Immunity In Tumor-bearing Mice Research Articles

Bioinspired ginsenoside Rg3 PLGA nanoparticles coated with tumor-derived microvesicles to improve chemotherapy efficacy and alleviate toxicity.

Breast cancer, a pervasive malignancy affecting women, demands a diverse treatment approach including chemotherapy, radiotherapy, and surgical interventions. However, the effectiveness of doxorubicin (DOX), a cornerstone in breast cancer therapy, is limited when used as a monotherapy, and concerns about cardiotoxicity persist. Ginsenoside Rg3, a classic compound of traditional Chinese medicine found in Panax ginseng C. A. Mey., possesses diverse pharmacological properties, including cardiovascular protection, immune modulation, and anticancer effects. Ginsenoside Rg3 is considered a promising candidate for enhancing cancer treatment when combined with chemotherapy agents. Nevertheless, the intrinsic challenges of Rg3, such as its poor water solubility and low oral bioavailability, necessitate innovative solutions. Herein, we developed Rg3-PLGA@TMVs by encapsulating Rg3 within PLGA nanoparticles (Rg3-PLGA) and coating them with membranes derived from tumor cell-derived microvesicles (TMVs). Rg3-PLGA@TMVs displayed an array of favorable advantages, including controlled release, prolonged storage stability, high drug loading efficiency and a remarkable ability to activate dendritic cells in vitro. This activation is evident through the augmentation of CD86+CD80+ dendritic cells, along with a reduction in phagocytic activity and acid phosphatase levels. When combined with DOX, the synergistic effect of Rg3-PLGA@TMVs significantly inhibits 4T1 tumor growth and fosters the development of antitumor immunity in tumor-bearing mice. Most notably, this delivery system effectively mitigates the toxic side effects of DOX, particularly those affecting the heart. Overall, Rg3-PLGA@TMVs provide a novel strategy to enhance the efficacy of DOX while simultaneously mitigating its associated toxicities and demonstrate promising potential for the combined chemo-immunotherapy of breast cancer.

A “2 + 2” strategy for tumor immune microenvironment remodeling based on complementary immune checkpoint blockade

Despite a clinical success of cancer immunotherapy, its efficacy is often limited due to immune resistance and insufficient immune response induced by the tumor immune microenvironment (TIME). Thus, further appropriate interventions are needed to reshape the TIME and to maintain an effective and durable immune response. Herein, we report that a strategy involving complementary immune checkpoint blockade of ADAR1 and PD-1 can enhance the immune response and decrease the rate of immune evasion by regulating the TIME. We first designed a charge-reversible nanosystem Au-PEI/shADAR1/PEI-citraconic anhydride (AAPC), to effectively deliver shADAR1 to inhibit expression of ADAR1, a potential immune checkpoint in tumor cells. Then, we demonstrated a simultaneous decrease in immune escape and activation of antitumor immunity in tumor-bearing mice upon combined treatment with AAPC and an anti-PD-1 antibody. The immune checkpoint blockade acts on T cells and we show that this combination therapy was more efficacious than individual treatments. Additionally, mechanistic and transcriptomic analysis confirmed that complementary immune checkpoint blockade regulated the abundance of immune cells. Finally, combined with X-ray irradiation, a “2 + 2” strategy involving combined dual normalization (“2”, dsRNA sensitization and anti-PD therapy) and dual enhancement (“2”, antigen presentation by radiotherapy and immunogenetic cell death induced by MDA5 activation) immunotherapy efficiently reshaped the TIME by regulating a variety of immune cells.

5-Fluorouracil Suppresses Colon Tumor through Activating the p53-Fas Pathway to Sensitize Myeloid-Derived Suppressor Cells to FasL+ Cytotoxic T Lymphocyte Cytotoxicity.

Myelosuppression is a major adverse effect of 5-fluorouracil (5-FU) chemotherapy. However, recent findings indicate that 5-FU selectively suppresses myeloid-derived suppressor cells (MDSCs), to enhance antitumor immunity in tumor-bearing mice. 5-FU-mediated myelosuppression may thus have a beneficial effect for cancer patients. The molecular mechanism underlying 5-FU's suppression of MDSCs is currently unknown. We aimed at testing the hypothesis that 5-FU suppresses MDSCs through enhancing MDSC sensitivity to Fas-mediated apoptosis. We observed that, although FasL is highly expressed in T cells, Fas is weakly expressed in myeloid cells in human colon carcinoma, indicating that downregulation of Fas is a mechanism underlying myeloid cell survival and accumulation in human colon cancer. 5-FU treatment upregulated expression of both p53 and Fas, and knocking down p53 diminished 5-FU-induced Fas expression in MDSC-like cells, in vitro. 5-FU treatment also increased MDSC-like cell sensitivity to FasL-induced apoptosis in vitro. Furthermore, we determined that 5-FU therapy increased expression of Fas on MDSCs, suppressed MDSC accumulation, and increased CTL tumor infiltration in colon tumor-bearing mice. In human colorectal cancer patients, 5-FU chemotherapy decreased MDSC accumulation and increased CTL level. Our findings determine that 5-FU chemotherapy activates the p53-Fas pathway, to suppress MDSC accumulation, to increase CTL tumor infiltration.

Well-Defined Mannosylated Polymer for Peptide Vaccine Delivery with Enhanced Antitumor Immunity.

Peptide-based cancer vaccines offer production and safety advantages but have had limited clinical success due to their intrinsic instability, rapid clearance, and low cellular uptake. Nanoparticle-based delivery vehicles can improve the in vivo stability and cellular uptake of peptide antigens. Here, a well-defined, self-assembling mannosylated polymer is developed for anticancer peptide antigen delivery. The amphiphilic polymer is prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization, and the peptide antigens are conjugated to the pH-sensitive hydrophobic block through the reversible disulfide linkage for selective release after cell entry. The polymer-peptide conjugates self-assemble into sub-100nm micelles at physiological pH and dissociate at endosomal pH. The mannosylated micellar corona increases the accumulation of vaccine cargoes in the draining inguinal lymph nodes and facilitates nanoparticle uptake by professional antigen presenting cells. In vivo studies demonstrate that the mannosylated micelle formulation improves dendritic cell activation and enhances antigen-specific T cell responses, resulting in higher antitumor immunity in tumor-bearing mice compared to free peptide antigen. The mannosylated polymer is therefore a simple and promising platform for the delivery of peptide cancer vaccines.

Immunological functional study by use of the chemotherapy drug plus immunologic hapten like penicillin on the fibrosis of local tumor inflammatory reaction and the immunity of tumor-bearing mice.

e15196 Background: Observe the antitumor effect of intratumoral injection of chemotherapy drugs or combined with immune adjuvant on Balb/c and c57 tumor bearing mice and the local inflammatory response, fibrosis and immune effect of tumor. Methods: H22 ascites tumor was inoculated in the armpit of BABL / c mice left forelimb to establish subcutaneous solid tumor model of liver cancer, B16 melanoma tumor filtrate was inoculated in the armpit of C57 mice left forelimb to establish subcutaneous solid tumor model of melanoma. The mice were randomly divided into DNP control group, penicillin control group, I group, II group, III group, IV group, V group, VI group, DOX control group, PYM control group, BLM control group and control group when the longest tumor diameter was about 5-8mm. On the first day of the experiment, the mice in each group were injected with corresponding of different group drugs. Three mice in each group were killed on day 1, day 4 and day 7 respectively and then the tumors were taken The C57 mice were used for immunohistochemistry BABL/c mice to detect the content changes of various fibroproliferative, inflammatory and immune related factors. Results: I group: The expression of COL1A1, CD4, IL12A, TGFB1, elastin, NFkB, COX 2, CD11c, CD8 and TNFa in group I was higher than that in other factors at day 1. After 4 and 7 days of administration, all factors decreased significantly; The results of C57 Mouse immunohistochemistry showed that the expression of CD4, cd11bc, Foxp3 in tumor tissue of group V was significantly increased on the first day after administration, and the expression of CD4, CD8, CD86, CD11bc in tumor tissue of group V was significantly increased on the seventh day. Conclusions: The results of immunohistochemistry and PCR showed that the content of fibroproliferative, inflammatory and immune factors in tumor tissue changed significantly after administration og intratumoral injection of combination drugs and penicillin with H2O2. All the above results has showed that chemotherapy drug and hapten was released in the tumor through H2O2 and induced the inflammatory reaction inside tumor (including various fiber proliferation and tumor growth inhibition) as well as the systemic immune response, this also is called abscopal effect.

Viral vaccines improve PD-1 therapy against cancer

Abstract Programmed cell death-1 (PD-1) therapy is effective against various types of cancer. However, only 30% of cancers respond to PD-1 therapy, highlighting the need to further improve its efficacy. In this study, we interrogated whether one could exploit the immunostimulatory properties of viral vaccines to enhance PD-1 therapy in a B16 melanoma model that is poorly responsive to PD-1 therapy. Immunization of tumor-bearing mice with clinically approved viral vaccines, such as the smallpox virus vaccine and the yellow fever virus vaccine (YFV-17D), resulted in an unprecedented improvement of PD-1 therapy. This synergistic effect on PD-1 therapy was more potent upon a secondary immunization, suggesting a positive effect of recruiting pre-existent immune memory to the tumor microenvironment. Interestingly, co-administration of viral vaccines together with PD-1 therapy did not increase tumor-specific CD8 T cell responses relative to PD-1 therapy alone. Mechanistically, the adjuvant effect of viral vaccines on PD-1 therapy seems to be dependent on innate immune responses. These findings may be important for improving the efficacy of checkpoint-based immunotherapies by exploiting the immunogenicity of viral vaccines. Altogether, our data suggest that viral vaccines could be repurposed as immunotherapy adjuvants, and that the beneficial effects of viral vaccines on PD-1 therapy can be further improved by repetitive boosting of bystander immunity. R21 AI132848-01A1 (NIAID); IRG-15-173-21 (ACS), and the Lurie Cancer Center.

Re-engineering of the Immunosuppressive Tumor Microenvironment by Antiangiogenic Therapy

Active immunotherapy of cancer is a new promising anticancer strategy. However, this approach is still limited by numerous challenges; an important one is the abnormal and immunosuppressive tumor microenvironment. It is proposed that combination of immunotherapy with an angiogenesis inhibitor can re-engineer the immunosuppressive tumor microenvironment, resulting in more efficient delivery of drugs to the targeted cancer cells. Recently, we demonstrated that an engineered peptide derived from endostatin, called ES-SS, could strongly inhibit angiogenesis and growth of breast adenocarcinoma in mice. In the present study, we evaluated the impact of ES-SS on the induction of immunity in both healthy and mammary carcinoma-bearing mice. In order to establish tumor, 4T-1 cell lines inoculated into the right flank of mice. Spleens of treated groups receiving 1 and 5 mg/kg/day ES-SS were isolated 14 and 25 days after treatment as well as control groups and the amount of different cytokines secreted from splenocytes were measured using ELISA method. In addition, tumor sizes and animal weights were measured. Results demonstrated that ES-SS is completely safe and no differences were observed in control and treated groups in any cytokine levels in healthy mice, however, the peptide could activate cellular and humoral immunity in tumor-bearing mice through increasing of TNF-α, IL-10, IFN-γ, IL-4, and IL-17 production compared to the control (p < 0.05). Therefore this anti-angiogenic peptide could be proposed as a promising adjuvant in immunotherapy of cancer and could enhance the efficiency of immuno/chemotherapy given conventionally or perhaps in a dose-dependent manner.

Integration of Oncogenes via Sleeping Beauty as a Mouse Model of HPV16+ Oral Tumors and Immunologic Control.

Human papillomavirus type 16 (HPV16) is the etiologic factor for cervical cancer and a subset of oropharyngeal cancers. Although several prophylactic HPV vaccines are available, no effective therapeutic strategies to control active HPV diseases exist. Tumor implantation models are traditionally used to study HPV-associated buccal tumors. However, they fail to address precancerous phases of disease progression and display tumor microenvironments distinct from those observed in patients. Previously, K14-E6/E7 transgenic mouse models have been used to generate spontaneous tumors. However, the rate of tumor formation is inconsistent, and the host often develops immune tolerance to the viral oncoproteins. We developed a preclinical, spontaneous, HPV16+ buccal tumor model using submucosal injection of oncogenic plasmids expressing HPV16-E6/E7, NRas G12V , luciferase, and sleeping beauty (SB) transposase, followed by electroporation in the buccal mucosa. We evaluated responses to immunization with a pNGVL4a-CRT/E7(detox) therapeutic HPV DNA vaccine and tumor cell migration to distant locations. Mice transfected with plasmids encoding HPV16-E6/E7, NRas G12V , luciferase, and SB transposase developed tumors within 3 weeks. We also found transient anti-CD3 administration is required to generate tumors in immunocompetent mice. Bioluminescence signals from luciferase correlated strongly with tumor growth, and tumors expressed HPV16-associated markers. We showed that pNGVL4a-CRT/E7(detox) administration resulted in antitumor immunity in tumor-bearing mice. Lastly, we demonstrated that the generated tumor could migrate to tumor-draining lymph nodes. Our model provides an efficient method to induce spontaneous HPV+ tumor formation, which can be used to identify effective therapeutic interventions, analyze tumor migration, and conduct tumor biology research. Cancer Immunol Res; 6(3); 305-19. ©2018 AACR.

A Genetically Modified attenuated Listeria Vaccine Expressing HPV16 E7 Kill Tumor Cells in Direct and Antigen-Specific Manner.

Attenuated Listeria monocytogenes (L. monocytogenes, LM) induces specific CD8+ and CD4+ T cell responses, and has been identified as a promising cancer vaccine vector. Cervical cancer is the third most common cancer in women worldwide, with human papillomavirus (HPV), particularly type 16, being the main etiological factor. The therapeutic HPV vaccines are urgently needed. The E7 protein of HPV is necessary for maintaining malignancy in tumor cells. Here, a genetically modified attenuated LM expressing HPV16 E7 protein was constructed. Intraperitoneal vaccination of LM4Δhly::E7 significantly reduced tumor size and even resulted in complete regression of established tumors in a murine model of cervical cancer. We provided evidence that recombinant LM strains could enter the tumor tissue and induce non-specific tumor cell death, probably via activation of reactive oxygen species and increased intracellular Ca2+ levels. LM4Δhly::E7 effectively triggered a strong antigen-specific cellular immunity in tumor-bearing mice, and elicited significant infiltration of T cells in the intratumoral milieu. In summary, these data showed LM4Δhly::E7 to be effective in a cervical cancer model and LM4Δhly::E7 induced an antitumor effect by antigen-specific cellular immune responses and direct killing of tumor cells, indicating a potential application against cervical cancer.

Chemoimmunotherapeutic effect of combined treatment with ex vivo generated antigen-presenting immune cells and conventional antitumor agents in a mouse neuroblastoma model

PurposeCombining antitumor immunotherapy with conventional intensive multimodal therapy may be considered for advanced neuroblastoma. We investigated combination therapy with ex vivo generated immunostimulatory cells and intraperitoneal doxorubicin. MethodsImmunogenic death of neuro-2a neuroblastoma cells was induced by doxorubicin or cisplatin (negative control). Mouse bone marrow cells were cultured with granulocyte-macrophage colony-stimulating factor, followed by addition of doxorubicin-killed neuro-2a cells with or without interleukin-4 and/or CpG-oligodeoxynucleotide to induce immunostimulatory cells. CD8α+ lymphocytes were cocultured with killed neuro-2a cells and immunostimulatory cells, and interferon-γ was measured in the supernatant. Furthermore, female A/J mice were injected with viable neuro-2a cells, followed by immunostimulatory cells and doxorubicin. Then intraabdominal tumor nodules were evaluated. ResultsBone marrow-derived immunostimulatory cells only promoted interferon-γ production by CD8α+ lymphocytes after first being stimulated by doxorubicin-killed neuro-2a cells and interleukin-4, followed by CpG-oligodeoxynucleotide. These cells had a surface antigen expression profile compatible with activated dendritic cells and suppressed tumors in mice intravenously injected with neuro-2a cells. Despite a similar surface antigen profile, the in vivo antitumor effect was stronger after injection of immunostimulatory cells induced by doxorubicin-killed neuro-2a cells compared with cells induced by cisplatin-killed neuro-2a cells. Moreover, interferon-γ production was greater when CD8α+ lymphocytes were cocultured with doxorubicin-killed neuro-2a cells and immunostimulatory cells rather than with cisplatin-killed cells. ConclusionCells with antitumor activity can be induced from bone marrow cells. Combining such cells with doxorubicin may activate antitumor immunity in tumor-bearing mice. Interactions between induced immunostimulatory cells and conventional chemotherapy might be important for antitumor immunity.

Adenovirus-mediated interleukin 21 gene transfer enhances antitumor immunity and reduces tumorigenicity of Hepa1-6 in mice.

In the present study, adenovirus-mediated interleukin 21 (Ad5-IL-21-EGFP) gene expression was induced in Hepa1–6 cells to investigate whether IL-21 was capable of enhancing antitumor immunity and reducing tumorigenicity of Hepa1–6 in a mouse model. Mice were inoculated intradermally into the right flank with Hepa1–6 cells or Hepa1–6 cells infected with Ad5 or Ad5-IL-21. Four weeks later, the mice were sacrificed humanely, and the tumor volume, tumor weight and mouse spleen index were measured. The levels of IL-21, IL-4 and interferon (IFN)-γ levels in mouse serum and tumor tissues were detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Cell counting kit-8 (CCK-8) assay was used to detect the killing ability of spleen T cells and natural killer (NK) cells, and the proliferation ability of T cells. The expression of IL-21 was confirmed by reverse transcription-polymerase chain reaction, western blot analysis and ELISA assay in Ad5-IL-21-EGFP-infected Hepa1–6 cells. The overexpression of IL-21 significantly reduced the tumorigenicity of Hepa1–6 cells. The tumor volumes and tumor weights in Ad5-IL-21-Hepa1–6 mice were much smaller than those in the Ad5-Hepa1–6 group and Hepa1–6 wild-type group. The immunohistochemistry and ELISA assay demonstrated that IL-21 and IFN-γ levels were much higher while the IL-4 level was much lower in the Ad5-IL-21-Hepa1–6 group than in the other two groups. CCK-8 assay revealed that the killing ability of NK cells and T cells, and the proliferation ability of T cells in Ad5-IL-21-Hepa1–6 mice were higher than in the other two groups; the spleen index of Ad5-IL-21-Hepa1–6 mice was also higher than in the other groups. The data had a significant difference (P<0.01). In conclusion, IL-21 reduces tumorigenicity of Hepa1–6 by a mechanism involving enhanced activation of cell-mediated immunity in tumor-bearing mice.

Recombinant Newcastle Disease Virus Encoding IL-12 and/or IL-2 as Potential Candidate for Hepatoma Carcinoma Therapy.

Interleukins as immunomodulators are promising therapeutic agents for cancer therapy. Previous studies showed that there was an improved antitumor immunity in tumor-bearing mice using recombinant Newcastle disease virus carrying for interleukin-2. Interleukin-12 is a promising antitumor cytokine too. So we investigated and compared the antitumor effect of genetically engineered Newcastle disease virus strains expressing both interleukin-12 and/or interleukin-2 (rClone30-interleukin-2, rClone30-interleukin-12, and rClone30-interleukin-12-interleukin-2). In vitro studies showed that rClone30s could efficiently infect tumor cells and express interleukin-12 and/or interleukin-2. 3-(4,5-Dimethylthiazol-2-y)-2,5-diphenyl-tetrazolium bromide results showed rClone30s possessed strong cytotoxic activities against multiple tumor cell lines (U251, HepG2, A549, and Hela). Animal studies showed that rClone30-interleukin-12-interleukin-2 was more effective in inhibition of murine hepatoma carcinoma tumors, with the mean tumor volume (day 14) of 141.70 mm(3) comparing 165.67 mm(3) of rClone30-interleukin-12 group, 210.47 mm(3) of rClone30-interleukin-2 group, 574.70 mm(3) of rClone30 group, and 1206.83 mm(3) of phosphate-buffered saline group. Moreover, the rClone30-interleukin-12-interleukin-2 treated mice secreted more interferon γ (333.518 pg/mL) and its downstream cytokine interferon-γ induced protein 10 (16.006 pg/mL) in tumor than the rClone30-interleukin-12 group (interferon γ: 257.548 pg/mL; interferon-γ induced protein 10: 13.601 pg/mL), rClone30-interleukin2 group (interferon γ: 124.601 pg/mL; interferon-γ induced protein 10: 9.779 pg/mL), or rClone30 group (interferon γ: 48.630 pg/mL; interferon-γ induced protein 10:1.650 pg/mL). For the survival study, rClone30-interleukin12-interleukin2 increased the survival rate (12 of 16) of the tumor-bearing mice versus 11 of 16 in rClone30-interleukin-12 group, 10 of 16 in rClone30-interleukin-2 group, 7 of 16 in Clone30 group, and 0/16 in phosphate-buffered saline group, respectively. To determine whether the mice treated with recombinant virus developed protective immune response, the mice were rechallenged with the same tumor cells. The results showed that viral-treated mice were significantly protected from rechallenge. These results suggest that expressing both interleukin-2 and/or interleukin-12 could be ideal approaches to enhance the antitumor ability of Newcastle disease virus, and rClone30-interleukin-12-interleukin-2 is slightly superior over rClone30-interleukin-12 and rClone30-interleukin-2 alone.

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.

PD-1 or PD-L1 Blockade Restores Antitumor Efficacy Following SSX2 Epitope-Modified DNA Vaccine Immunization.

DNA vaccines have demonstrated antitumor efficacy in multiple preclinical models, but low immunogenicity has been observed in several human clinical trials. This has led to many approaches seeking to improve the immunogenicity of DNA vaccines. We previously reported that a DNA vaccine encoding the cancer-testis antigen SSX2, modified to encode altered epitopes with increased MHC class I affinity, elicited a greater frequency of cytolytic, multifunctional CD8(+) T cells in non-tumor-bearing mice. We sought to test whether this optimized vaccine resulted in increased antitumor activity in mice bearing an HLA-A2-expressing tumor engineered to express SSX2. We found that immunization of tumor-bearing mice with the optimized vaccine elicited a surprisingly inferior antitumor effect relative to the native vaccine. Both native and optimized vaccines led to increased expression of PD-L1 on tumor cells, but antigen-specific CD8(+) T cells from mice immunized with the optimized construct expressed higher PD-1. Splenocytes from immunized animals induced PD-L1 expression on tumor cells in vitro. Antitumor activity of the optimized vaccine could be increased when combined with antibodies blocking PD-1 or PD-L1, or by targeting a tumor line not expressing PD-L1. These findings suggest that vaccines aimed at eliciting effector CD8(+) T cells, and DNA vaccines in particular, might best be combined with PD-1 pathway inhibitors in clinical trials. This strategy may be particularly advantageous for vaccines targeting prostate cancer, a disease for which antitumor vaccines have demonstrated clinical benefit and yet PD-1 pathway inhibitors alone have shown little efficacy to date.

Requirement of Innate Immunity in Tumor-Bearing Mice Cured by Adoptive Immunotherapy Using Tumor-Draining Lymph Nodes

Background. The purpose of this study was to determine the cellular effectors of both the adoptively transferred cells and the tumor-bearing host that participate in the antitumor response to adoptive immunotherapy using culture-activated tumor-draining lymph nodes (TDLNs). Methods. TDLNs harvested from mice with 4T1 carcinoma cells were fractionated to derive the L-selectinlow subpopulation and activated ex vivo prior to in vitro cytokine release assays and adoptive transfer into BALB/c mice bearing 3-day established subcutaneous tumors. Tumor-bearing recipients were SCID (lacking T, B, and NK cells), Rag2 deficient (lacking T and B cells), and wild-type BALB/c mice. Results. Culture-activated L-selectinlow 4T1 TDLN from BALB/c mice secreted significant levels of interferon-gamma in response to 4T1 but not control tumor cells in vitro. CD4 cells within the adoptively transferred effector cell population contributed significantly to the antitumor effect in vivo. Culture-activated L-selectinlow TDLNs from BALB/c wild-type mice were able to cure Rag2 deficient but not SCID mice bearing 4T1 subcutaneous tumors, suggesting a requirement of NK cells within the innate immune system of the tumor-bearing host during the antitumor response. Conclusions. These results identify the cellular effectors involved in tumor regression following adoptive transfer and demonstrate the requirement for intact innate immunity within the tumor-bearing host.

Abstract 2593: Multitargeted receptor tyrosine kinase inhibitors synergize with folate-hapten mediated immunotherapy in folate receptor positive cancers

Abstract The folate receptor (FR) which binds the vitamin folic acid with high affinity is overexpressed by a variety of human cancers including ovarian, endometrial, renal, and lung cancers. Consequently, the receptor has been exploited for the delivery of imaging agents and highly potent drugs exclusively to FR expressing tumors while avoiding uptake by the surrounding healthy tissue. This targeting mechanism can also be utilized for immunotherapy by vaccinating mice against a particular hapten in order to induce the production of anti-hapten antibodies and subsequently treating tumor bearing mice with a folate-hapten conjugate. The resulting reduction in tumor burden is achieved by antibody-dependent cellular cytotoxicity (ADCC). However, hapten mediated immunotherapy alone has been ineffective at controlling tumor progression over longer periods of time and requires supplementation with immunomodulatory cytokines for optimum therapeutic results. Several multitargeted receptor tyrosine kinase (RTK) inhibitors have recently shown promising synergy in a variety of different cancers when combined with other immunotherapies. Therefore, we examined the effect of combining the RTK inhibitors sunitinib and axitinib with folate-fluorescein therapy in tumor bearing mice that had previously been immunized against fluorescein. We report here that sunitinib synergizes with folate-fluorescein in both the L1210A lymphoma and the M109 lung cancer models resulting in slowed tumor progression, inhibition of tumor vasculature, and increased levels of circulating CD4+ and CD8+ T cells. The combination of axitinib with folate-fluorescein was only evaluated in mice bearing L1210A tumors and these mice have also demonstrated lower average tumor volumes than mice treated with either drug alone or not treated at all. These results suggest that RTK inhibitors play an important role in modulating the anti-tumor immunity of tumor bearing mice and could be a potent addition to ADCC mediated therapeutic approaches for cancers. Citation Format: Nimalka A. Bandara, Philip S. Low. Multitargeted receptor tyrosine kinase inhibitors synergize with folate-hapten mediated immunotherapy in folate receptor positive cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2593. doi:10.1158/1538-7445.AM2014-2593

Experimental study of Dai medicine Yapa Tang on inhibiting the lewis lung carcinoma in mice growth and metastasis and effects on immune function

Objective To study the Dai medicine Yapa Tang inhibition on mice Lewis lung carcinoma growth and metastasis,and to explore the impact of the drug on mice immune function. Methods Subcutaneous injection LLC（Lewis lung carcinoma cells）in C57BL/6 mice’s right armpit was to establish rat models. 60 successful tumor-bearing mice were selected and thenafter inoculating LLC were randomly divided into 6 groups, namely the control group,Yapa Tang low-dose group,Yapa Tang mid-dose group, Yapa Tang high-dose group,cyclophosphamide group and Yapa Tang mid-dose combining cyclophosphamide group.Then,after inoculated 3 days,each group was administrated with medicines.The first four groups wereintragastricly administrated with 0.9% saline, low-dose, mid-dose and high-dose Yapa Tang with0.4ml respectively;the cyclophosphamide group was administrated with cyclophosphamide,and Yapa Tang mid-dose combining cyclophosphamide group was administrated with middle dose Yapa Tang and cyclophosphamide. After 21days, blood was taken from eyeballs. Tumor tissue, spleen, thymus were gotten and weighted to calculate inhibitory rate,spleen and thymus index, enzyme-linked immunosorbent assay （Elisa） was adopted to detect Interleukin-2（IL-2）, Interleukin-10（IL-10）and their contents. Results The tumor weight of Yapa Tang high-dose group, cyclophosphamide group and Yapa Tang mid-dose combining cyclophosphamide group was 3.46±0.39, 2.39±1.04 and 2.30±0.76 respectively,all lower than the control group, which was 5.21±0.50, showing significant difference（P〈0.05）, with the inhibition rate were33.69%, 54.12%, 56.00%. The thymus and spleen index of Yapa Tang low-dose group, middle-dose group and high-dose group was2.16±0.69, 2.24± 0.76, 2.23 ± 0.63, 16.82 ± 3.14, 15.82 ± 1.72, 17.08 ± 3.65, significantly higher than that of the control group,which was1.94±0.6, 15.17±3.53. The IL-2, IL-10 level of control group were（883.54±181.49）ng/L, （1 106.86±343.79）ng/L. Yapa Tanggroupsshowed an increase in IL-2（1 732.29±100.52）ng/L, （1 813.33± 168.32）ng/L, （2 275.63 ± 394.76）ng/L and the decrease in level of IL-10, respectively were（834.02 ± 271.97）ng/L, （636.83±270.56）ng/L, （682.08±147.85）ng/L, with significant difference. Conclusion Yapa Tang can inhibit lewis lung cancer grow and reduce the number of lung metastases, regulate immune cytokines IL-2 and IL-10, and promote the immunity of tumor-bearing mice. Key words: Yapa Tang; Lewis lung cancer

Immunotherapy-induced CD8+ T Cells Instigate Immune Suppression in the Tumor

Despite clear evidence of immunogenicity, cancer vaccines only provide a modest clinical benefit. To evaluate the mechanisms that limit tumor regression following vaccination, we have investigated the weak efficacy of a highly immunogenic experimental vaccine using a murine melanoma model. We discovered that the tumor adapts rapidly to the immune attack instigated by tumor-specific CD8+ T cells in the first few days following vaccination, resulting in the upregulation of a complex set of biological networks, including multiple immunosuppressive processes. This rapid adaptation acts to prevent sustained local immune attack, despite continued infiltration by increasing numbers of tumor-specific T cells. Combining vaccination with adoptive transfer of tumor-specific T cells produced complete regression of the treated tumors but did not prevent the adaptive immunosuppression. In fact, the adaptive immunosuppressive pathways were more highly induced in regressing tumors, commensurate with the enhanced level of immune attack. Examination of tumor infiltrating T-cell functionality revealed that the adaptive immunosuppression leads to a progressive loss in T-cell function, even in tumors that are regressing. These novel observations that T cells produced by therapeutic intervention can instigate a rapid adaptive immunosuppressive response within the tumor have important implications for clinical implementation of immunotherapies.

Immunomodulation of macrophages by methylglyoxal conjugated with chitosan nanoparticles against Sarcoma-180 tumor in mice

Methylglyoxal (MG), the potent anticancer agent has been conjugated to a nontoxic, biocompatible polymer, chitosan, to protect it from in vivo enzymatic degradation. This polymeric complex, ‘Nano-MG’ shows remarkable antitumor property and elicits macrophage-mediated immunity in tumor bearing mice on intravenous (0.4mg/kg bodywt/day) treatment more efficiently than MG (20mg/kg bodywt/day). These activated macrophages appear more in numbers in the peritoneum and produce more superoxide and nitrite. Moreover, immunomodulatory cytokines and surface receptors of these macrophages like iNOS, IFN-γ, TNF-α, IL-1β, IL-6, M-CSF, TLR-4 and TLR-9 also exhibit marked up-regulation in Sarcoma-180 tumor bearing mice after Nano-MG treatment compared to untreated tumor bearing counterpart. Hence, Nano-MG acts as an immunostimulant in tumor bearing mice to combat cancer at conspicuously lower dose, probably due to its longer circulation time in blood.

Aptamer-targeted inhibition of mTOR in T cells enhances antitumor immunity

Recent studies have underscored the importance of memory T cells in mediating protective immunity against pathogens and cancer. Pharmacological inhibition of regulators that mediate T cell differentiation promotes the differentiation of activated CD8(+) T cells into memory cells. Nonetheless, pharmacological agents have broad targets and can induce undesirable immunosuppressive effects. Here, we tested the hypothesis that aptamer-targeted siRNA inhibition of mTOR complex 1 (mTORC1) function in CD8(+) T cells can enhance their differentiation into memory T cells and potentiate antitumor immunity more effectively than the pharmacologic inhibitor rapamycin. To specifically target activated cells, we conjugated an siRNA targeting the mTORC1 component raptor to an aptamer that binds 4-1BB, a costimulatory molecule that is expressed on CD8(+) T cells following TCR stimulation. We found that systemic administration of the 4-1BB aptamer-raptor siRNA to mice downregulated mTORC1 activity in the majority of CD8(+) T cells, leading to the generation of a potent memory response that exhibited cytotoxic effector functions and enhanced vaccine-induced protective immunity in tumor-bearing mice. In contrast, while treatment with the general mTORC1 inhibitor rapamycin also enhanced antigen-activated CD8(+) T cell persistence, the cytotoxic effector functions of the reactivated memory cells were reduced and the alloreactivity of DCs was diminished. Consistent with the immunological findings, mice treated with rapamycin, but not with 4-1BB aptamer-raptor siRNA, failed to reject a subsequent tumor challenge.