anti-tumor immunity by overcoming PDL1-induced immune suppression from within cancer cells and this approach may also be applicable to other cancer targets.
RESUMEN
Therapeutic treatment of large established tumors using immunotherapy has yielded few promising results. We investigated whether adoptive transfer of tumor-specific CD8(+) T cells, together with tumor-specific CD4(+) T cells, would mediate regression of large established B16BL6-D5 melanomas in lymphopenic Rag1(-/-) recipients devoid of regulatory T cells. The combined adoptive transfer of subtherapeutic doses of both TRP1-specific TCR transgenic Rag1(-/-) CD4(+) T cells and gp100-specific TCR transgenic Rag1(-/-) CD8(+) T cells into lymphopenic recipients, who received vaccination, led to regression of large (100-400 mm(2)) melanomas. The same treatment strategy was ineffective in lymphoreplete wild-type mice. Twenty-five percent of mice (15/59) had tumors recur (15-180 d postregression). Recurrent tumors were depigmented and had decreased expression of gp100, the epitope targeted by the CD8(+) T cells. Mice with recurrent melanoma had increased CD4(+)Foxp3(+) TRP1-specific T cells compared with mice that did not show evidence of disease. Importantly, splenocytes from mice with recurrent tumor were able to suppress the in vivo therapeutic efficacy of splenocytes from tumor-free mice. These data demonstrate that large established tumors can be treated by a combination of tumor-specific CD8(+) and CD4(+) T cells. Additionally, recurrent tumors exhibited decreased Ag expression, which was accompanied by conversion of the therapeutic tumor-specific CD4(+) T cell population to a Foxp3(+)CD4(+) regulatory T cell population.
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Antígenos de Neoplasias/metabolismo , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/patología , Linfocitos T Reguladores/inmunología , Regulación hacia Arriba/inmunología , Traslado Adoptivo , Animales , Antígenos de Neoplasias/biosíntesis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/trasplante , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/trasplante , Línea Celular Tumoral , Femenino , Recuento de Linfocitos , Melanoma Experimental/terapia , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Recurrencia Local de Neoplasia/terapia , Oxidorreductasas/biosíntesis , Oxidorreductasas/deficiencia , Linfocitos T Reguladores/patología , Antígeno gp100 del Melanoma/metabolismoRESUMEN
Few immunotherapists would accept the concept of a single vaccination inducing a therapeutic anticancer immune response in a patient with advanced cancer. But what is the evidence to support the "more-is-better" approach of multiple vaccinations? Because we are unaware of trials comparing the effect of a single vaccine versus multiple vaccinations on patient outcome, we considered that an anticancer immune response might provide a surrogate measure of the effectiveness of vaccination strategies. Because few large trials include immunologic monitoring, the majority of information is gleaned from smaller trials in which an evaluation of immune responses to vaccine or tumor, before and at 1 or more times following the first vaccine, was performed. In some studies, there is convincing evidence that repeated administration of a specific vaccine can augment the immune response to antigens contained in the vaccine. In other settings, multiple vaccinations can significantly reduce the immune response to 1 or more targets. Results from 3 large adjuvant vaccine studies support the potential detrimental effect of multiple vaccinations as clinical outcomes in the control arms were significantly better than that for treatment groups. Recent research has provided insights into mechanisms that are likely responsible for the reduced responses in the studies noted above, but supporting evidence from clinical specimens is generally lacking. Interpretation of these results is further complicated by the possibility that the dominant immune response may evolve to recognize epitopes not present in the vaccine. Nonetheless, the Food and Drug Administration approval of the first therapeutic cancer vaccine and recent developments from preclinical models and clinical trials provide a substantial basis for optimism and a critical evaluation of cancer vaccine strategies.
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Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Vacunación/métodos , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/uso terapéutico , Epítopos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Humanos , Inmunización Secundaria , Melanoma/patología , Melanoma/terapia , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Linfocitos T/inmunología , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/uso terapéuticoRESUMEN
PURPOSE: Tumor-specific antigens of 3-methylcholanthrene (MCA)-induced sarcomas were defined by the narrow immune responses they elicited, which uniquely rejected the homologous tumor, with no cross-reactions between independently derived syngeneic MCA-induced tumors. This study examines whether an autophagosome-enriched vaccine derived from bortezomib-treated sarcomas can elicit an immune response that cross-reacts with other unique sarcomas. EXPERIMENTAL DESIGN: Mice were vaccinated with either MCA-induced sarcomas or autophagosomes derived from those tumors and later challenged with either homologous or nonhomologous sarcomas. In addition, 293 cells expressing a model antigen were used to understand the necessity of short-lived proteins (SLiP) in this novel vaccine. These findings were then tested in the sarcoma model. Autophagosomes were characterized by Western blotting and fluorescent microscopy, and their ability to generate immune responses was assessed in vitro by carboxyfluorescein succinimidyl ester dilution of antigen-specific T cells and in vivo by monitoring tumor growth. RESULTS: In contrast to a whole-cell tumor vaccine, autophagosomes isolated from MCA-induced sarcomas treated with a proteasome inhibitor prime T cells that cross-react with different sarcomas and protect a significant proportion of vaccinated hosts from a nonhomologous tumor challenge. Ubiquitinated SLiPs, which are stabilized by proteasome blockade and delivered to autophagosomes in a p62/sequestosome-dependent fashion, are a critical component of the autophagosome vaccine, as their depletion limits vaccine efficacy. CONCLUSION: This work suggests that common short-lived tumor-specific antigens, not physiologically available for cross-presentation, can be sequestered in autophagosomes by p62 and used as a vaccine to elicit cross-protection against independently derived sarcomas.