Established B16 Melanoma Tumors Research Articles

Abstract 5079: Intratumoral CD40 agonist enhances the antitumor effect of anti-PD1 immunotherapy by activation of antigen-presenting cells and selective expansion of effector CD8+ T cells

Abstract Agonistic CD40 antibodies have shown promise when used in combination with checkpoint inhibitors in clinical trials for the treatment of malignancies. However, the mechanisms driving antitumor immune responses in patients are not well understood. The aim of this study was to use a preclinical melanoma model to evaluate the impact of intratumoral anti-CD40 administration on treatment efficacy and the tumor immune landscape in the context of systemic anti-PD1 therapy. Mice bearing 8-day established B16 melanoma tumors were injected with CD40 agonist intratumorally, either alone or in combination with anti-PD1 Ab, every 3 days for a total 4 doses. All mice treated with the combination therapy exhibited tumor growth arrest while progressive tumor growth was observed in control mice and mice treated with anti-PD1 alone. At day 15, tumor weights were 7- and 3-fold reduced in mice treated with the combination therapy as compared to control IgG or PD1 monotherapy, respectively. CyTOF analysis showed a 4-fold increase in the frequency of tumor-infiltrating immune cells in mice treated with either CD40 agonist alone or the combination therapy. Interestingly, CD40 agonistic Ab selectively expanded CD8+ T cells and the combination therapy exhibited a more pronounced effect compared to treatment with anti-PD1 Ab alone. Moreover, CD39+ CD8 T cells, representing tumor antigen-specific cytotoxic T cells, were 14- and 3-fold higher in tumors from mice treated with the combination therapy as compared to control or PD1-treated mice, respectively. This effect correlated with increases in the frequency of antigen-presenting cells, including cDC1 (6-fold) and B cells expressing CD40 (3-fold) in response to the combination therapy. In addition, the combination therapy significantly decreased myeloid cell population resulting in an 8-fold reduction in the ratio of myeloid cells to T cells. Amongst myeloid cells, the density of the monocytic population was diminished, with a selective 10-fold reduction of CD206+ M2-macrophages in tumors from mice treated with both Abs. Our findings provide evidence that combining systemic anti-PD1 therapy with intratumoral CD40 agonist enhanced antitumor immune responses by selectively expanding tumor antigen-specific effector CD8+ T cells, which was associated with increased infiltration of antigen-presenting cells and attenuation of immunosuppressive myeloid cells. Citation Format: Barbara Pazdrak, Heather M. Sonnemann, Salah-Eddine Bentebibel, Barbara M. Nassif, Greg Lizee, Adi Diab. Intratumoral CD40 agonist enhances the antitumor effect of anti-PD1 immunotherapy by activation of antigen-presenting cells and selective expansion of effector CD8+ T cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5079.

Antigen epitope-TLR7/8a conjugate as self-assembled carrier-free nanovaccine for personalized immunotherapy

Epitope-based vaccine is a promising personalized cancer immunotherapy; however, a simple and effective approach for its bulk manufacturing is challenging. Current vaccination strategies complicate the process by introducing unnecessary components such as additional delivery carriers, and assembly units. Herein, a type of toll-like receptor 7/8 agonist-epitope conjugate (termed as TLR7/8a-epitope) has been developed as a self-assembled and carrier-free nano vaccine platform, which effectively introduces the antigen and adjuvant with maximum precision, resulting in significantly enhanced dendritic cells (DCs) activation through the MyD88-dependent TLR signaling pathway. TLR7/8a-epitope nanovaccine can prolong the local retention and increase drainage efficiency into the lymph node, eliciting a significantly higher level of CD8 T-cell immunity than those of conventional vaccine formulations. The immunization with TLR7/8a-epitope nanovaccine in mice can not only resist the invasion of B16 cancer cells, but also produce significant therapeutic effects against established B16 melanoma tumors. Therefore, the TLR7/8a-epitope nanovaccine, developed by the direct chemical conjugation of antigen peptide with immunoadjuvant, has great advantages of clear and leanest compositions, controllable and definite preparation process, and remarkable therapeutic effects, representing a new appraoch for personalized cancer immunotherapy. Statement of significanceHerein, a kind of toll-like receptor 7/8 agonist-epitope conjugate was developed and spontaneously self-assemble into nanostructure in aqueous solution without the use of any additional constituents, which can be termed as unique carrier-free nanovaccine platform, providing effectually the leanest vaccine components with maximally and precisely loading of antigen and adjuvant.Significantly, the nanovaccine augmented the immunogenicity of antigenic peptide by increasing DCs activation through MyD88-mediated TLR signaling pathways and promoting T-cell priming. Moreover, nanovaccines could prolong the local retention and further increase the efficiency of drainage into dLNs, which was contributing to efficient initiation of epitope-specific memory and effector T-cell immune responses, leading to effective prophylactic and therapeutic antitumor effects.

Host conditioning with IL-1β improves the antitumor function of adoptively transferred T cells

Host conditioning has emerged as an important component of effective adoptive cell transfer-based immunotherapy for cancer. High levels of IL-1β are induced by host conditioning, but its impact on the antitumor function of T cells remains unclear. We found that the administration of IL-1β increased the population size and functionality of adoptively transferred T cells within the tumor. Most importantly, IL-1β enhanced the ability of tumor-specific T cells to trigger the regression of large, established B16 melanoma tumors in mice. Mechanistically, we showed that the increase in T cell numbers was associated with superior tissue homing and survival abilities and was largely mediated by IL-1β-stimulated host cells. In addition, IL-1β enhanced T cell functionality indirectly via its actions on radio-resistant host cells in an IL-2- and IL-15-dependent manner. Our findings not only underscore the potential of provoking inflammation to enhance antitumor immunity but also uncover novel host regulations of T cell responses.

Biodegradable STING agonist nanoparticles for enhanced cancer immunotherapy

Therapeutic cancer vaccines require adjuvants leading to robust type I interferon and proinflammatory cytokine responses in the tumor microenvironment to induce an anti-tumor response. Cyclic dinucleotides (CDNs), a potent Stimulator of Interferon Receptor (STING) agonist, are currently in phase I trials. However, their efficacy may be limited to micromolar concentrations due to the cytosolic residence of STING in the ER membrane. Here we utilized biodegradable, poly(beta-amino ester) (PBAE) nanoparticles to deliver CDNs to the cytosol leading to robust immune response at >100-fold lower extracellular CDN concentrations in vitro. The leading CDN PBAE nanoparticle formulation induced a log-fold improvement in potency in treating established B16 melanoma tumors in vivo when combined with PD-1 blocking antibody in comparison to free CDN without nanoparticles. This nanoparticle-mediated cytosolic delivery method for STING agonists synergizes with checkpoint inhibitors and has strong potential for enhanced cancer immunotherapy.

Abstract 2994: Engineered STING-targeting immunotherapy delays B16 melanoma tumor growth and significantly improves animal survival as compared to an immune-checkpoint inhibitor

Abstract Background: The stimulator of interferon genes (STING) is a cytosolic innate sensor activated by cyclic di-nucleotides (CDNs) including the bacterially derived bis-(3′-5′)-cyclic-dimeric-guanosine monophosphate (c-di-GMP), leading to the induction of type I interferons. We recently published that an engineered adenovirus (Ad) vector delivering a potent, bacterially derived diguanylate cyclase (AdVCA0848) in vivo can generate sustained c-di-GMP levels in vivo and induce beneficial immune responses, including induction of IFN-β, activation of NK cells and DCs, as well as enhance antigen-specific T cell responses. Here, we determined if in vivo use of AdVCA0848 can also induce potent anti-tumor responses. Methods: Experiments to be presented include those that subcutaneously placed 150,000 B16F10 melanoma cells into C57BL/6 mice. On day 7 post tumor implant (PTI), tumor-bearing mice were randomly divided into control groups (receiving intra-tumoral (i.t.) vehicle treatments or Ad controls, ± i.p administration of non-specific antibodies) and experimental groups, (single i.t. administrations with 2x1010 vps of AdVCA0848 alone, or in combination with repeated i.p. administrations of 200 μg of an anti-PD1 antibody (RMP-1-14) on days 7,11,15 and 19 PTI. Results: As compared to appropriate controls, we have repeatedly found that a single i.t. injection of AdVCA0848 significantly slowed B16 tumor growth (p<0.01), a result that correlated with high levels of animal survival (p<0.001). These levels of efficacy significantly exceeded those achieved with lone use of a PD-1 inhibitor. Under these same conditions, combined use of the AdVCA0848 therapy with anti-PD1 treatments trended to further improve animal survival (50% survival at day 27 PTI), as compared to lone use of either therapy (36.4% and 26.7% survival at day 27 PTI for AdVCA0848 and PD-1 treatments, respectively). Multiple innate and adaptive immune response readouts and immunohistological evaluations will be presented. Conclusions: The growth of established B16 melanoma tumors was rapidly inhibited after a single i.t. treatment with the engineered STING activator AdVCA0848, and this efficacy resulted in significant improvements in animal survival, at rates that were also significantly better than those achieved using a PD-1 antagonist. Furthermore, the i.t. administration of AdVCA0848 trended to show further improved efficacy when combined with a PD-1 antagonist, suggesting synergy. The AdVCA0848 platform is a novel, off the shelf immune-therapeutic that can now be justified for use to improve anti-tumor responses in melanoma, augment currently approved FDA immune therapies for melanoma (such as PD-1 targeted therapies), as well may show efficacy in other aggressive cancers. Citation Format: Andrea Amalfitano, Fadel S. Alyaqoub, Yasser A. Aldhamen, Abdulraouf Abbas, Cristiane Pereira-Hicks, Sarah Godbehere, Christopher M. Waters. Engineered STING-targeting immunotherapy delays B16 melanoma tumor growth and significantly improves animal survival as compared to an immune-checkpoint inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2994. doi:10.1158/1538-7445.AM2017-2994

Abstract 4272: Potent in situ cancer immunotherapy with synthetic human STING-activating cyclic dinucleotides

Abstract Spontaneous tumor-initiated T cell priming is dependent on IFN-β production by tumor-resident dendritic cells. Based on recent observations indicating that IFN-β expression was dependent upon activation of the host STING (Stimulator of Interferon Genes) pathway, we hypothesized that direct engagement of STING through intratumoral administration of specific agonists would result in effective anti-tumor therapy. To this end, we generated novel synthetic cyclic dinucleotide (CDN) derivatives with superior STING-activating and anti-tumor properties. The most potent molecule contains a 2′-5′ and 3′-5′ phosphate bridge that increases its affinity for STING at least 10-fold. Additionally, sulfur atoms at the non-bridging oxygens of the internucleotide phosphate bridge enable both superior STING binding affinity, as well as resistance to host cell phosphodiesterases. Importantly, the lead molecule activates PBMCs from a panel of human donors representing all known STING alleles, including homozygotes for the most refractory allele. Intratumoral injection of the lead CDN molecule induced profound regression of established B16 melanoma tumors, generated substantial systemic immune responses capable of rejecting distant metastases and provided long-lived immunologic memory. Cures in entire cohorts of mice bearing well-established 4T1 mammary and CT26 colon carcinoma models correlated with induction of cytokine-mediated innate and adaptive antigen-specific T cell immunity, both systemically and in the tumor microenvironment. Immune activation and anti-tumor efficacy was lost in mice encoding non-functional STING. In vivo activation of STING with the lead CDN administered by multiple routes demonstrated a high safety and tolerability margin in preclinical toxicology studies. In addition, pharmacokinetic analysis indicates that the lead CDN compound was rapidly cleared from systemic circulation, consistent with pulsatile STING activation and transient systemic cytokine and hematological changes. These results demonstrate that CDNs have high translation potential for treatment strategies to induce activation of the tumor microenvironment, leading to effective tumor-initiated T cell priming and anti-tumor immunity. Citation Format: Laura Hix Glickman, David B. Kanne, Kelsey E. Gauthier, George E. Katibah, Justin J. Leong, Ken Metchette, Thomas W. Dubensky, Sarah M. McWhirter. Potent in situ cancer immunotherapy with synthetic human STING-activating cyclic dinucleotides. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4272. doi:10.1158/1538-7445.AM2015-4272

Simultaneous Targeting of Tumor Antigens and the Tumor Vasculature Using T Lymphocyte Transfer Synergize to Induce Regression of Established Tumors in Mice

Most systemic cancer therapies target tumor cells directly, although there is increasing interest in targeting the tumor stroma that can comprise a substantial portion of the tumor mass. We report here a synergy between two T-cell therapies, one directed against the stromal tumor vasculature and the other directed against antigens expressed on the tumor cell. Simultaneous transfer of genetically engineered syngeneic T cells expressing a chimeric antigen receptor targeting the VEGF receptor-2 (VEGFR2; KDR) that is overexpressed on tumor vasculature and T-cells specific for the tumor antigens gp100 (PMEL), TRP-1 (TYRP1), or TRP-2 (DCT) synergistically eradicated established B16 melanoma tumors in mice and dramatically increased the tumor-free survival of mice compared with treatment with either cell type alone or T cells coexpressing these two targeting molecules. Host lymphodepletion before cell transfer was required to mediate the antitumor effect. The synergistic antitumor response was accompanied by a significant increase in the infiltration and expansion and/or persistence of the adoptively transferred tumor antigen-specific T cells in the tumor microenvironment and thus enhanced their antitumor potency. The data presented here emphasize the possible beneficial effects of combining antiangiogenic with tumor-specific immunotherapeutic approaches for the treatment of patients with cancer.

A role for the GITR pathway in anaphylaxis (P4326)

Abstract Immune modulation using monoclonal antibodies has a significant impact on the overall survival of patients with metastatic melanoma. Monoclonal antibodies that engage members of the tumor necrosis factor receptor (TNFR) superfamily have shown promising tumor protection in preclinical models. Glucocorticoid-induced TNFR-related protein (GITR) is a particularly potent target for tumor immunotherapy. A single dose of GITR agonist antibody results in 60% protection from established B16 melanoma tumors. Therefore, several doses of anti-GITR may be necessary in order to fully eradicate established tumors. Because GITR is expressed throughout the immune system, we investigated whether multiple doses of anti-GITR result in adverse immune-related events. We observed that three doses of anti-GITR cause anaphylaxis, a side effect that has not been reported using this or other TNFR superfamily agonist antibodies. Anaphylaxis was not observed upon the first or second doses of anti-GITR or in GITR knockout mice. Anti-GITR-induced anaphylaxis is associated with a severe drop in body temperature and elevated serum proinflammatory cytokine levels. We are further investigating the causes of toxicity resulting from repetitive anti-GITR treatment and whether this effect can be reversed or prevented while preserving the antitumor activity of anti-GITR, an important step given the translational potential of this therapy.

Abstract 2835: Combined stimulation of TLR9 and 4.1BB receptors enhances Trp2 peptide-mediated B16 melanoma regression by increasing Ag-specific CTL activity and infiltration to tumor sites.

Abstract We previously reported that intratumoral electroporation (IT-EP) with IL-12 cDNA (IT-EP/IL12) led to the eradication of established B16 melanoma tumors in mice. Tumor eradication required the participation of CD8+ T cells, but not CD4+ T cells and NK cells. IT-EP/IL12 induced antigen-specific CD8+ T cell responses against the immunodominant Trp2180-188 epitope and generated a systemic response, resulting in significant therapeutic effects against distal, untreated tumors. In this study, we observed that when Trp2180-188 self-peptides were combined with the TLR9 agonist, CpG-oligodeoxynucleotide (ODN) for antitumor immune therapy, self-tolerance was broken, giving rise to induction of Trp2-specific CTL responses and some antitumor activity against established melanoma (7 mm in mean diameter). A combination of anti-4.1BB antibodies with Trp2 peptides plus CpG-ODN increased the antitumor regression rate from 0 to 75%. This effect was concomitant with greater induction of Ag-specific CD8+ CTLs and their infiltration into the tumor sites, highlighting the importance of combined stimulation of TLR9 and 4.1BB for achieving tumor eradication. These findings may have implications for designing peptide-based therapeutic vaccines for cancer-patients. Citation Format: Jeong-Im Sin, Hun Mo Ryoo. Combined stimulation of TLR9 and 4.1BB receptors enhances Trp2 peptide-mediated B16 melanoma regression by increasing Ag-specific CTL activity and infiltration to tumor sites. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2835. doi:10.1158/1538-7445.AM2013-2835

Ongoing priming of naïve melanoma/melanocyte specific CD8 T cells occurs in hosts with vitiligo (P225) (162.6)

Abstract Vitiligo, the autoimmune destruction of melanocytes, correlates with improved survival in melanoma patients although it is often disregarded as a side effect of robust anti-tumor T cell responses. We have shown that antigen provided by vitiligo is required for long-lived CD8 T cell memory against melanoma-expressed self-antigens. However, the possibility that autoimmune melanocyte destruction itself may prime CD8 T cells able to cross-react against melanoma has not been investigated. Previously, we reported vitiligo development in 60% of mice depleted of regulatory CD4 T cells while bearing established B16 melanoma tumors. To probe the immunogenicity of vitiligo, we transferred naïve melanocyte-antigen specific pmel CD8 T cells in to actively depigmenting hosts. We find that CD8 T cell activation and proliferation occurs in an antigen-specific manner only in hosts with vitiligo. Furthermore, although the pmel cells do not produce IFN-γ, the cells produce Granzyme B and express LAMP-1 and P selectin ligand. Interestingly, although vitiligo-primed CD8 T cells appear strongly activated, the cells do not form robust memory populations in the presence of CD4 T regulatory cells. Ongoing studies aim to investigate the anti-melanoma capabilities of vitiligo-primed CD8 T cells. These studies will further enhance our understanding of the direct relationship between tumor immunity and autoimmunity, and the contributions of these cells in protecting against recurrent tumor growth.

Intratumoral electroporation of IL-12 cDNA eradicates established melanomas by Trp2(180-188)-specific CD8+ CTLs in a perforin/granzyme-mediated and IFN-γ-dependent manner: application of Trp2(180-188) peptides.

Intratumoral electroporation (IT-EP) with IL-12 cDNA (IT-EP/IL12) can lead to the eradication of established B16 melanoma tumors in mice. Here, we explore the immunological mechanism of the antitumor effects generated by this therapy. The results show that IT-EP/IL12 applied only once resulted in eradication in 70% animals with large established B16 tumors. Tumor eradication required the participation of CD8+ T cells, but not CD4+ T cells and NK cells. IT-EP/IL12 induced antigen-specific CD8+ T cell responses against the immunodominant Trp2(180-188) epitope and generated a systemic response, resulting in significant therapeutic effects against distal, untreated tumors. The therapeutic effect of IT-EP/IL12 was absent in perforin-deficient mice, indicating that tumor elimination occurred through conventional perforin/granzyme lysis by CTLs. Moreover, this therapy induced some degree of immunological memory that protected approximately one-third of the cured mice against a subsequent tumor challenge. Moreover, antitumor efficacy and long-term protection against B16 were significantly improved by concurrent Trp2 peptide immunization through more induction of Ag-specific CTL responses and more attraction of IFN-γ-expressing CD8+ T cells into tumor sites. The antitumor effect of IT-EP/IL12 required the participation of IFN-γ, which was shown to induce MHC class I expression on B16 cells and increase the lytic activity of the CD8+ CTL generated by IT-EP/IL12. The results from these animal studies may help in the development of IT-EP/IL12 for cancer patients.

Determinants of Successful CD8+ T-Cell Adoptive Immunotherapy for Large Established Tumors in Mice

Adoptive cell transfer (ACT) of tumor infiltrating or genetically engineered T cells can cause durable responses in patients with metastatic cancer. Multiple clinically modifiable parameters can comprise this therapy, including cell dose and phenotype, in vivo antigen restimulation, and common gamma-chain (γ(c)) cytokine support. However, the relative contributions of each these individual components to the magnitude of the antitumor response have yet to be quantified. To systematically and quantitatively appraise each of these variables, we employed the Pmel-1 mouse model treating large, established B16 melanoma tumors. In addition to cell dose and magnitude of in vivo antigen restimulation, we also evaluated the relative efficacy of central memory (T(CM)), effector memory (T(EM)), and stem cell memory (T(SCM)) subsets on the strength of tumor regression as well as the dose and type of clinically available γ(c) cytokines, including IL-2, IL-7, IL-15, and IL-21. We found that cell dose, T-cell differentiation status, and viral vaccine titer each were correlated strongly and significantly with the magnitude of tumor regression. Surprisingly, although the total number of IL-2 doses was correlated with tumor regression, no significant benefit to prolonged (≥6 doses) administration was observed. Moreover, the specific type and dose of γ(c) cytokine only moderately correlated with response. Collectively, these findings elucidate some of the key determinants of successful ACT immunotherapy for the treatment of cancer in mice and further show that γ(c) cytokines offer a similar ability to effectively drive antitumor T-cell function in vivo.

Natural Splice Variant of MHC Class I Cytoplasmic Tail Enhances Dendritic Cell-Induced CD8+ T-Cell Responses and Boosts Anti-Tumor Immunity

Dendritic cell (DC)-mediated presentation of MHC class I (MHC-I)/peptide complexes is a crucial first step in the priming of CTL responses, and the cytoplasmic tail of MHC-I plays an important role in modulating this process. Several species express a splice variant of the MHC-I tail that deletes exon 7-encoding amino acids (Δ7), including a conserved serine phosphorylation site. Previously, it has been shown that Δ7 MHC-I molecules demonstrate extended DC surface half-lives, and that mice expressing Δ7-Kb generate significantly augmented CTL responses to viral challenge. Herein, we show that Δ7-Db-expressing DCs stimulated significantly more proliferation and much higher cytokine secretion by melanoma antigen-specific (Pmel-1) T cells. Moreover, in combination with adoptive Pmel-1 T-cell transfer, Δ7-Db DCs were superior to WT-Db DCs at stimulating anti-tumor responses against established B16 melanoma tumors, significantly extending mouse survival. Human DCs engineered to express Δ7-HLA-A*0201 showed similarly enhanced CTL stimulatory capacity. Further studies demonstrated impaired lateral membrane movement and clustering of human Δ7-MHC-I/peptide complexes, resulting in significantly increased bioavailability of MHC-I/peptide complexes for specific CD8+ T cells. Collectively, these data suggest that targeting exon 7-encoded MHC-I cytoplasmic determinants in DC vaccines has the potential to increase CD8+ T-cell stimulatory capacity and substantially improve their clinical efficacy.

Abstract 473: CD4+ T cell help differentially influences anti-tumor immunity as a function of T cell avidity

Abstract One of the major hurdles for cancer immunotherapy is that most tumors express self-antigens to which the immune system is unresponsive due to self-tolerance. Our lab and others have demonstrated that high-avidity tumor-specific CD8+ T cells are superior in anti-tumor activity to low avidity T cells. However, adoptive transfer of high-avidity, tumor-specific CTL populations may fail due to loss of CTL effector functions upon tumor infiltration. Therefore, strategies to maintain effector functions of high avidity CTL, or enhancement of low avidity CTL are desirable. To investigate whether provision of CD4+ T cell help will enhance the effectiveness of high-avidity CTLs in anti-tumor immunity as compared to lower avidity T cells, we utilized two transgenic mouse lines developed in our lab that bear different TCR transgenes specific for a common melanoma differentiation antigen, tyrosinase-related protein 2 (TRP-2(180-188)). T cells from clone 24 TCR Tg mice (TCRhi) show higher avidity than those from clone 37 (TCRlo) mice based on tetramer dissociation and in vitro effector function assays. Here we report that co-activation of CD4+ T cells (OVA323-339 specific, CD4-OVA) with TRP-2-specific CD8+ T cells can boost the in vitro function of TCRhi cells, including proliferation, IFN-γ secretion, Gr-B secretion as well as cell-mediated cytotoxicity, but had minimal effect on TCRlo cells. We observed that only adoptive co-transfer of CD4-OVA cells with TCRhi cells suppressed tumor growth of (5 day) established B16 melanoma tumors. Furthermore, those mice receiving co-transferred CD4-OVA cells with TCRhi cells developed autoimmune depigmentation at the sites of vaccination and tumor challenge. Our finding may have important implications for enhancement of cancer immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 473. doi:10.1158/1538-7445.AM2011-473

Agonist Anti-GITR Monoclonal Antibody Induces Melanoma Tumor Immunity in Mice by Altering Regulatory T Cell Stability and Intra-Tumor Accumulation

In vivo GITR ligation has previously been shown to augment T-cell-mediated anti-tumor immunity, yet the underlying mechanisms of this activity, particularly its in vivo effects on CD4+ foxp3+ regulatory T cells (Tregs), have not been fully elucidated. In order to translate this immunotherapeutic approach to the clinic it is important gain better understanding of its mechanism(s) of action. Utilizing the agonist anti-GITR monoclonal antibody DTA-1, we found that in vivo GITR ligation modulates regulatory T cells (Tregs) directly during induction of melanoma tumor immunity. As a monotherapy, DTA-1 induced regression of small established B16 melanoma tumors. Although DTA-1 did not alter systemic Treg frequencies nor abrogate the intrinsic suppressive activity of Tregs within the tumor-draining lymph node, intra-tumor Treg accumulation was significantly impaired. This resulted in a greater Teff:Treg ratio and enhanced tumor-specific CD8+ T-cell activity. The decreased intra-tumor Treg accumulation was due both to impaired infiltration, coupled with DTA-1-induced loss of foxp3 expression in intra-tumor Tregs. Histological analysis of B16 tumors grown in Foxp3-GFP mice showed that the majority of GFP+ cells had lost Foxp3 expression. These “unstable” Tregs were absent in IgG-treated tumors and in DTA-1 treated TDLN, demonstrating a tumor-specific effect. Impairment of Treg infiltration was lost if Tregs were GITR−/−, and the protective effects of DTA-1 were reduced in reconstituted RAG1−/− mice if either the Treg or Teff subset were GITR-negative and absent if both were negative. Our results demonstrate that DTA-1 modulates both Teffs and Tregs during effective tumor treatment. The data suggest that DTA-1 prevents intra-tumor Treg accumulation by altering their stability, and as a result of the loss of foxp3 expression, may modify their intra-tumor suppressive capacity. These findings provide further support for the continued development of agonist anti-GITR mAbs as an immunotherapeutic strategy for cancer.

Efficient Immunization and Cross-Priming by Vaccine Adjuvants Containing TLR3 or TLR9 Agonists Complexed to Cationic Liposomes

Complexing TLR9 agonists such as plasmid DNA to cationic liposomes markedly potentiates their ability to activate innate immunity. We therefore reasoned that liposomes complexed with DNA or other TLR agonists could be used as effective vaccine adjuvants. To test this hypothesis, the vaccine adjuvant effects of liposomes complexed to TLR agonists were assessed in mice. We found that liposomes complexed to nucleic acids (liposome-Ag-nucleic acid complexes; LANAC) were particularly effective adjuvants for eliciting CD4(+) and CD8(+) T cell responses against peptide and protein Ags. Notably, LANAC containing TLR3 or TLR9 agonists effectively cross-primed CD8(+) T cell responses against even low doses of protein Ags, and this effect was independent of CD4(+) T cell help. Ag-specific CD8(+) T cells elicited by LANAC adjuvants were functionally active and persisted for long periods of time in tissues. In a therapeutic tumor vaccine model, immunization with the melanoma peptide trp2 and LANAC adjuvant controlled the growth of established B16 melanoma tumors. In a prophylactic vaccine model, immunization with the Mycobacterium tuberculosis protein ESAT-6 with LANAC adjuvant elicited significant protective immunity against aerosol challenge with virulent M. tuberculosis. These results suggest that certain TLR agonists can be combined with cationic liposomes to produce uniquely effective vaccine adjuvants capable of eliciting strong T cell responses against protein and peptide Ags.

Immunosensitization of Tumor Cells to Dendritic Cell-Activated Immune Responses with the Proteasome Inhibitor Bortezomib (PS-341, Velcade)

Proteasome inhibition results in proapoptotic changes in cancer cells, which may make them more sensitive to immune effector cells. We established a murine model to test whether the proteasome inhibitor bortezomib could sensitize established B16 melanoma tumors to dendritic cell (DC)-activated immune effector cells. Day 3-established s.c. B16 tumors had significantly decreased tumor outgrowth when treated with a combination of bortezomib and DC, regardless of whether the DC were loaded or not with a tumor Ag. In vivo Ab-depletion studies demonstrated that the effector cells were NK and CD8+ cells, but not CD4+ cells. NF-kappaB nuclear transcription factor assay and gene-expression profiling of B16 treated with bortezomib was consistent with inhibition of NF-kappaB target genes leading to a proapoptotic phenotype. In vitro lytic assays demonstrated that TNF-alpha, but not perforin, Fas-ligand, or TRAIL, was responsible for bortezomib-sensitized B16 cytotoxicity. In conclusion, the proteasome inhibitor bortezomib can pharmacologically sensitize tumor cells to the lytic effects of DC-activated immune effector cells.