Immunologic Effects of a Peptide Vaccine with Intralesional IFNgamma For Treatment of Melanoma
ABSTRACT: Introduction: Optimal approaches to induce T-cell infiltration of tumors are not known. Chemokines CXCL9, CXCL10, and CXCL11 support effector T-cell recruitment, and may be induced by IFNgamma. This study tests the hypothesis that intratumoral administration of IFNgamma will induce CXCL9-11, and will induce T-cell recruitment and anti-tumor immune signatures in melanoma metastases. Patients and Methods: Nine eligible patients were immunized with a vaccine comprised of 12 class I MHC-restricted melanoma peptides (12MP) and received IFNgamma intratumorally. Effects on the tumor microenvironment (TME) were evaluated in sequential tumor biopsies. Adverse events (AE; CTCAE v4) were recorded. T-cell responses to vaccination were assessed in peripheral blood (PBMC) by IFNgamma ELIspot assay. Tumor biopsies were evaluated for immune cell infiltration, chemokine protein expression and gene expression. Results: Vaccination and intratumoral administration of IFNgamma were well tolerated. Circulating T-cell responses to vaccine were detected in 6 of 9 patients. IFNgamma increased production of chemokines CXCL10, CXCL11, and CCL5 in patient tumors. Neither vaccination alone nor the addition of IFNgamma promoted immune cell infiltration or induced anti-tumor immune gene signatures. Conclusion: The cancer vaccine did not significantly increase T-cell infiltration of tumors. This study provides intriguing findings highlighting some of the limitations of intratumoral IFNgamma treatment. Although IFNgamma is pivotal in anti-tumor immunity, single intratumoral injection may induce secondary immune regulation that paradoxically limits immune infiltration and effector functions. Therefore, alternate dosing strategies or additional combinatorial treatments may be needed to optimally promote trafficking and retention of T-cells in tumor, which merit further study. Overall design: Biopsies (incisional, core or excisional biopsies) of cutaneous or subcutaneous metastatic melanoma were obtained on day 1 (baseline, pre-treatment) (n=7), day 22 (1 week after the third vaccine) (n=7) and day 24 (48 hrs after intratumoral injection of IFNgamma (n=7). When possible, biopsies at day 24 included both a tumor injected with IFNgamma (n=7) and one uninjected (n=3).
INSTRUMENT(S): [HuGene-2_0-st] Affymetrix Human Gene 2.0 ST Array [transcript (gene) version]
Project description:Introduction: Infiltration of cancers by T-cells is associated with improved patient survival and response to immune therapies; however, optimal approaches to induce T-cell infiltration of tumors are not known. This study tests the hypothesis that topical treatment of melanoma metastases with the TLR7 agonist imiquimod treatment plus administration of a multipeptide cancer vaccine will improve immune cell infiltration of melanoma metastases. Patients and Methods: Eligible patients were immunized with a vaccine comprised of 12 melanoma peptides and a tetanus toxoid-derived helper peptide, and imiquimod was applied topically to tumors daily. Adverse events (AE; CTCAE v4.03) were recorded and effects on the tumor microenvironment (TME) were evaluated from sequential tumor biopsies. T-cell responses were assessed by IFNgamma ELIspot assay, and T-cell tetramer staining. Patient tumors were evaluated for immune cell infiltration, cytokine and chemokine production, and gene expression. Results and Conclusions: Four eligible patients were enrolled, and administration of imiquimod and vaccination was well tolerated in these patients. Circulating T-cell responses to the vaccine were detected by ex vivo ELIspot assay in 3 of 4 patients. Treatment of metastases with imiquimod induced immune cell infiltration and favorable gene signatures in the patients with circulating T-cell responses. This study supports further study of topical imiquimod combined with vaccines or other immune therapies for the treatment of melanoma. Precis: This clinical trial tested topical application of imiquimod to melanoma metastases combined with a melanoma vaccine. The regimen dramatically upregulated immune rejection gene signatures in melanoma metastases and increased T-cell infiltrate. Overall design: Biopsies (incisional, core or excisional biopsies) of cutaneous or subcutaneous metastatic melanoma were obtained on day 1 (baseline, pre-treatment) (n=3), day 22 (after 3 weeks of imiquimod treatment, and 1 week after the third vaccine) (n=5) and day 43 (after 6 weeks of imiquimod, and 1 week after the 4th vaccine) (n=3).
Project description:Clinical approaches to treat advanced melanoma include immune therapies, whose benefits depend on tumor-reactive T-cells to infiltrate metastases. However, most tumors lack significant immune infiltration prior to therapy, and some immune therapies are hindered by a persistent lack of immune cell infiltration. CXCL10 has been implicated as a critical chemokine supporting T-cell migration into tumors; thus agents that induce CXCL10 in tumors may improve patient responses to systemic immune therapy. We find that melanoma cells treated with TLR2/6 agonists (MALP-2 or FSL-1) and interferon-gamma (IFNgamma) upregulate CXCL10 production, when compared to IFNgamma treatment alone or no treatment. Gene profiling of melanoma cells lines treated with TLR2/6 agonists and IFNgamma demonstrate that a selective profile of genes are induced which may be favorable for promoting immune cell infiltration of tumors. TLR2 and TLR6 are widely expressed on human melanoma cells, and treatment of melanoma cells with TLR2/6 agonists and IFNgamma does not hinder melanoma cell apoptosis or promote proliferation. Furthermore, melanoma cells from surgically resected patient tumors upregulate CXCL10 production after treatment with TLR2/6 agonists and IFNgamma when compared to treatment with either agent alone. Collectively, these data identify TLR2/6 agonists and IFNgamma as a novel target for promoting CXCL10 production directly from melanoma cells. Samples from four human melanoma cell lines, VMM1 (n=6), DM13 (n=6), DM93 (n=6) and VMM39 (n=6), were treated with media alone, MALP-2 (TLR2/6 agonist), FSL-1 (TLR2/6 agonist), IFNgamma alone, MALP-2 and IFNgamma, or FSL-1 and IFNgamma.
Project description:Immunotherapy provides an alternative approach for cancer treatment. However, in-depth analyses of the effects of immunotherapy on the tumor microenvironment (TME) have not been conducted in non-melanoma tumors. Here we describe changes in the pancreatic ductal adenocarcinoma (PDAC) TME following immunotherapy treatment, and show for the first time that vaccine-based immunotherapy directly alters the TME, inducing neogenesis of tertiary lymphoid structures that convert immunologically quiescent tumors into immunologically active tumors. Alterations in five pathways important for immune modulation and lymphoid structure development (TH17/Treg, NFkB, Ubiquitin-proteasome, Chemokines/chemokine receptors, and Integrins/adhesion molecules) in vaccine-induced intratumoral lymphoid aggregates were associated with improved post-vaccination responses. Additional studies in other cancers and patients treated with other forms of immunotherapy are warranted to further develop signatures defined in intratumoral lymphoid structures into biomarkers that predict effective anti-tumor immune responses. These signatures may also expose therapeutic targets for promoting more robust antitumor immune responses in the TME. Between July 2008 and September 2012, 59 patients were enrolled into an ongoing study of an irradiated, allogeneic GM-CSF-secreting pancreatic tumor vaccine (GVAX) administered intradermally either alone or in combination with immune modulatory doses of cyclophophamide (Cy) as neoadjuvant and adjuvant treatment for patients with resectable pancreatic ductal adenocarcinoma (PDAC). Patients were randomized 1:1:1 to 3 treatment arms. In Arm A, patients received GVAX alone; in Arm B, patients received GVAX plus a single intravenous dose of Cy at 200 mg/m2 1 day prior to each vaccination; in Arm C, patients received GVAX plus oral Cy at 100 mg once daily for 1 week on and 1 week off. Up to 6 GVAX treatments were administered and all of the patients remained in their initial treatment arms throughout the duration of the study. All 59 of the patients received the 1st GVAX treatment 2 weeks +/-4 days prior to surgery. Formalin-fixed paraffin-embedded (FFPE) tissue blocks of surgically resected PDAC were obtained from the pathology archive. FFPE tissue blocks from each subject were stained by H&E immediately before the vaccine therapy-induced lymphoid aggregates were microdissected . To better understand the functional status of these vaccine therapy induced lymphoid aggregate structures, gene microarray analysis on RNA isolated from microdissected lymphoid aggregates was performed. Gene expression was compared among samples grouped according to patient overall survival, post-vaccination induction of enhanced mesothelin-specific T cell responses in peripheral blood lymphocytes (PBL), and the intratumoral CD8+ T effector to FoxP3+ Treg ratio. Post-vaccination induction of enhanced mesothelin-specific T cell responses has been reported to correlate with longer survival in patients treated with Panc GVAX.
Project description:To understand why cancer vaccine-induced T cells often fail to eradicate tumors, we studied immune responses in mice vaccinated with gp100 peptide emulsified in incomplete Freund's adjuvant (IFA), commonly used in clinical cancer vaccine trials. After gp100 peptide/IFA vaccination, tumor-specific CD8+ T cells (adoptively transferred from gp100-specific TCR-transgenic pmel-1 mice) accumulated not in tumors but at the persisting, antigen-rich vaccination site. Once there, primed T cells became dysfunctional and underwent antigen-driven, IFN-γ and FasL-mediated apoptosis, resulting in systemic hyporesponsiveness to subsequent vaccination. Provision of anti-CD40 antibody, TLR7 agonist and interleukin-2 (covax) reduced T cell apoptosis but did not prevent vaccination site sequestration. A non-persisting vaccine formulation shifted T cell localization towards tumors, inducing superior anti-tumor activity. Short-lived formulation also reduced systemic T cell dysfunction and promoted memory formation, as shown by gene expression profiling and other measures. Persisting peptide/IFA vaccine depots, currently used to vaccinate cancer patients, can induce specific T cell sequestration at vaccination sites followed by dysfunction and deletion; short-lived depot formulations may overcome these limitations and result in greater therapeutic efficacy of peptide-based cancer vaccines. Overall design: To study the fate of melanoma-specific CD8+ T cells after peptide vaccination, we tracked T cell receptor-transgenic pmel-1 T cells in mice vaccinated with heteroclitic gp100_25-33 peptide emulsified in IFA. Splenic pmel-1 CD8+ T cells were purified at 6 and 21 days after vaccination with either gp100/IFA/covax or gp100/saline/covax, and then their total RNA was extracted and used for comparison by gene expression profiling.
Project description:To understand why cancer vaccine-induced T cells often fail to eradicate tumors, we studied immune responses in mice vaccinated with gp100 peptide emulsified in incomplete Freund's adjuvant (IFA), commonly used in clinical cancer vaccine trials. After gp100 peptide/IFA vaccination, tumor-specific CD8+ T cells (adoptively transferred from gp100-specific TCR-transgenic pmel-1 mice) accumulated not in tumors but at the persisting, antigen-rich vaccination site. Once there, primed T cells became dysfunctional and underwent antigen-driven, IFN-γ and FasL-mediated apoptosis, resulting in systemic hyporesponsiveness to subsequent vaccination. Provision of anti-CD40 antibody, TLR7 agonist and interleukin-2 (covax) reduced T cell apoptosis but did not prevent vaccination site sequestration. A non-persisting vaccine formulation shifted T cell localization towards tumors, inducing superior anti-tumor activity. Short-lived formulation also reduced systemic T cell dysfunction and promoted memory formation, as shown by gene expression profiling and other measures. Persisting peptide/IFA vaccine depots, currently used to vaccinate cancer patients, can induce specific T cell sequestration at vaccination sites followed by dysfunction and deletion; short-lived depot formulations may overcome these limitations and result in greater therapeutic efficacy of peptide-based cancer vaccines. To study the fate of melanoma-specific CD8+ T cells after peptide vaccination, we tracked T cell receptor-transgenic pmel-1 T cells in mice vaccinated with heteroclitic gp100_25-33 peptide emulsified in IFA. Splenic pmel-1 CD8+ T cells were purified at 6 and 21 days after vaccination with either gp100/IFA/covax or gp100/saline/covax, and then their total RNA was extracted and used for comparison by gene expression profiling.
Project description:Both targeted inhibition of oncogenic driver mutations and immune-based therapies show efficacy in treatment of patients with metastatic cancer but responses are either short-lived or incompletely effective. Oncogene inhibition can augment the efficacy of immune-based therapy but mechanisms by which these two interventions might cooperate are incompletely resolved. Using a novel transplantable BRAFV600E-mutant murine melanoma model (SB-3123), we explore potential mechanisms of synergy between the selective BRAFV600E inhibitor vemurafenib and adoptive cell transfer (ACT)-based immunotherapy. We found that vemurafenib cooperated with ACT to delay melanoma progression but surprisingly did not enhance tumor infiltration or effector function of endogenous or adoptively transferred CD8+ T cells as previously observed. Instead, we found that the T cell cytokines IFN-gamma and TNF-alpha synergized with vemurafenib to induce cell cycle arrest of tumor cells in vitro. This was recapitulated in vivo as continuous vemurafenib administration was required to delay melanoma progression following ACT. The unexpected finding that immune cytokines synergize with oncogene inhibitors to induce growth arrest have major implications for understanding cancer biology at the intersection of oncogenic and immune signaling and provides a basis for design of combinatorial therapeutic approaches for patients with metastatic cancer. SB-3123p cells were treated in triplicate (biological replicates) under the following conditions for 96 hours: DMSO vehicle (control) (n=3); mouse IFNgamma (2.4 ng/ml) and mouse TNFalpha (0.24 ng/mL) (n=3); Vemurafenib (1uM) (n=3); and mouse IFNgamma (2.4 ng/ml), mouse TNFalpah (0.24 ng/mL) and Vemurafenib (1uM) (n=3).
Project description:Gene expression analysis in lymph nodes and site of injection (intradermal) after vaccination with adenovirus (Ad), modified vaccinia Ankara (MVA) or a mixed formulation of Ad+MVA. The hypothesis tested in the present study was that co-administration of two viral vectors induce a differential gene expression in the site of vaccination (dermis) and the draining lymph nodes that ultimately influences the protective ability of a vaccine against pre-erythrocytic malaria. Total RNA was isolated from the vaccination site (dermis) and lymph nodes after vaccination with adenovirus, MVA or Ad+MVA mixed co-administration after 6h and 24h (ear biopsies) and 9h, 24h and 72h for lymph nodes. Differential gene expression was assessed between vaccinated and non-immunized mice. Four ear biopsy samples did not pass quality control, and are not included in this submission.
Project description:Cancer immunotherapies utilize distinct mechanisms to harness the power of the immune system to eradicate cancer cells. Therapeutic vaccines, aimed at inducing active immune responses against an existing cancer, are highly dependent on the immunological microenvironment, where many immune cell types display high levels of plasticity and, depending on the context, promote very different immunological outcomes. Among them, plasmacytoid dendritic cells (pDC), known to be highly immunogenic upon inflammation, are maintained in a tolerogenic state by the tumor microenvironment. Here we report that intratumoral (i.t.) injection of established solid tumors with CpG oligonucleotides-B (CpG-B) inhibits tumor growth. Interestingly, control of tumor growth was independent of tumor-associated (TA) pDC, which remained refractory to CpG-B stimulation and whose depletion did not alter the efficacy of the treatment. Instead, tumor growth inhibition subsequent to i.t. CpG-B injection depended on the recruitment of neutrophils into the milieu, resulting in the activation of conventional dendritic cells (cDC), subsequent increased anti-tumor T cell priming in draining lymph nodes, and enhanced effector T cell infiltration in the tumor microenvironment. These results reinforce the concept that intratumoral delivery of TLR9 agonists alters the tumor microenvironment by improving the anti-tumor activity of both innate and adaptive immune cells. Overall design: mRNA profiles of tumor-associated plasmacytoid and conventional dendritic cells after intratumoral injection of CpG-B were generated by RNAseqencing, using Illumina HiSeq, 3-4 replicates per condition were used
Project description:Tumor-induced immunosuppression remains a major challenge for immunotherapy of cancer patients. To further elucidate why an allogeneic gene-modified (Interleukin-7(IL-7)/CD80 co-transfected) renal cell cancer vaccine failed to induce clinically relevant TH1-polarized immune responses, peripheral blood mononuclear cells (PBMCs) from enrolled study patients were analyzed by gene expression profiling (GEP) both prior and after vaccination. At baseline before vaccination, a profound downregulation of gene signatures associated with antigen presentation, immune response/T cells, cytokines/chemokines and signaling/transcription factors was observed in renal cell cancer patients as compared to healthy controls. Vaccination led to a partial reversion of preexisting immunosuppression, however, GEP indicated that an appropriate TH1 polarization could not be achieved. Most interestingly, our results suggest that the nuclear factor kappa B (NF-κB) signaling pathway might be involved in the impairment of immunological responsiveness and the observed TH2 deviation. In summary, our data suggest that GEP might be a powerful tool for the prediction of immunosuppression and the monitoring of immune responses within immunotherapy trials. Gene expression was profiled using Affymetrix Human Gene v1.1 ST microarrays in the following settings: 9 RCC patients were profiled before and after vaccination (pairs of measurements) and additionally 9 healthy control samples were profiled.
Project description:Whole blood transcriptomic profiling indicated that CHAd63-KH induced innate immune responses characterized by an interferon signature and the presence of activated dendritic cells. Overall design: Whole transcriptome using paired RNA samples with a high dose of the vaccine, pre vaccination and 24 hour post vaccination, and a subsequent experiment showing RNAseq from a low dose of the vaccine pre vaccination, 2hours post, 24 hours post and 14 days post vaccination.