Vaccine-induced Antitumor Immunity Research Articles

RNA-encoded interleukin-2 with extended bioavailability amplifies RNA vaccine-induced antitumor T-cell immunity.

Interleukin-2 (IL-2) is a crucial cytokine in T-cell immunity and a promising combination partner to boost cancer vaccine efficacy. However, therapeutic application of IL-2 is hampered by its short half-life and substantial toxicities. Herein, we report preclinical characterization of a mouse serum albumin-IL-2 fusion protein (Alb-IL2) encoded on nucleoside-modified RNA delivered via a nanoparticle formulation (Alb-IL2 RNA-NP) mediating prolonged cytokine availability. Alb-IL2 RNA-NP was combined with RNA-lipoplex (RNA-LPX) vaccines to evaluate its effect on the expansion of vaccine-induced antigen-specific T-cell immunity. In mice dosed with Alb-IL2 RNA-NP, translated protein was shown to be systemically available up to two days, with an albumin-dependent preferred presence in the tumor and tumor-draining lymph node. Alb-IL2 RNA-NP administration prolonged serum availability of the cytokine compared to murine recombinant IL-2 (rIL-2). In combination with RNA-LPX vaccines, Alb-IL2 RNA-NP administration highly increased expansion of RNA-LPX vaccine-induced CD8+ T cells in the spleen and blood. The combination enhanced and sustained the fraction of IL-2 receptor (IL-2R)α-positive antigen-specific CD8+ T cells and ameliorated the functional capacity of the CD8+ T-cell population. Alb-IL2 RNA-NP strongly improved the antitumor activity and survival of concomitant RNA-LPX vaccination and PD-L1 blockade in a subcutaneous mouse tumor model. The favorable pharmacokinetic properties of Alb-IL2 RNA-NP render it an attractive modality for rationally designed combination immunotherapy. RNA vaccines that induce tumor-specific T-cell immunity for Alb-IL2 RNA-NP to further amplify are particularly attractive combination partners.

Abstract 5296: Enhancing the strength and durability of vaccine-induced anti-tumor immunity with CD27 agonism of CD4+ T cells

Abstract Despite encouraging early phase clinical trials, decades of research have yet to yield effective cancer vaccine immunotherapies. To determine potential markers for success, we performed a follow-up study of a HER2-pulsed dendritic cell cancer vaccine Phase I trial performed in advanced HER2+ cancer patients. Surprisingly, we found that all treated patients were alive at over 18 years post-treatment. These patients still possessed significant HER2-specific memory T cell responses, typified by CD27 expression. The expansion of HER2-specific CD27+ memory T cells was also observed in patients from a separate clinical trial, who received a HER2-expressing viral vaccine. To validate these clinical responses, we vaccinated human CD27 transgenic mice with a HER2 viral vector again observing HER2-specific CD27+ memory T cells, suggesting the significance of this axis in eliciting and maintaining effective vaccine-specific memory T cell responses. We hypothesized that CD27 expression was not just a marker of memory T cells, but represented a critical activation pathway during memory formation. To determine the potential impact of CD27 agonism in vaccination, we combined HER2 vaccination with an anti-CD27 agonist antibody (Varlilumab). The combination treatment elicited a robust increase in HER2-specific CD4+ memory T cell responses that was maintained for at least 300 days post-vaccination. Using therapeutic HER2 vaccination in combination with anti-CD27 of HER2+ tumor bearing mice, we show significantly increased anti-tumor efficacy and further enhancement in combination with PD1 inhibition. The anti-tumor response was triggered by anti-CD27 antibody during the vaccine priming phase, which enhanced HER2-specific CD4+ T cells. Critically, we found that HER2 specific CD4+ T cell responses allowed for anti-tumor responses that persisted after CD8+ T cell depletion, indicating a direct role for antigen-specific CD4+ T cells independent of CD8+ T cells. Collectively, our results support a central role for antigen-specific CD27+ CD4+ T cells in cancer vaccination. These data support the addition of CD27 agonism to improve the therapeutic index of cancer vaccines and potentially enhance responses to immune checkpoint blockade in refractory cancers. Citation Format: Bin-Jin Hwang, Erika Crosby, Timothy Trotter, Li-Chung Tsao, Tao Wang, Congxiao Liu, Xiao Yang, Gangjun Lei, Junping Wei, Xingru Ma, Bushanqing Liu, Amy Hobeika, Michael Morse, Tibor Keler, Li-Zhen He, Herbert Kim Lyerly, Zachary Hartman. Enhancing the strength and durability of vaccine-induced anti-tumor immunity with CD27 agonism of CD4+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5296.

Spatiotemporal Controllable Sono-Nanovaccines Driven by Free-Field Based Whole-Body Ultrasound for Personalized Cancer Therapy.

Therapeutic cancer vaccines fail to produce satisfactory outcomes against solid tumors since vaccine-induced anti-tumor immunity is significantly hampered by immunosuppression. Generating an in situ cancer vaccine targeting immunological cold tumor microenvironment (TME) appears attractive. Here, a type of free-field based whole-body ultrasound (US)-driven nanovaccines are constructed, named G5-CHC-R, by conjugating the sonosensitizer, Chenghai chlorin (CHC) and the immunomodulator, resiquimod (R848) on top of a super small-sized dendrimeric nanoscaffold. Once entering tumors, R848 can be cleaved from a hypoxia-sensitive linker, thus modifying the TME via converting macrophage phenotypes. The animals bearing orthotopic pancreatic cancer with intestinal metastasis and breast cancer with lung metastasis are treated with G5-CHC-R under a free-field based whole-body US system. Benefit from the deep penetration capacity and highly spatiotemporal selectiveness, G5-CHC-R triggered by US represented a superior alternative for noninvasive irradiation of deep-seated tumors and magnification of local immune responses via driving mass release of tumor antigens and "cold-warm-hot" three-state transformation of TME. In addition to irradiating primary tumors, a robust adaptive anti-tumor immunity is potentiated, leading to successful induction of systemic tumor suppression. The sono-nanovaccines with good biocompatibility posed wide applicability to a broad spectrum of tumors, revealing immeasurable potential for translational research in oncology.

Melanoma stem cell vaccine induces effective tumor immunity against melanoma

ABSTRACT Melanoma stem cells (MSCs)-based vaccine strategies have been a potent immunotherapeutic approach for melanoma treatment, which aimed at inducing specific anti-tumor immunity and targeting cancer stem-like cells. As the main cancer-fighting immune cells, CD8+T cells play an important role in vaccine-induced antitumor immunity. Here, we developed a novel MSC vaccine that induces CD8+T cells to target melanoma stem cells specifically. The MSC vaccine was prepared for our study in order to determine the effectiveness of antitumor immunity. The proportion and activity of CD8+T cells were examined in the spleen after immunization, in particular, the expression and cytotoxicity of the immune checkpoint of spleen lymphocytes were detected by flow cytometry and ELISA, moreover, tumor size and the number of lung metastasis nodules were observed and the specific killing effect of the vaccine was evaluated in immunized mice. We found that the MSC vaccine could promote DCs maturation, activate CD8+T cells, suppress the expression of CTLA-4, PD-1, and Tim-3, and increase the expression of IFN-γ and GzmB of CD8+T cells. Melanoma growth and metastasis were inhibited by the vaccine’s specific targeted killing effect. The vaccines based on melanoma stem cells (MSCs) delay the progression of melanoma by inducing anti-tumor immune responses in CD8+T cells.

Abstract SY17-02: Multiantigen vaccines for colon cancer prevention

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the US when men and women are combined. While overall CRC incidence has declined over the years, there has been an increase in CRC diagnosis in young and middle-aged adults. The US Preventive Services Task Force has recently issued draft recommendations for CRC screening to begin at 45 years of age instead of the previously recommended age of 50 for asymptomatic healthy individuals, who are at average risk of CRC, i.e. without a family history of hereditary CRC syndromes such as Lynch syndrome and familial adenomatous polyposis, or with no prior diagnosis of CRC, adenomatous polyps, inflammatory bowel disease. While the evidence supports that CRC-screening and early detection confers a substantial benefit, additional CRC prevention strategies can greatly help reduce the cancer burden in the US and around the world. A number of previous studies have shown that the use of nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of cancers, including CRC. However, their extended use can be associated with serious side effects, including gastrointestinal bleeding. Safer and more efficacious preventive approaches are needed for durable long-term CRC prevention. The majority of newly diagnosed CRC are non-familial (sporadic) and non-hypermutated/microsatellite-stable (MSS). Compared to CRC with high microsatellite instability, MSS CRC and adenomas have significantly lower somatic mutation frequencies, thus lower neoantigen load. Furthermore, MSS CRC have generally lower levels of tumor-infiltrating lymphocytes (TILs), higher density of myeloid derived suppressor cells (MDSC), and downregulation of HLA molecules. These are characteristic of immunologically “cold” tumors, making it highly challenging to immunologically control tumor growth. If cancer vaccines can successfully prime the immune system, in particular before the development of invasive cancer, vaccine-induced adaptive antitumor immunity may render “cold” tumors “hot” and achieve a favorable tumor control. One of the key questions, however, is what tumor antigens can be targeted by vaccines if tumors have lower levels of neoantigens that are known to elicit robust antitumor immune responses. Another approach for cancer vaccine-mediated immune priming is to target tumor associated antigens (TAAs). Unlike tumor-specific neoantigens, TAAs are self-antigens that are overexpressed in tumors, but may also be expressed in normal cells at various levels. Therefore, it is highly critical to identify T cell epitopes that break tolerance and help elicit effective antitumor immunity. Clinically beneficial antitumor immunity is characterized by high-density infiltrates of effector CD8+ cytotoxic T lymphocytes and effector memory T cells and the low abundance of immunosuppressive elements (e.g. regulatory T cells, MDSC, and M2-polarized tumor-associated macrophages) in the tumor microenvironment. Disis and colleagues from University of Washington have previously established a systematic and stepwise approach to identify Th1-selective epitopes for efficacious cancer vaccines while eliminating Th2-epitopes. They have shown cancer vaccines targeting TAAs thus developed could elicit robust Th1 immune response associated with antitumor effects. The NCI PREVENT Cancer Preclinical Drug Development Program (PREVENT) is a peer-reviewed agent development program focused on preclinical development of innovative interventions, including cancer vaccines and drugs, for precision cancer prevention-interception towards clinical applications (https://prevention.cancer.gov/major-programs/prevent-cancer-preclinical). PREVENT and Disis team collaborated on the development of a multi-antigen multi-peptide colon cancer vaccine, named Colovac, for immunoprevention of CRC. It consists of highly immunogenic Th1-promoting epitopes from three TAAs that are selected through CRC genomic database and literature search. TAAs selected are overexpressed in CRC, known to play a role in CRC oncogenesis, and associated with poor disease outcomes. This presentation will cover the background of Colovac development, preclinical antitumor efficacy data on Colovac used alone or in combination with naproxen, characterization of immune correlates of protection, and current ongoing development efforts toward clinical trials. Citation Format: Shizuko Sei. Multiantigen vaccines for colon cancer prevention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY17-02.

Antigen folding improves loading efficiency and antitumor efficacy of PC7A nanoparticle vaccine

Cancer vaccines hold great promise to produce antigen-specific T cell immunity for personalized therapy of cancer. Previously, we reported an ultra-pH-sensitive nanoparticle, PC7A, capable of priming an efficacious immune response without significant systemic toxicity. Despite the early success, the relationship between antigen properties and encapsulation efficiency for downstream immune activation remains poorly understood. In this study, we investigated a small library of melanoma antigens and the effects of several formulation methods on the efficiency of peptide loading inside PC7A nanoparticles. Results show loading efficiency is not highly dependent on the formulation methods, but instead mainly driven by the peptide antigen properties. In particular, we identified a phase transition event, namely the folding of antigenic peptides from random coils to α-helical structure, is important for antigen loading inside PC7A nanoparticles. Mutation of a peptide that abrogates the formation of helical structure resulted in poor loading efficiency. Antitumor efficacy studies in melanoma-bearing mice demonstrate the importance of peptide loading in vaccine-induced antitumor immunity. This study highlights the contribution of phase transition of peptide antigens on vaccine formulation in order to make widespread use of personalized nanoparticle vaccines feasible.

Integrity of plasma DNA is inversely correlated with vaccine-induced antitumor immunity in ovarian cancer patients

Cancer immunotherapy including vaccine therapy is a promising modality for cancer treatment, but few patients show its clinical benefits currently. The identification of biomarkers that can identify patients who will benefit from cancer immunotherapy is thus important. Here, we investigated the potential utility of the circulating cell-free DNA (cfDNA) integrity—a ratio of necrotic cell-derived, longer DNA fragments versus apoptotic cell-derived shorter fragments of Alu gene—as a biomarker of vaccine therapy for patients with ovarian cancer. We analyzed plasma samples from 39 patients with advanced or recurrent ovarian cancer enrolled in clinical trials for personalized peptide vaccinations. We observed that (1) the cfDNA integrity was decreased after the first cycle of vaccination, and (2) the decreased levels of cfDNA integrity were correlated with vaccine-induced immune responses; i.e., decreased cfDNA integrity was observed in 91.7% and 59.3% of the IgG-positive and negative patients, respectively (p = 0.0445). Similarly, decreased cfDNA integrity was observed in 92.9% and 56.0% of CTL response-positive and negative patients, respectively (p = 0.0283). These results suggest that the circulating cfDNA integrity is a possible biomarker for cancer vaccine therapy.

Abstract A054: Inhibition of TGF-beta isoforms 1 and 2 enhances therapeutic tumor vaccine efficacy

Abstract Transforming growth factor-beta (TGF-beta) is a pleiotropic cytokine comprised of three isoforms, TGF-beta1, 2, and 3, that exhibits immunosuppressive functions. In the context of immunosuppression, TGF-beta dampens activation and maturation of both innate and adaptive immune cells, including natural killer cells, macrophages, and dendritic cells, as well as CD4+ and CD8+ T cells. Thus, TGF-beta inhibition, like blockade of other negative regulators of immunity, provides a potential modality of cancer treatment that can be used in combination therapy. Although we have previously demonstrated that blockade of all three isoforms of TGF-beta using a monoclonal antibody increases tumor vaccine efficacy, as measured by the reduction in tumor growth and increase in the number of antigen-specific CTLs, the function of individual isoforms of TGF-beta in the framework of tumor immunity is not well understood. In this study, we examined whether it is necessary to block TGF-beta3, in addition to the two other isoforms of TGF-beta, to improve vaccine-induced tumor immunity against the syngeneic TC1 tumor model, a C57BL/6 lung epithelial cell line transfected with HPV-16 E6 and E7 genes. When the subcutaneous tumors became approximately 5 mm in diameter, the mice were immunized with a peptide-based vaccine that targets the E7 oncogene with or without the simultaneous administration of anti-TGF-beta antibodies with unique specificities for the three isoforms. Mice immunized with the vaccine alone exhibited slightly delayed tumor growth relative to the untreated control. This effect could be enhanced by the combination of anti-TGF-beta antibodies that neutralize all three isoforms of TGF-beta. Interestingly, blockade of only TGF-beta1 & 2 also improved vaccine efficacy to a similar degree as the blockade of all three isoforms, though neutralization of TGF-beta alone could not suppress tumor growth. Therefore, it is not necessary to inhibit TGF-beta3 to generate significant vaccine-induced anti-tumor immunity. Systemic depletion of CD8+ T cells using anti-CD8-depleting antibodies completely abrogated the tumor protection observed in mice immunized with vaccine alone or vaccine in combination with TGF-beta blockade, suggesting that the mechanism of anti-tumor immunity in these conditions relies entirely on CD8+ T cells. In vivo CTL assays revealed that anti-TGF-beta antibodies tended to increase the quality of CTL activity induced by the vaccine. To better understand the effect of treatment on the tumor microenvironment, we used flow cytometric analysis to examine T cells in the tumor and tumor draining lymph nodes. While the vaccine significantly increased the number of tumor antigen-specific CD8+ T cells and IFN-gamma producing T cells in both lymph nodes and tumors, TGF-beta inhibition at two weeks, but not one week, after vaccination further augmented the increase in the number of T cells infiltrating tumors. Vaccine in combination with blockade of TGF-beta1 & 2, regardless of TGF-beta3 inhibition, also led to the greatest increase in the number of T-bet-expressing CD4+ and CD8+ T cells in tumors. This observation implies that blockade of TGF-beta1 & 2 alone may be sufficient to improve therapeutic tumor vaccines by facilitating the infiltration of more Th1/Tc1-type T cells into tumors. Because several studies suggest that TGF-beta3 actually plays a protective role against tumorigenesis in a range of tissues, developing TGF-beta blockade strategies that do not inhibit this isoform could be beneficial in certain settings. Citation Format: Katharine Clark, Faith C. Robertson, Emma De Ravin, Shingo Kato, Anja Bloom, Amer Mirza, Jay A. Berzofsky, Masaki Terabe. Inhibition of TGF-beta isoforms 1 and 2 enhances therapeutic tumor vaccine efficacy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A054.

Abstract PR11: Potentiating immunological memory in mice using aptamer targeted siRNA delivery to inhibit mediators of effector differentiation in CD8+ cytotoxic T lymphocytes

Abstract Recent studies in mice, nonhuman primates, and clinical trials in human patients have emphasized the importance of the persistence of the vaccine-induced immune response, immunological memory, in mediating protective immunity against infectious diseases and cancer. Inhibition of mediators of effector differentiation like mTOR, Blimp-1, CD25, glycolysis, or GSK3β, using genetic means or whenever available pharmacological agents, not only prevented the accumulation of the short-lived effectors but also redirected the activated T cells to differentiate along the memory pathway. For example, pharmacological inhibition of mTOR, or GSK3β with rapamycin, or TSW119, respectively, led to the differentiation of antigen activated CD8+ T cells into long lasting memory cells that exhibited enhanced antiviral and antitumor immunity. Nevertheless, pharmacological agents often exhibit undesirable effects reflecting the broad distribution of their targets. For example, rapamycin inhibition of mTOR promotes the development of Treg, and GSK3β inhibition polarizes dendritic cells to a tolerogenic state, arguably counterproductive in the setting of vaccination. In addition, development of pharmacological agents to modulate the function of intracellular targets that are not accessible to antibodies (undruggable targets) is highly challenging, and their availability especially for clinical use is limited. We are developing a versatile, broadly applicable, and clinically feasible approach to promote the generation of memory T cell responses that addresses the main limitations of pharmacological agents. RNAi is used to downregulate intracellular mediators of effector differentiation, that are targeted to CD8+ T cells by conjugation to an oligonucleotide aptamer ligand. We have shown that a CD8+ T cell targeted 4-1BB aptamer-raptor siRNA conjugate administered to mice by tail vein injection downregulated mTORC1, while preserving mTORC2, activity in at least 60% of adoptively transferred OVA-specific transgenic OT-I cells while sparing host cells. Both rapamycin and aptamer-siRNA conjugate led to the development of an enhanced memory response in mice. Whereas the aptamer-raptor siRNA generated CTL exhibited normal cytotoxic effector functions and enhanced vaccine-induce protective antitumor immunity in both prophylactic and therapeutic tumor models, the rapamycin generated CTL were defective in their cytotoxic effector functions and failed to elicit protective immunity against a tumor challenge. Underscoring the lack of cell specificity of rapamycin action, and providing a potential mechanisms underlying the effector defect of rapamycin generated memory CD8+ T cells, dendritic cells from mice treated with rapamycin, but not with aptamer-raptor siRNA conjugate, exhibited reduced alloMLR activity, consistent with the known suppressive effects of rapamycin induced mTOR inhibition on DC. Given that inhibition of mTORC1 may not be the optimal way of enhancing memory, we have used the aptamer targeting platform to inhibit other mediators of effector differentiation in activated CD8+ T cells. In preliminary studies 4-1BB aptamer-targeted inhibition of Axin-1, to promote wnt signaling, or reducing IL-2 signaling by downregulating CD25 expression, enhanced vaccine-induced protective antitumor immunity in tumor-bearing mice. Using the OT-I adoptive transfer model, in preliminary experiments we have shown that 4-1BB aptamer targeted co-inhibition of both raptor and PD-1 increased the formation of memory OT-I cells; while not superior to that of inhibiting raptor alone, the OT-I cells expressed significantly less PD-1 on cell surface, and were, therefore, resistant to PD-1-mediated immune suppression. Overall, these studies demonstrate the feasibility and efficiency of aptamer targeted delivery of siRNAs to immune cells, and underscore the potential advantages of aptamer-targeted siRNA delivery over nontargeted administration of pharmacological agents. This abstract is also presented as Poster A23. Citation Format: Alexey Berezhnoy, Rajagopalan Anugraha, Thomas Malek, Eli Gilboa. Potentiating immunological memory in mice using aptamer targeted siRNA delivery to inhibit mediators of effector differentiation in CD8+ cytotoxic T lymphocytes. [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 PR11.

Irradiation of necrotic cancer cells, employed for pulsing dendritic cells (DCs), potentiates DC vaccine-induced antitumor immunity against high-grade glioma

ABSTRACTDendritic cell (DC)-based immunotherapy has yielded promising results against high-grade glioma (HGG). However, the efficacy of DC vaccines is abated by HGG-induced immunosuppression and lack of attention toward the immunogenicity of the tumor lysate/cells used for pulsing DCs. A literature analysis of DC vaccination clinical trials in HGG patients delineated the following two most predominantly applied methods for tumor lysate preparation: freeze-thaw (FT)-induced necrosis or FT-necrosis followed by X-ray irradiation. However, from the available clinical evidence, it is unclear which of both methodologies has superior immunogenic potential. Using an orthotopic HGG murine model (GL261-C57BL/6), we observed that prophylactic vaccination with DCs pulsed with irradiated FT-necrotic cells (compared to FT-necrotic cells only) prolonged overall survival by increasing tumor rejection in glioma-challenged mice. This was associated, both in prophylactic and curative vaccination setups, with an increase in brain-infiltrating Th1 cells and cytotoxic T lymphocytes (CTL), paralleled by a reduced accumulation of regulatory T cells, tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC). Further analysis showed that irradiation treatment of FT-necrotic cells considerably increased the levels of carbonylated proteins — a surrogate-marker of oxidation-associated molecular patterns (OAMPs). Through further application of antioxidants and hydrogen peroxide, we found a striking correlation between the amount of lysate-associated protein carbonylation/OAMPs and DC vaccine-mediated tumor rejection capacity thereby suggesting for the first time a role for protein carbonylation/OAMPs in at least partially mediating antitumor immunity. Together, these data strongly advocate the use of protein oxidation-inducing modalities like irradiation for increasing the immunogenicity of tumor lysate/cells used for pulsing DC vaccines.

218. Enhanced Antitumor Immunity Induced By HPV16 and 18 E6/E7 DNA Vaccine in Combination with IL-12 as an Immuno-Adjuvant

Human papillomavirus (HPV) infection is associated with 99% of cervical cancers and approximately 20% of head and neck squamous cell carcinomas. HPV E6- and E7-specific T cell immunity has been considered a critical characteristic of an effective therapeutic vaccine designed to control and eliminate established pre-existing HPV infections and associated cancers. The use of molecular adjuvants is considered an important tool to increase the potency of DNA vaccines further. IL-12 has shown to drive cellular immune responses by aiding the priming and expansion of CD8 T cells. We have previously demonstrated that a highly optimized engineered DNA vaccine targeting HPV 16 and 18 E6/E7 (pGX3001 and pGX3002) elicited potent TH1 and cytotoxic cellular immune responses in a Phase I clinical trial. Here, we sought to determine whether the HPV vaccine-induced antitumor immunity could be further enhanced by the addition of IL12 as an adjuvant.We first demonstrated that IL-12 could enhance HPV16 E6 and E7-specific cellular responses measured by ELISpot in mice (average of 1037 vs 504 SFU/106 splenocytes). Then we performed an in vivo tumor therapy study to determine whether IL-12 could enhance the therapeutic efficacy of pGX3001 in TC-1 tumor bearing C57BL/6 mice. The results induced that mice immunized with pGX3001+IL-12 exhibited smaller tumors compared to those in the no adjuvant group. Thirty days post tumor transplantation, 9 out of 10 mice in the pGX3100+IL-12 group showed complete tumor regression, while there were only 4 out of 10 mice in the pGX3001 group that showed complete tumor regression, indicating that the addition of plasmid-based mouse IL-12 enhanced the vaccine-induced antitumor immunity in a murine model. Furthermore, an additional study in a more relevant, non-human primate model was performed to ensure that the vaccine-induced cellular immune responses could also be augmented by using a rhesus macaque IL-12 plasmid. The data indicated that the addition of IL-12 increased the magnitude of vaccine-induced cellular immune responses about two fold post third immunization (average of 2711 SFU vs 1400 SFU/106 PBMCs as measured by ELISpot). The HPV antigen-specific memory responses in the HPV+IL-12 group were increased more than four-fold compared to the responses in the unadjuvanted group (average of 2570 vs 577 SFU/106 PBMCs). Flow cytometric analysis revealed the induction of both HPV-specific CD4 and CD8 T cells that efficiently increased IFN-γ responses. Both adjuvant and unadjuvanted groups showed an increase in effector and central memory CD8 T cells after immunization, while the IL-12 group exhibited stronger effector memory CD8 T cell responses. Taken together, these data support the further development of this HPV DNA vaccine in combination with IL12 as a cancer immunotherapy candidate.

Irradiation of necrotic tumor cells used to pulse dendritic cells (DCs) potentiates DC vaccine-induced anti-tumor immunity in a mouse model of high-grade glioma

Meeting abstracts The prognosis of high-grade glioma (HGG) is poor despite advancements in neurosurgery, radio-and chemotherapy. DC-based immunotherapy has emerged as a promising and feasible treatment approach due to its high degree of selectivity and its ability to induce an antigen-specific

Abstract IA27: Combinatorial immunotherapy using whole tumor antigen: Evidence from phase I trials

Abstract Novel therapeutic strategies are warranted in recurrent ovarian cancer. We report two independent consecutive studies of combinatorial immunotherapy comprising dendritic cell (DC)-based autologous whole tumor antigen vaccination in combination with antiangiogenesis therapy. Thirty one patients with recurrent progressive stage III and IV ovarian cancer with available tumor lysate from debulking surgery enrolled in two different vaccination studies. First six underwent priming with intravenous bevacizumab and oral metronomic cyclophosphamide followed by vaccination with an autologous DC preparation pulsed with freeze-thaw autologous tumor lysate while the other 25 underwent vaccination with an enhanced vaccine of autologous DCs loaded with HOCl-oxidized autologous tumor lysate administered intranodally in combination with antiangiogenesis therapy. Both studies were followed by lymphodepletion and transfer of autologous vaccine-primed, ex vivo CD3/CD28-costimulated peripheral blood T-cells, in combination with antiangiogenesis therapy and vaccination. Feasibility, safety, biological and clinical efficacy were evaluated. Eleven patients have completed vaccination and T cell transfer to date, while twenty-three additional patients completed vaccination only. Vaccination was well tolerated and elicited tumor-specific T cell responses against various ovarian tumor antigens. Preliminary results demonstrate that patients' DCs loaded with HOCl-oxidized lysate elicited strong tumor-specific IFN-gamma; secretions and produced high levels of Th1-priming cytokines and chemokines, including IL-12. A clinical benefit of 65% correlated with the immune response with some patients experiencing prolonged progression free survival. Following lymphodepletion, adoptive transfer of vaccine-primed T-cells was well tolerated and resulted in durable reduction of T-regulatory cells and restoration of vaccine-induced antitumor immunity in patients who experienced clinical benefit. One patient exhibited a complete response at end of study and stable disease was observed in 7 out of the 11 patients who completed vaccination and T cell transfer. Data on additional patients will be presented at the meeting. Our results suggest the use of combinatorial cellular immunotherapy comprising DC vaccination with whole tumor antigen and adoptive lymphocyte transfer using tumor antigen-specific T cells for the treatment of patients with recurrent ovarian cancer is promising yet warrants further investigation. Citation Format: Lana E. Kandalaft, Janos Tanyi, Cheryl Chiang, George Coukos. Combinatorial immunotherapy using whole tumor antigen: Evidence from phase I trials. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA27.

Abstract LB-335: Autologous whole-tumor antigen vaccination in combination with adoptive T cell therapy for patients with recurrent ovarian cancer.

Abstract Novel therapeutic strategies are warranted in recurrent ovarian cancer. We report two independent consecutive studies of combinatorial immunotherapy comprising dendritic cell (DC)-based autologous whole tumor antigen vaccination in combination with antiangiogenesis therapy. 31 patients with recurrent progressive stage III and IV ovarian cancer with available tumor lysate from secondary debulking surgery enrolled in two different studies. First 6 underwent priming with intravenous bevacizumab and oral metronomic cyclophosphamide followed by vaccination with an autologous DC preparation pulsed with freeze-thaw autologous tumor lysate while the other 25 underwent vaccination with an enhanced vaccine where autologous DCs were loaded with HOCl-oxidized autologous tumor lysate administered intranodally every 2 weeks in combination with intravenous bevacizumab. Both studies were followed by lymphodepletion and transfer of autologous vaccine-primed, ex vivo CD3/CD28-costimulated peripheral blood T-cells, in combination with antiangiogenesis therapy and vaccination. Feasibility, safety, and biological and clinical efficacy were evaluated. Eleven subjects have completed vaccination and T cell transfer to date, while twenty-three additional subjects completed vaccination only. Vaccination was well tolerated and elicited tumor-specific T cell responses against various ovarian tumor antigen in both studies and clinical benefit of 65% correlated with the immune response with some experiencing prolonged progression free survival. Following lymphodepletion, adoptive transfer of vaccine-primed T-cells was well tolerated and resulted in durable reduction of T-regulatory cells and restoration of vaccine-induced antitumor immunity in patients who experienced clinical benefit. One patient exhibited no evidence of disease at end of study. Stable disease was observed in 8 subjects to date. Data on additional patients will be presented at the meeting. Our results suggest the use of combinatorial cellular immunotherapy comprising DC vaccination with whole tumor antigen and adoptive lymphocyte transfer using tumor antigen-specific T cells for the treatment of patients with recurrent ovarian cancer is promising yet warrants further investigation. Citation Format: Lana E. Kandalaft, Janos Tanyi, Cheryl Chiang, Daniel Powell, George coukos. Autologous whole-tumor antigen vaccination in combination with adoptive T cell therapy for patients with recurrent ovarian cancer. [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 LB-335. doi:10.1158/1538-7445.AM2013-LB-335

Abstract A25: Targeting costimulation to the tumor stroma with bispecific oligonucleotide aptamers.

Abstract The paucity of costimulation at the tumor site compromises the ability of tumor-specific T cells to eliminate the tumor. The recent FDA approval of Ipilumimab, an antibody that blocks the inhibitory action of CTLA-4, has underscored the therapeutic potential of using monoclonal antibodies as ligands to stimulate protective immunity in human patients. Nonetheless, systemic administration of immunostimulatory ligands carries the risk of activating low avidity autoreactive T cells that could result in immune related pathologies. Indeed, treatment with Ipilimumab is associated with significant dose-limiting autoimmune toxicities. Similarly, treatment with an agonistic antibody that binds to 4-1BB, an immune stimulatory receptor expressed on activated CD8+ T cells, while demonstrating antitumor efficacy in cancer patients was associated with severe hepatic toxicity that led to the suspension of the clinical trial. Arguably, targeting costimulatory ligands to the disseminated tumor lesions of the patient would reduce drug associated toxicities. We have previously shown that systemic delivery of a bispecific aptamer composed of an agonistic 4-1BB binding aptamer, the therapeutic aptamer, conjugated to a an aptamer that bound to a product expressed on the surface of a tumor cell, the targeting aptamer, led to inhibition of tumor growth, was more effective than, and synergized with, vaccination, and exhibited a superior therapeutic index compared to nontargeted costimulation with 4-1BB antibodies or 4-1BB aptamers. The chemically synthesized oligonucleotide aptamers offer significant advantages over antibodies in terms of synthesis, cost, and conjugation chemistry to generate bispecific dimeric ligands. Nonetheless, the main current limitation of tumor targeted costimulation stems from the fact that the therapeutic cargo, the costimulatory (aptamer) ligand, has to be displayed on the surface of the targeted tumor cells. Consequently one is limited to target receptors that do not internalize upon ligand binding. While such receptor-ligand interactions exist, case in point is Rituximab, an anti-CD19 antibody that kills their targets via antibody-dependent cell cytotoxicity, most receptor-ligand complexes are internalized, and consequently the targeting choices are severely limited. Here we describe an approach that addresses this limitation and broadens the scope of tumor-targeted costimulation whereby the therapeutic aptamer ligand is targeted not to a product expressed on the surface of the tumor cells, but rather to a product that is secreted into the tumor stroma. Thus, the tumor infiltrating T cells will encounter the ligand and receive immune stimulation in advance to engaging their cognate MHC-restricted antigen on the tumor cells. In support of this approach we have shown that agonistic 4-1BB aptamers conjugated to aptamers which bind to either of two secreted products, VEGF or osteopontin, potentiated vaccine-induced protective antitumor immunity in mice that was superior to that of the non-targeted 4-1BB aptamers. Targeting secreted products will also significantly broaden the scope of tumor-targeted costimulation because secreted products like VEGF or osteopontin are broadly expressed in many tumors of disparate origin, unlike cell surface expressed products like PSMA or Her2 that exhibit a much more restricted expression pattern. Citation Format: Brett Schrand, Randall Brenneman, Alexey Berezhnoy, Eli Gilboa. Targeting costimulation to the tumor stroma with bispecific oligonucleotide aptamers. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A25.

Abstract PR15: Autologous whole-tumor antigen combinatorial immunotherapy for recurrent ovarian cancer.

Abstract Novel therapeutic strategies are warranted in recurrent ovarian cancer. We report two independent consecutive studies of combinatorial immunotherapy comprising dendritic cell (DC)-based autologous whole tumor antigen vaccination in combination with antiangiogenesis therapy. Twenty-one patients with recurrent progressive stage III and IV ovarian cancer with available tumor lysate from secondary debulking surgery enrolled in two different studies. First 6 underwent priming with intravenous bevacizumab and oral metronomic cyclophosphamide (bev/cy x 2 doses), followed by vaccination with an autologous DC preparation pulsed with freeze-thaw autologous tumor lysate while the other fifteen underwent vaccination with an enhanced more immunogenic vaccine where autologous DCs were loaded with HOCl-oxidized autologous tumor lysate administered intranodally every 2 weeks in combination with intravenous bev. Both studies were followed by lymphodepletion using high-dose outpatient cyclophosphamide and fludarabine and transfer of 5-30 x10e9 autologous vaccine-primed, ex vivo CD3/CD28-costimulated peripheral blood T cells, in combination with antiangiogenesis therapy and vaccination. Feasibility, safety, and biological and clinical efficacy were evaluated. Eight subjects have completed vaccination and T cell transfer to date, while thirteen additional subjects completed vaccination only. Vaccination was well tolerated and produced limited grade 1 toxicities and elicited tumor-specific T cell responses against various ovarian tumor antigen in both studies and clinical responses correlated with the immune response with ~66% of patients achieving clinical benefit with some experiencing prolonged progression free survival and remission inversion. Following lymphodepletion, adoptive transfer of vaccine-primed, CD3/CD28-costimulated autologous T cells produced grade 1 toxicity and resulted in durable reduction of CD4+FoxP3+ T regulatory cells, increased total lymphocyte counts and restoration of vaccine-induced antitumor immunity in patients who experienced clinical benefit at end of study. One patient exhibited no evidence of disease at end of study. Stable disease was observed in three subjects to date. Interestingly in a subject where adoptive T cell transfer was not followed by restoration of vaccine-induced antitumor immunity, disease progression was observed. Data on additional patients will be presented at the meeting. Our results suggest the use of combinatorial cellular immunotherapy comprising DC vaccination with whole tumor antigen and adoptive lymphocyte transfer using tumor antigen-specific T cells for the treatment of patients with recurrent ovarian cancer is promising yet warrants further investigation. This abstract is also presented as Poster A30. Citation Format: Lana E. Kandalaft, Janos Tanyi, Cheryl Lai-Lai Chiang, Drew Torigian, Daniel J. Powell, Jr., George Coukos. Autologous whole-tumor antigen combinatorial immunotherapy for recurrent ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR15.

Nondominant CD8 T Cells Are Active Players in the Vaccine-Induced Antitumor Immune Response

We previously reported that CD8(+) T cells are directed predominantly toward the immunodominant Her-2/neu (neu) epitope RNEU(420-429) in nontolerized FVB/N but not tolerized HER-2/neu (neu-N) mice. In this study, we screened overlapping peptides of the entire neu protein and identified six new epitopes recognized by vaccine-induced neu-N-derived T cells. Evaluation of individual nondominant responses by tetramer staining and IFN-γ secretion demonstrate that this repertoire is peripherally tolerized. To address the role that the complete CD8(+) T cell repertoire plays in vaccine-induced antitumor immunity, we created a whole-cell vaccine-expressing neu cDNA that has been mutated at the RNEU(420-429) anchor residue, thereby abrogating activation of immunodominant epitope responses. Studies comparing the mutated and nonmutated vaccines indicate that nondominant CD8(+) T cells can induce antitumor immunity when combined with regulatory T cell-depleting agents in both neu-N and FVB/N mice. Collectively, these studies demonstrate that the neu-directed T cell repertoire is not intrinsically incapable of eradicating tumors. Rather, they are suppressed by mechanisms of peripheral tolerance. Thus, these studies provide new insights into the function of the complete T cell repertoire directed toward a clinically relevant tumor Ag in tumor-bearing hosts.

TNFRSF25 agonists as potent cancer vaccine adjuvants (131.25)

Abstract Signaling through TNFRSF25 by its natural ligand, TNFSF15, or agonistic antibodies enhances cytokine production and effector function by antigen-activated T cells. Paradoxically, we recently discovered that TNFRSF25 signaling induces rapid and specific proliferation of CD4+FoxP3+ regulatory T cells (Treg) in vivo in the absence of exogenous antigen. When TNFRSF25 agonistic antibody, clone 4C12, was used in combination with heat shock protein (gp96-Ig) based vaccination, 4C12 treatment led to synergistic antigen-specific CTL expansion when given together with the vaccine, which occurred simultaneously with Treg expansion in vivo. The frequency of CTL and Treg reached 65% of total CD8+ cells and 45% of total CD4+ cells, respectively, within the same tissue following combined administration of 4C12 and gp96-Ig. TNFR25 driven synergism with gp96-Ig vaccination was dependent upon mTOR signaling. In a therapeutic setting, combined administration of 4C12 and gp96-Ig enhanced rejection of established EG7, LLC and 3-MCA induced sarcoma tumors. These data indicate that TNFRSF25 agonists enhance vaccine-induced anti-tumor immunity which correlates with increased magnitude and duration of tumor-specific CTL expansion, systemically. These findings illustrate that TNFRSF25 signaling may enhance either inflammatory or regulatory immunity depending on the temporal availability of its ligand and the inflammatory milieu.

Tumor-Induced Suppression of CTL Expansion and Subjugation by gp96-Ig Vaccination

Established tumors suppress antitumor immune responses and induce tolerance by incompletely characterized mechanisms, and this phenomenon is an important barrier to tumor immunotherapy. Single vaccination with tumor cells expressing gp96-Ig stimulates robust expansion of tumor-specific CTLs in tumor-naïve mice and this expansion is inhibited by established tumors. Interestingly, frequent vaccinations restore antitumor immune responses in the presence of established tumors. Syngeneic EG7 tumor-bearing mice have heterogeneous responses to frequent vaccination with EG7-gp96-Ig, with 32% complete responders and 68% partial responders. Comparison of responders to nonresponders revealed an inverse correlation between tumor-specific CTL expansion in the peripheral blood and tumor size. To identify immune cells and molecules associated with effective antitumor immune responses, reverse transcription-PCR arrays were performed using cells isolated from the vaccination site. ELISAs, cellular phenotyping, and tumor immunohistochemistry were also performed comparing vaccine responders to nonresponders. These data show that up-regulation of T-bet, RORgammat, IFNgamma, CCL8, CXCL9, and CXCL10 at the vaccination site are associated with vaccine-induced antitumor immunity. These data correlate with increased CTL expansion in the peripheral blood of responders, increased infiltration of responder tumors by CD8+ cells and interleukin-17+ cells, and decreased infiltration of responder tumors by CD11b+Gr-1+ cells and FoxP3+ cells. Furthermore, serum ELISAs revealed a significant elevation of transforming growth factor-beta in nonresponders as compared with responders. Interestingly, CD8+ T cells isolated from responders and nonresponders have equivalent cytotoxic activity in vitro. Taken together, our data suggest that established tumors may escape immunosurveillance by preventing clonal expansion of tumor-specific CTL without inducing anergy.

Combination Chemotherapy and ALVAC-CEA/B7.1 Vaccine in Patients with Metastatic Colorectal Cancer

The combination of vaccines and chemotherapy holds promise for cancer therapy, but the effect of cytotoxic chemotherapy on vaccine-induced antitumor immunity is unknown. This study was conducted to assess the effects of systemic chemotherapy on ALVAC-CEA/B7.1-induced T-cell immunity in patients with metastatic colorectal cancer. Patients with metastatic colorectal cancer were treated with fluorouracil, leucovorin, and irinotecan and were also given ALVAC-CEA/B7.1 vaccine with or without tetanus toxoid adjuvant. Eligible patients were randomized to ALVAC followed by chemotherapy and booster vaccination (group 1), ALVAC and tetanus toxoid followed by chemotherapy (group 2), or chemotherapy alone followed by ALVAC in patients without disease progression (group 3). Humoral immune responses were measured by standard ELISA assay, and carcinoembryonic antigen (CEA)-specific T-cell responses were measured by IFN-gamma enzyme-linked immunospot assay. One hundred eighteen patients were randomized to receive either ALVAC before and concomitantly with chemotherapy (n = 39), ALVAC with tetanus adjuvant before and concomitantly with chemotherapy (n = 40), or chemotherapy followed by ALVAC (n = 39). Serious adverse events were largely gastrointestinal (n = 30) and hematologic (n = 24). Overall, 42 patients (40.4%) showed objective clinical responses. All patients developed antibody responses against ALVAC, but increased anti-CEA antibody titers were detected in only three patients. Increases in CEA-specific T cells were detected in 50%, 37%, and 30% of patients in groups 1, 2, and 3, respectively. There were no differences in clinical or immune responses between the treatment groups. The combination of ALVAC-CEA/B7.1 vaccine and systemic chemotherapy has an acceptable safety profile in patients with metastatic colorectal cancer. Systemic chemotherapy did not affect the generation of CEA-specific T-cell responses following vaccination.