Tumor Antigen-specific CTLs Research Articles

Utility of the RIG-I Agonist Triphosphate RNA for Melanoma Therapy

The pattern recognition receptor RIG-I plays an important role in the recognition of nonself RNA and antiviral immunity. RIG-I's natural ligand, triphosphate RNA (ppp-RNA), is proposed to be a valuable addition to the growing arsenal of cancer immunotherapy treatment options. In this study, we present comprehensive data validating the concept and utility of treatment with synthetic RIG-I agonist ppp-RNA for the therapy of human cancer, with melanoma as potential entry indication amenable to intratumoral treatment. Using mRNA expression data of human tumors, we demonstrate that RIG-I expression is closely correlated to cellular and cytokine immune activation in a wide variety of tumor types. Furthermore, we confirm susceptibility of cancer cells to ppp-RNA treatment in different cellular models of human melanoma, revealing unexpected heterogeneity between cell lines in their susceptibility to RNA agonist features, including sequence, secondary structures, and presence of triphosphate. Cellular responses to RNA treatment (induction of type I IFN, FasR, MHC-I, and cytotoxicity) were demonstrated to be RIG-I dependent using KO cells. Following ppp-RNA treatment of a mouse melanoma model, we observed significant local and systemic antitumor effects and survival benefits. These were associated with type I IFN response, tumor cell apoptosis, and innate and adaptive immune cell activation. For the first time, we demonstrate systemic presence of tumor antigen-specific CTLs following treatment with RIG-I agonists. Despite potential challenges in the generation and formulation of potent RIG-I agonists, ppp-RNA or analogues thereof have the potential to play an important role for cancer treatment in the next wave of immunotherapy.

Abstract 5024: Treatment with synthetic RIG-I agonist triphosphate RNA leads to local and systemic anti-tumor effects in a mouse melanoma tumor model

Abstract RIG-I is a highly important cytosolic pattern recognition receptor (PRR) involved in sensing RNA virus infection and inducing interferon (IFN) production. RIG-I’s natural ligand, triphosphate RNA (ppp-RNA), is proposed to be a valuable addition to the growing arsenal of cancer immunotherapy treatment options. This study validates the use of intratumoral treatment with synthetic RIG-I agonist ppp-RNA for the therapy of human cancer, with melanoma as potential entry indication amenable to intratumoral treatment. Firstly, we demonstrate that RIG-I expression is closely correlated to cellular and cytokine immune activation in a wide variety of tumor types. Secondly, cellular models of human melanoma confirm susceptibility of cancer cells to ppp-RNA treatment, revealing unexpected heterogeneity between cell lines in their selectivity for RNA features, including sequence, secondary structures and presence of triphosphate. Cellular RNA treatment responses (type I IFN, FasR, MHC-I, cytotoxicity) were demonstrated to be RIG-I dependent using RIG-I KO cells. Thirdly, we show that ppp-RNA treatment of a mouse melanoma tumor model, leads to significant local and systemic anti-tumor effects and survival benefits, associated with a type I IFN response, tumor cell apoptosis and innate and adaptive immune cell activation. For the first time, we demonstrate systemic presence of tumor antigen specific CTLs following treatment with RIG-I agonist. Overall our study demonstrates that ppp-RNA or analogs thereof have the potential to play an important role for cancer treatment in the next wave of immunotherapy. However, potential challenges in the generation and formulation of potent RIG-I agonists remain to be solved. Citation Format: Mike W. Helms, Eric Parmantier, Kerstin Jahn-Hofmann, Felix Gnerlich, Lars König, Christiane Metz-Weidmann, Monika Braun, Gabriele Dietert, Kaj Grandien, Joachim Theilhaber, Hui Cao, Tim Wagenaar, Max Schnurr, Stefan Endres, Dmitri Wiederschain, Sabine Scheidler, Simon Rothenfusser, Bodo Brunner. Treatment with synthetic RIG-I agonist triphosphate RNA leads to local and systemic anti-tumor effects in a mouse melanoma tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5024.

Abstract P4-06-27: LncRNA NKILA promotes tumour immune evasion by sensitizing tumour-specific t cells to activation-induced cell death

Abstract Activation induced cell death (AICD) of T lymphocytes is critical to maintain T-cell homeostasis, which is adopted by malignant tumors to convey immune evasion by eliminating tumor-reactive cytotoxic T cells. In this study, we demonstrated excessive apoptosis of tumor antigen-specific CTLs in breast and lung cancers. However, the mechanism involved in AICD of tumor-specific T cells remains obscure. Here, we demonstrated that NF-κB activity in tumor-specific T cells is high at the early phase of CTL activation induced by breast tumor antigens, but is suppressed at the later phase. This results in massive apoptosis of tumor-specific CTLs challenged by tumor cells. Interestingly, NKILA, an NFκB interacting lncRNA, sensitizes CTLs to AICD by inhibiting NF-κB activities after their activation, leading to tumor immune evasion. In vivo, administering CTLs with NKILA silencing into immunocompromised mice with breast cancer patient derived xenografts (PDXs) effectively inhibits PDX growth by increasing CTL infiltration. Clinically, NKILA was overexpressed in the tumor specific CTLs of breast and lung cancers, which was associated with less CTL infiltration in the tumors and shorter patient survival. Our findings present the first evidence that AICD in tumor-specific CTLs is crucial to cancer immune evasion, and targeting NKILA in CTLs emerges as a novel anti-tumor immunotherapy. Citation Format: Song E, Su S, Huang D. LncRNA NKILA promotes tumour immune evasion by sensitizing tumour-specific t cells to activation-induced cell death [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-27.

Abstract A79: Cancer immunotherapy with recombinant poliovirus induces IFN-dominant activation of antigen-presenting cells and tumor antigen-specific CTLs

Abstract The tumor microenvironment favors tumor immune escape by suppressing production, activation and/or function of antitumor T cells. Our group has developed a recombinant Rhino-Poliovirus chimera, PVSRIPO, currently being evaluated in Phase-II clinical trial against recurrent glioma. PVSRIPO therapy has afforded durable clinical responses in recurrent glioma patients. Clinical and pre-clinical findings suggest that PVSRIPO primarily acts through an immunological mechanism, rather than via cancer selective cytotoxicity. Here we define the immune adjuvant potential of intratumoral-delivered PVSRIPO that ultimately leads to the engagement/production of antitumor T cells. Sub-lethal infection of human dendritic cells (DCs) with PVSRIPO yields potent, sustained type I interferon dominant activation. PVSRIPO infection of macrophages induces similar activation; however, virus translation and the resulting type I Interferon/TNF-α production by macrophages is strongly enhanced in the presence of the Th2 cytokine IL-4. Compared to conventional adjuvants, e.g. poly(I:C) and LPS, PVSRIPO stimulation of APCs withstands tumor-associated immunosuppression, particularly in macrophages, and achieves more durable activation of DCs. Therapy of B16 melanoma tumors with PVSRIPO led to early production of type I interferon, IL-12, and IFN-γ; which was associated with the recruitment of neutrophils to the tumor site. Neutrophil influx was followed by DC and T cell infiltration. Mice treated with PVSRIPO developed tumor-antigen specific, cytotoxic T cells that were present in both tumor draining lymph nodes and spleens. These events correlated with delay in tumor growth and increase in survival following PVSRIPO therapy. Thus, PVSRIPO’s immune adjuvancy stimulates canonical innate inflammatory responses within the tumor microenvironment that culminates in tumor antigen-specific T cell responses. Citation Format: Michael C. Brown, Eda K. Holl, David Boczkowski, Elena Dobrikova, Mubeen Mosaheb, Vidya Chandramohan, Darell D. Bigner, Matthias Gromeier, Smita K. Nair. Cancer immunotherapy with recombinant poliovirus induces IFN-dominant activation of antigen-presenting cells and tumor antigen-specific CTLs [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A79.

Abstract B79: Identification and analysis of tumor-reactive CTLs in ovarian and cervical cancer tissues for developing a personalized immunotherapy

Abstract Cancer immunotherapy that target individual mutated neoantigens can mediate tumor regression. Ovarian cancer and cervical cancer are known to have relatively many somatic mutations and are considered to be good indications for immunotherapy. In order to develop personalized immunotherapy, we aimed to clarify the mutated neoantigens of individual patients with these cancers and to characterize the tumor antigen specific CTLs infiltrating into the tumor tissues. We obtained resected tumor tissues and normal tissues from about 30 patients with ovarian cancer or cervical cancer. In ex vivo assay, we investigated the cell surface markers of CD8+ T cell in tumor and normal tissues and identified that the expression of CD107a or co-inhibitory molecules on CD8+ T cells in tumor tissues was higher than that in normal tissues. These tumor-infiltrating lymphocytes were cultured for 14 days and we could expand the numbers of TILs from all patients’ tumors. At the same time, we tried to establish patient derived xenografts (PDX) mouse model or generate tumor cell lines from these patients. In cases where we could obtain tumor cells by these ways, TILs and autologous tumor cells were co-cultured and we evaluated the tumor reactive CTLs by CD107a assay. Tumor reactive CTLs were enriched by sorting for CD107a+ PD-1+ CD8+ T cells and expanded in vitro. The expanded CD 107a+ PD-1+ CD8+ T cells were co-cultured with autologous tumor cells and expression of CD 107a was analyzed. As a result, the proportion of CD 107a+ cells increased after expansion. Therefore, we could prove that many tumor-reactive CTLs are contained in population of CD107a+ PD-1+ CD8+ T cells. These results may provide the marker to isolate tumor reactive TILs ex vivo. We will identify candidates of mutated neoantigens by the next generation sequencing combined with bioinformatics prediction and will find the tumor reactive CTLs and these TCRs that recognize these mutated neoantigens. Citation Format: Kazuto Nosaka, Yoshiaki Yoshikawa, Toshihiro Suzuki, Keigo Saito, Manami Shimomura, Shoichi Mizuno, Shiro Suzuki, Fumitaka Kikkawa, Tetsuya Nakatsura. Identification and analysis of tumor-reactive CTLs in ovarian and cervical cancer tissues for developing a personalized immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B79.

Abstract 3570: Adoptive transfer of tumor antigen-specific CTLs requires anti-CTLA-4 and anti-PD-1 to drive tumor eradication

Abstract Adoptive transfer of tumor-reactive T cells (ACT) has led to significant clinical responses in hematologic malignancies. However, its efficacy in solid tumors has been dismal, likely due to the immunosuppression imposed by the tumor microenvironment (TME) of solid tumors. A proven effective strategy to revert the immunosuppressive TME is immune checkpoint blockades (ICBs), including anti-CTLA-4 and anti-PD-1/PD-L1, which have been recently approved by FDA for cancer therapy. Here, using the orthotopic B16-F10 melanoma model, we combine ACT of PMEL-1 Tg+ CTLs that recognize the endogenous melanoma antigen gp100 with dual blockade of CTLA-4 and PD-1, the highly pursued effective combinatorial ICB, to treat established tumors. Although ACT alone transiently suppresses tumor growth, complete tumor regression requires additional dual anti-CTLA-4 and anti-PD-1 blockade. Mechanistically, we demonstrate that the ICOS-Eomes-IFN-γαμμα axis, but not the ICOS-T-bet-IFN-γαμμα axis, in CTLs mediates the therapeutic effects of ACT and ICBs. Our data provide direct evidence that combining ACT of CTLs with ICBs confers long-term protection to tumor challenges, which largely depends on CTL-intrinsic expression of ICOS. Citation Format: Lewis Zhichang Shi, Jianjun Gao, Luis Vence, Jorge Brando, James Allison, Padmanee Sharma. Adoptive transfer of tumor antigen-specific CTLs requires anti-CTLA-4 and anti-PD-1 to drive tumor eradication [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3570.

Cancer immunotherapy with recombinant poliovirus induces IFN-dominant activation of dendritic cells and tumor antigen-specific CTLs.

Tumors thrive in an immunosuppressive microenvironment that impedes antitumor innate and adaptive immune responses. Thus, approaches that can overcome immunosuppression and engage antitumor immunity are needed. This study defines the adjuvant and cancer immunotherapy potential of the recombinant poliovirus/rhinovirus chimera PVSRIPO. PVSRIPO is currently in clinical trials against recurrent World Health Organization grade IV malignant glioma, a notoriously treatment-refractory cancer. Cytopathogenic infection of neoplastic cells releases the proteome and exposes pathogen- and damage-associated molecular patterns. At the same time, sublethal infection of antigen-presenting cells, such as dendritic cells and macrophages, yields potent, sustained type I interferon-dominant activation in an immunosuppressed microenvironment and promotes the development of tumor antigen-specific T cell responses in vitro and antitumor immunity in vivo. PVSRIPO's immune adjuvancy stimulates canonical innate anti-pathogen inflammatory responses within the tumor microenvironment that culminate in dendritic cell and T cell infiltration. Our findings provide mechanistic evidence that PVSRIPO functions as a potent intratumor immune adjuvant that generates tumor antigen-specific cytotoxic T lymphocyte responses.

Immunotherapy of WAP-TNP mice with early stage mammary gland tumors.

The SV40 transgenic BALB/c mouse based WAP-T/WAP-TNP model for triple-negative breast cancer allows the analysis of parameters influencing immunotherapeutic approaches. Except for WAP-TNP tumors expressing the immune-dominant LCMV NP-epitope within SV40 T-antigen (T-AgNP) which is not expressed by T-Ag of WAP-T tumors, the tumors are extremely similar. Comparative anti-PD1/PD-L1 immunotherapy of WAP-T and WAP-TNP mice supported the hypothesis that the immunogenicity of tumor antigen T-cell epitopes strongly influences the success of immune checkpoint blockade therapy, with highly immunogenic T-cell epitopes favoring rapid CTL exhaustion. Here we analyzed the immune response in NP8 mice during early times of tumor development. LCMV infection of lactating NP8 mice induced lifelong tumor protection by memory CTLs. Immunization with LCMV after involution and appearance of T-AgNP expressing parity-induced tumor progenitor cells could not cure the mice, as memory CTLs became exhausted. However, immunization significantly prolonged the time of tumor outgrowth. Elimination of exhausted CTLs and of immunosuppressive cells by sub-lethal γ-irradiation, followed by adoptive transfer of NP-epitope specific CTLs into NP8 tumor mice with early lesions, completely prevented tumor outgrowth, when lymphocytes obtained after injection of weakly immunogenic NP8 tumor-derived cells into BALB/c mice were transferred. Transfer of lymphocytes obtained after infection of BALB/c mice with highly immunogenic LCMV into such mice delayed tumor outgrowth for a significant period, but could not prevent it. We conclude that eliminating exhausted CTLs and immune-suppressive cells followed by transfer or generation of low-avidity tumor antigen-specific CTLs might be a promising approach for curative tumor immunotherapy.

Abstract B34: Signal transducing adaptor protein 2 (STAP2) has the crucial role in maintaining the CTL function of memory T cells

Abstract Purpose: Memory CD8+ T cells characterized the long survival after the strong activation followed by the appropriate antigenic stimulation have been believed to be an effective tumoricidal effectors in patients with cancer because they are possible to attack tumor cells repeatedly for a long time. Despite its importance, analysis of differentiation and function of memory CD8+ T cells is insufficient. In this study, we compared the memory CD8+ T cells induced by two murine vaccine systems to identify mechanism(s) participating in maintenance of effector function of tumoricidal long survival memory CTLs. Methods and Results: Full-length human NY-ESO-1 cDNA or CTL epitope peptide of NY-ESO-1 (p81-88) was immunized subcutaneously (s.c.) in BALB/c mice twice at 2 weeks interval or 3 times at weekly interval, respectively. At 44 days after the final vaccination, NY-ESO-1-expressing CT26 cells (CT26/NY-ESO-1) were inoculated s.c. of immunized mice, and subsequent tumor growth was measured. In addition, IFN-γ-expressing CD8+ T cell ratio in spleen of the immunized mice was monitored to evaluate vaccination efficacy. As a result, the inhibition of tumor growth was surprisingly shown in the NY-ESO-1 cDNA vaccination group more vigorously than the NY-ESO-1 CTL epitope peptide-treated group in comparison with untreated group. In addition, significant elevation of vaccine-specific IFN-γ-expressing CD8+ T cell ratio was observed in spleen of the NY-ESO-1 cDNA-treated mice. These results indicated that quality of memory CTLs was fully maintained in DNA vaccination group, but not peptide-treated group. To address this potent issue of superiority of DNA vaccination on the generation of long survival memory CTLs, vaccine-specific CTLs from DNA and peptide vaccination groups were purified and subjected to the total RNA micro array analysis. Memory CTLs from DNA-treated mice expressed signal transducing adaptor protein 2 (STAP2) over a 200 fold change compared with peptide-induced CTLs. Moreover, we clearly demonstrated by the study using STAP2 siRNA that STAP2 is essential for the maintenance of the tumoricidal effector function and cytokine expression of DNA-induced long survival memory CTLs. Conclusion: In this study, our murine vaccination system using human NY-ESO-1 gene and peptide clearly indicated that DNA vaccination is effective for the generation of tumoricidal long survival memory CTLs. In addition, we demonstrated for the first time that STAP2 plays as a crucial factor in the long-term maintenance of memory CTLs by probably the lasting care of cytokine expression and cytotoxicity. Since the maintenance of the effector function of the tumor antigen-specific memory CTLs has been thought to influence directly in the outcome of caner immunotherapy, our results regarding correlation between the generation of long survival memory CTLs and STAP2 have a possibility to be useful in the future treatment of patients with cancer. Citation Format: Daisuke Muraoka, Naohiro Seo, Tae Hayashi, Naozumi Harada, Hiroshi Shiku. Signal transducing adaptor protein 2 (STAP2) has the crucial role in maintaining the CTL function of memory T cells. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B34.

Adjuvant effects of formalin‐inactivated HSV through activation of dendritic cells and inactivation of myeloid‐derived suppressor cells in cancer immunotherapy

Use of adequate adjuvant is necessary for induction of effective antitumor immune responses. To develop an effective adjuvant for cancer immunotherapy, we selected formalin-inactivated (f)-HSV as an adjuvant component, and analyzed the mechanisms underlying its adjuvant effects. First, we found that f-HSV can induce the tumor antigen-specific CTLs by enhancing antigen cross-presentation by dendritic cells (DCs), mainly through TLR2, but not TLR9. Next, f-HSV was also found to prevent the accumulation of myeloid-derived suppressor cells (MDSCs). We demonstrated that the expansion of MDSCs in the blood and spleen during tumor progression required B cells producing the inflammatory angiogenesis factors, vascular endothelial growth factor (VEGF)-A and neuropilin-1 (NRP-1), a co-receptor for VEGF receptor-2 (VEGFR-2). Interestingly, the transmembrane-type NRP-1 on B cells changed to soluble-type NRP-1 (sNRP-1) by f-HSV treatment. We further showed that the sNRP-1 and VEGF-A secreted from B cells by f-HSV treatment could abrogate the immunosuppressive ability of MDSCs. These results suggest that f-HSV can enhance antitumor immune responses as an adjuvant, not only through activation of DCs, but also inactivation of MDSCs via B cells.

Abstract 1939: Augmenting effector function and abrogating Treg function by OX40 costimulation enhances adoptive transfer tumor-specific CTL response

Abstract We have recently shown that OX40 (CD134) costimulation provided with GM-CSF-secreting vaccine can bypass CD8+ T cell tolerance. OX40 costimulation can also abrogate Foxp3+ regulatory T cell (Treg)-mediated suppression of antitumor immunity by reducing Foxp3 expression on Tregs. In addition, OX40 costimulation can directly augment tumor-specific CTL function without CD4+ T cell help in vivo. Here we show that pre-treatment with agonistic anti-OX40 mAb augments the antitumor immune effects of adoptive CD8+ T cell therapy in the clinically relevant wild-type model, not TCR-transgenic, mouse model. CD8+ T cells stimulated with tumor antigen (HER2/neu immunodominant peptide and T2 cells), including a small number of tumor antigen-specific effector CD8+ T cells, were transferred into tumor-bearing FVB/N mice. We could detect the response of tumor antigen-specific CD8+ T cells that were adoptively transferred into the recipient mice, however, the response was not sufficient to eradicate an established tumor. In addition, we showed that pre-treatment with OX40 costimulation in tumor-bearing recipient in order to inhibit the Treg-mediated suppressive function enhanced tumor specific immune response of adoptively transferred CD8+ T cells in the recipients, thus resulting in the eradication of an established tumor. Moreover, adoptively transferred CTLs, with OX40 costimulation in vitro before transfer, significantly enhanced a tumor-specific immune response in the tumor-bearing recipient. Furthermore, when tumor antigen-specific CTLs costimulated through OX40 in vitro were adoptively transferred into the tumor-bearing recipients that had been pre-treated with OX40 costimulation to inhibit Treg function, transferred tumor-specific CTLs were effectively maintained in this recipient 7 times more than in the recipient with CTL transfer alone without OX40 treatment, and about 3 times more than in the recipient with a single treatment of OX40 costimulation. These findings therefore suggest that both direct enhancement of adoptive transfer CTL function and pre-inhibition of Treg-mediated suppressive function in recipients with OX40 costimulation are useful strategy for antitumor adoptive CTL therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1939.

Phase I study of immunization with the analogue peptide NY-ESO-1 157–165V in combination with CpG (PF-3512676) and Montanide ISA 720 in patients with stage III/IV melanoma

20011 Background: Analogue peptides represent a promising tool to further optimize peptide-based vaccines in promoting the expansion of tumor antigen-specific CTLs. Here, we report the results of a pilot trial designed to study the safety/toxicity and immunogenicity of the analogue peptide NY-ESO-1 157–165V in combination with CpG 7909/PF3512676 and Montanide ISA 720 in patients with NY-ESO-1- expressing melanoma. Methods: Eight HLA-A2+ patients with stage III/IV measurable melanoma were immunized with a stable emulsion composed of 3 ml Montanide in combination with either 2 mg CpG (arm 1), 400 μg analogue peptide NY-ESO-1 157–165V (arm 2) or CpG and peptide NY-ESO-1 157–165V (arm 3). Patients were vaccinated every other week for 8 immunizations prior to clinical and immunological evaluation. Results: In all patients, we observed grade I/II skin toxicity at the injection site with redness and pain. Only the three patients immunized with Montanide, CpG and peptide NY-ESO-1 157–165V in arm 3 developed a rapid and strong increase of effector- memory NY-ESO-1-specific CD8+ T cells that were detectable ex vivo. The majority of these T cells exhibited an intermediate/late-stage differentiated phenotype (CD28-). This study has further demonstrated that vaccination stimulated spontaneous tumor-reactive NY- ESO-1-specific CD8+ T cells in two patients with advanced disease, but failed to prime tumor-reactive NY-ESO-1-specific T cells in a patient without spontaneous tumor-reactive CD8+ T cells directed against NY-ESO-1. No objective clinical responses have been noted to date. Conclusions: Collectively, these data demonstrate that the peptide analogue NY-ESO-1 157–165V combined with CpG and Montanide promotes strong expansion of NY-ESO-1-specific CD8+ T cells in the blood of patients with advanced cancer. This study also demonstrates that the presence of tumor-induced NY-ESO-1-specific T cells of well-defined clonotypes is critical for expansion of tumor-reactive NY-ESO-1- specific CD8+ T cells following peptide vaccine strategies. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Coley Pharmaceutical Group Coley Pharmaceutical Group

IFN-γ– and TNF-dependent bystander eradication of antigen-loss variants in established mouse cancers

Tumors elicit antitumor immune responses, but over time they evolve and can escape immune control through various mechanisms, including the loss of the antigen to which the response is directed. The escape of antigen-loss variants (ALVs) is a major obstacle to T cell-based immunotherapy for cancer. However, cancers can be cured if both the number of CTLs and the expression of antigen are high enough to allow targeting of not only tumor cells, but also the tumor stroma. Here, we showed that IFN-gamma and TNF produced by CTLs were crucial for the elimination of established mouse tumors, including ALVs. In addition, both BM- and non-BM-derived stromal cells were required to express TNF receptors and IFN-gamma receptors for the elimination of ALVs. Although IFN-gamma and TNF were not required by CTLs for perforin-mediated killing of antigen-expressing tumor cells, the strong inference is that tumor antigen-specific CTLs must secrete IFN-gamma and TNF for destruction of tumor stroma. Therefore, bystander killing of ALVs may result from IFN-gamma and TNF acting on tumor stroma.

Induction of tumor antigen-specific cytotoxic T cell responses in naïve mice by latex microspheres-based artificial antigen-presenting cell constructs

Latex microspheres-based artificial antigen-presenting cell constructs (aAPCs) are proved to be valuable tools to expand T cells ex vivo for adoptive cell therapy, but little is known about their potential for active immunization. In this report, HLA-A2/peptide tetramers were generated and co-coated with anti-mouse CD28 monoclonal antibody onto surface of cell-sized latex microspheres followed by immunization of naïve HLA-A2/K b transgenic mice. Five- to six-fold expansion of tumor antigen-specific CTLs was observed in the spleen after three rounds of immunization. The consequent splenocytes can efficiently recognize endogenously expressed tumor antigen on the surface of human target cells and cytolyze the tumor cells in an antigen-specific manner. This report provides initially the experimental evidence that latex microspheres-based aAPCs can effectively prime antigen-specific CTL proliferation and cytolysis in naïve mice. This may contribute to a better insight into the potential of microspheres-based aAPCs for active immunization.

Immune Escape Associated with Functional Defects in Antigen-Processing Machinery in Head and Neck Cancer

Recent revival of interest in the role of immune surveillance in the pathogenesis and control of malignant diseases has focused attention on escape mechanisms used by tumor cells to evade immune recognition. Defects in the host's tumor antigen-specific immune responses and abnormalities in tumor cell expression of HLA class I molecules and tumor antigen are known to contribute to tumor progression. However, the mechanism(s) responsible for the lack of tumor cell recognition by functional HLA class I antigen-restricted, tumor antigen-specific CTLs despite expression of the restricting HLA class I allele and targeted tumor antigen by tumor cells remain(s) unexplained. In squamous cell carcinomas of the head and neck (SCCHN), this type of tumor escape is a rule rather than the exception. Here, we discuss evidence pointing to functional defects in the antigen-processing machinery as one mechanism underlying resistance of SCCHN cells to recognition and lysis by HLA class I antigen-restricted, tumor antigen-specific CTL. In addition, based on the restoration by IFN-gamma of SCCHN cell sensitivity to recognition by these CTL, we suggest strategies that may improve the clinical course of the disease by enhancing susceptibility of malignant cells to immune recognition.

Adoptive Transfer of Type 1 CTL Mediates Effective Anti–Central Nervous System Tumor Response: Critical Roles of IFN-Inducible Protein-10

The development of effective immunotherapeutic strategies for central nervous system (CNS) tumors requires a firm understanding of factors regulating the trafficking of tumor antigen-specific CTLs into CNS tumor lesions. Using C57BL/6 mice bearing intracranial (i.c.) ovalbumin-transfected melanoma (M05), we evaluated the efficacy and tumor homing of i.v. transferred type 1 or 2 CTLs (Tc1 or Tc2, respectively) prepared from ovalbumin-specific T-cell receptor-transgenic OT-1 mice. We also tested our hypothesis that intratumoral (i.t.) delivery of dendritic cells that had been transduced with IFN-alpha cDNA (DC-IFN-alpha) would enhance the tumor-homing and antitumor effectiveness of adoptively transferred Tc1 via induction of an IFN-gamma-inducible protein 10 (IP-10). In vitro, DC-IFN-alpha induced IP-10 production by M05 and enhanced the cytolytic activity of Tc1. In vivo, i.v. transferred Tc1 trafficked efficiently into i.c. M05 and mediated antitumor responses more effectively than Tc2, and their effect was IP-10 dependent. I.t. injections of DC-IFN-alpha remarkably enhanced the tumor homing, therapeutic efficacy, and in situ IFN-gamma production of i.v. delivered Tc1, resulting in the long-term survival and persistence of systemic ovalbumin-specific immunity. These data suggest that Tc1-based adoptive transfer therapy may represent an effective modality for CNS tumors, particularly when combined with strategies that promote a type 1 polarized tumor microenvironment.

425. Translation of Enhanced Dendritic Cell Vaccines Using a CID-Inducible CD40 Receptor

Top of pageAbstract Dendritic cell (DC)-based vaccination is a promising strategy to induce cancer immunity. Nevertheless, clinical applications have been almost universally disappointing. One potential limitation of current strategies is the use of maturation cocktails (MC) that include PGE2, which upregulate chemotaxis to lymph nodes, but may inhibit the ultimate activation state of the resulting DC product. Herein, we describe a novel DC-based vaccine that circumvents the use of standard MC and instead utilizes a genetically engineered inducible CD40 (iCD40) receptor 1. Our platform contains human monocyte-derived DCs (MoDCs) transduced with adenovector Ad5/f35-ihCD40 (iCD40-MoDC) and incubated for 2 days in the presence of monophosphoryl lipid A (MPL) and dimerizing drug, AP20187. Here we show that MoDC transduction with Ad5/f35-iCD40 effectively enhances co- stimulatory and migratory functions, including upregulation of IL-6, activation marker CD83, chemokine receptor CCR7 and augmentation of chemotaxis to CCL19/MIP3|[beta]|. Furthermore, the migratory abilities of iCD40-MoDCs were comparable to MoDCs matured with standard maturation cocktail (i.e. TNF-|[alpha]|, IL-1|[beta]|, IL-6 and PGE2). Unlike murine bone marrow-derived DCs expressing iCD40, IL-12 production by human MoDCs required additional TLR-4-ligand agonists, such as LPS or clinical analog MPL, which led to high (ng) levels of IL-12p70 in ihCD40-MoDCs, but not MC-stimulated or control MoDCs. Moreover, iCD40 and TLR4 agonists additively enhanced activation marker expression. We further show that iCD40 and MPL enhanced the immunostimulatory functions of MoDC, such as improving the stimulation and tumor-specific killing activity of MAGE-3 tumor antigen-specific CTL (shown by IFN-|[gamma]| ELISPOT and 51Cr-release assays) and facilitated Th1 polarization of na|[iuml]|ve CD4+ T-helper cells in vitro. Moreover, we have developed a novel in vivo systemof tracking human DCs transduced with adenovirus expressing red-shifted luciferase. MPL-treated iCD40-MoDCs readily migrated to the lymph nodes of SCID mice and were detectable for over 1 week after inoculation. In addition, we appraised the systemic toxicity of the iCD40-DC vaccine in na|[iuml]|ve C57BL/6 male mice. Mouse DCs were pulsed with TRP2 melanoma antigen, transduced with Ad- iCD40 or control Ad-GFP and injected 3 times bi-weekly. Mice were monitored for 3 months and routine CBC and serum chemistry (for liver and kidney functions) was tested regularly. We detected no significant toxicity of the iCD40-DCs vaccine and highly significant long-lived elevation of tumor antigen specific CTLs. This data suggest the potency and safety of this immunotherapeutic approach that combines non-inflammatory TLR stimulation with synthetic activation of iCD40 in human MoDCs. Unlike standard DC maturation approaches, this combination of reagents leads to high IL-12p70 levels, enhanced chemotactic response to lymph node-derived chemokines and augmented tumor antigen-specific CTL and Th1 responses. Inducible CD40-enhanced DCs represent a promising new strategy for cancer immunotherapy.

Ex vivo expanded human CD4+ regulatory NKT cells suppress expansion of tumor antigen-specific CTLs

NKT cells can produce large amounts of both Th1- and Th2-type cytokines and are an important regulatory cell type. To elucidate their role in acquired immunity, we examined the effect of human Valpha24+Vbeta11+ NKT cells or CD1d-specific ligand alpha-galactosylceramide (alphaGalCer) on the in vitro generation of antigen-specific CTLs from PBMCs using autologous MART-1(26-35) peptide-pulsed dendritic cells as stimulators. Flow cytometry using tetramer for MART-1(26-35) peptide revealed that NKT cells have inhibitory effects on CTL generation. Cytokine analysis using cytometric bead array assay and ELISA showed higher IL-4 and IL-10 secretion in the alphaGalCer(+) and/or NKT cell(+) culture setting, whereas IL-13 secretion in the culture was not affected by the presence of alphaGalCer. The CD4+ NKT cell subset seemed to play a major role in this inhibitory effect by secreting large amounts of Th2-type cytokines. Interestingly however, unlike recent reports utilizing mouse models, IL-13 was not a main effector molecule in our human system. Culture with alphaGalCer in the presence of cytokine-neutralizing antibodies for the Th2 cytokines, IL-4, IL-5 and IL-10, resulted in enhanced CTL generation, suggesting the dominant role of Th2 cytokines over Th1 cytokines. Thus, CD4+ NKT cells can work as immunoregulatory T cells that suppress anti-tumor immune response and, therefore, NKT cells or alphaGalCer could be used as therapeutic modalities to modulate systemic immune responses, such as autoimmune diseases. Conversely, the use of NKT cells along with anti-Th2 cytokine-neutralizing antibodies or CD4-negative NKT cell subset could enhance the generation of antigen-specific CTLs for adoptive immunotherapy.