Abstract
Immune checkpoint inhibitors (ICIs) have been demonstrated to have significant clinical benefits. Effectiveness of ICIs have been limited by both primary and acquired resistance. In ...addition, absence of T cell infiltration within the tumor site is one of the major obstacles limiting ICIs efficacy against solid tumors. Interleukin-12 (IL-12) is a potent immune stimulator that has been shown to stimulate growth and survival of T cells as well as NK cells. Utilizing a B16.F10 melanoma model in C57Bl/6 mice, we observed a significant reduction in T regulatory cells and myeloid derived suppressor cells while T effector cells were significantly increased within the tumor microenvironment (TME) following intratumor delivery of a plasmid encoding IL-12 using gene electrotransfer. Monotherapy with plasmid IL-12 delivered with gene electrotransfer (pIL-12 GET) resulted in prolonged disease-free survival as well as long term immune memory. To determine the robustness of this therapeutic approach and to evaluate the responsiveness in combination with anti-PD1, we used a two-tumor model consisting of a subcutaneous B16F10 tumor and B16F10 cells expressing luciferase injected via the intraperitoneal route in a C57Bl/6 mouse. Intratumor delivery of pIL-12 GET as a monotherapy resulted in reduction or elimination of the subcutaneous tumor. However, the monotherapy approach was only successful in reducing the peritoneal spread in about 50% of the mice. When pIL-12 GET was combined with anti-PD1 administered via an intraperitoneal injection not only was there an elimination of the subcutaneous tumor, but it resulted in the elimination of intraperitoneal metastatic growth. The elimination of peritoneal spread was confirmed utilizing an In Vivo Imaging System. Observations on day 60 revealed background levels of luminescence in 90% of mice treated with the combination therapy thus confirming long-term disease-free survival of these mice. This level of response was not seen in mice treated with anti-PD-1 alone or with simple injection of pIL-12. In addition, the combination therapy resulted in an enhanced memory response which protected against growth of new tumors following challenge. In addition, exposure of tumor cells to IL-12 resulted in upregulation of MHC class 1 and PDL1 expression. The results have this study suggest that the combination of pIL-12 GET combined with ICIs could be utilized as an effective combination therapy for melanoma.
Citation Format: Richard Heller, Megan Scott, Cathryn Mangiamele, Jody Synowiec, Guilan Shi. Gene therapy approach to suppress metastasis in a mouse melanoma model abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4210.
Abstract
Inflammation is driven by inflammatory cell death and the secretion of pro-inflammatory cytokines like IL-1β and alarmins like High Mobility Group Box Protein 1 (HMGB1) by the inflammasome. ...The inflammasome is a cytosolic multiprotein complex that typically contains a Nod-like receptor (NLR), like NLRP3 or NLRC4, and the adaptor ASC which links to caspase-1 for activation. How signals from the cytoplasmic NLR induce nuclear ASC and HMGB1 translocation into the cytosol for inflammasome function remains elusive. Here we hypothesize that Dnase1L3 (DNase γ), a Ca2+/Mg2+-dependent endonuclease, facilitates inflammasome-mediated release of cytokines and alarmins. When we treated murine macrophages with the Dnase1L3 inhibitors Fmoc-D-cyclohexylalanine (FCA) or pontacyl violet 6R (PV), HMGB1 and IL-1β release were inhibited following NLRP3 or NLRC4 stimulation. FCA and PV did not directly inhibit Casp1. We confirmed the specificity of the inhibitors using RNAi silencing of Dnase1L3 in THP-1 cell lines. Interestingly, pyroptosis was only slightly blocked by Dnase1L3 inhibition. Mechanistically, we found that ASC nuclear translocation and speck formation was impaired following FCA treatment, as was Casp1 cleavage. These data suggest that Dnase1L3 inhibition prevents Asc release from the nucleus. This demonstrates that pyroptosis and cytokine release can be mechanistically separated. Taken all together, these data show that Dnase1L3 is necessary for inflammasome-mediated release of cytokines and alarmins.
Abstract
Malignant melanoma is the deadliest form of skin cancer. New immunotherapy approaches including anti-CTLA4, anti-PD-1 and anti-PD1-L1 have improved this prognosis for several patients. While ...the results obtained with these therapies are encouraging, there is still a need to establish improved therapies. IL-12 is a potent cytokine mediating antitumor activity. However, it has only achieved modest antitumor effects in clinical trials, often accompanied by unacceptable adverse events. We evaluated if intratumoral delivery of a plasmid encoding IL-12 using gene electrotransfer (GET) could be used to induce an anti-tumor response. Effective protocols delivering plasmids with GET directly into tumors induced not only local immune response, but a systemic one as well. We have been evaluating the specific response following pIL-12 delivery as well as the potential to combine with checkpoint inhibitors to determine if the response could be further augmented. Using a B16F10 mouse melanoma model, we show that melanoma exhibited unique immune cell composition within the tumor microenvironment after intratumoral injection of pIL12 with GET. The total number of memory immune cells was markedly increased in pIL12 GET melanoma groups compared with control group. pIL12 GET displayed significant regulation of multiple immune cell types, including CD8+ cells, regulatory T cells and myeloid cells, which were induced to mount a CD8+ immune response. It was also observed that there was an increase in PD-1 expression following pIl-12 delivery. Taken together, these findings suggest a sequence of immune activity following pIL12 GET. Further, these results suggest that pretreatment or simultaneous treatment with IL-12 may augment anti-PD1 therapy.
Abstract Immune checkpoint inhibitors (ICIs) have significant clinical benefits, but are limited by both primary and acquired resistance. The absence of T cells within the tumor microenvironment ...(TME) is a significant hurdle, reducing the efficacy of ICIs against solid tumors. Multiple studies have been initiated to modify the TME to enhance the effectiveness of ICIs. We previously demonstrated that the intratumor delivery of a plasmid encoding interleukin-12 (pIL-12) using gene electrotransfer (GET) resulted in a significant increase in T effector cells and a reduction in T regulatory cells and myeloid derived suppressor cells within the TME, inducing a robust immune response. Combining pIL-12 GET with ICIs resulted in local and systemic responses in both preclinical and clinical studies. While encouraging results were obtained, improvements were possible. For example, the established GET technology requires high applied voltage for plasmid delivery. In addition, satisfactory delivery cannot be immediately detected. Our current research has focused on developing the next generation GET technology to overcome these issues. This new system includes a pulse generator, a heat source and a tissue impedance measuring device. An array that incorporates independently addressable electrodes was also developed. A moderate elevation of the tissue temperature coupled with the electrode array enabled a 75% reduction in the applied voltage. We tested the approach in the B16.F10 mouse melanoma model. Delivery of pIL-12 with the new system resulted in 100% long-term complete regression and 100% protection from challenge. In addition, monitoring impedance within each independent section of the electrode array revealed different pulse numbers were needed to achieve delivery in each section. We next evaluated a combination therapy, pIL-12 plasmid combined with a plasmid encoding a PD1 peptide, delivered with the next-gen device. In a multi-tumor model, the plasmids were delivered to the subcutaneous tumor; tumors generated with an intraperitoneal injection were untreated. This new therapy induced complete regression of the subcutaneous tumor and blockage of peritoneal tumor growth as assessed via in vivo imaging. Peritoneal tumor growth was not affected by single plasmid delivery or when both plasmids were delivered with the established GET technology. Work is ongoing to translate this approach to clinical evaluation. Citation Format: Richard Heller, Loree C. Heller, Guilan Shi, Jody Synowiec, Julie Singh, Alex Otten, Mark J. Jaroszeski. Intratumor delivery of plasmid DNA encoding immune stimulating agents induces both local and systemic responses 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 7240.
The cancer-testis (CT) family of antigens are expressed in multiple types of malignant neoplasm and are silent in normal tissues, apart from the testis. Immunotherapy targeting CT antigens is a ...promising therapeutic strategy for treatment of solid tumors. One member of this family, melanoma-associated antigen A4 (MAGE-A4), has been demonstrated to be expressed in melanomas and lung cancer. Patients with tumors expressing the MAGE-A4 antigen exhibit specific cellular and humoral immune responses to the antigen, resulting in a favorable prognosis. Conversely, the expression of MAGE-A4 is associated with poor survival in lung cancer. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immunosuppressive cells, which are upregulated in the cancer microenvironment. Little is known regarding any potential correlation between the expression of MAGE-A4 antigens and the accumulation of MDSCs. The present study aimed to examine the association between circulating MDSC levels and MAGE-A4 expression in a mouse model of Lewis lung cancer. The expression of MAGE-A4 in tumor cells or tissues was evaluated using western blotting, while the percentage of MDSCs (CD11b+Gr-1+) in the blood was detected by flow cytometry. In addition, the suppressive capacity of MDSCs and the effectiveness of MDSC depletion were assessed in C57BL/6 tumor-bearing mice. MDSCs were demonstrated to upregulate MAGE-A4 expression via the phosphosphorylated-signal transducer and activator of transcription 3705 pathway, while depletion of MDSCs decreased the tumor growth rate, prolonged median survival and enhanced the recognition of MAGE-A4 by CD8+ T cells. These findings indicated that immunotherapeutic strategies involving induction of cytotoxic T lymphocytes that target MAGE-A4, in combination with MDSC depletion, may be an effective approach to immunotherapy for cancer types with high expression of MAGE-A4.
Abstract
In this study, we evaluated effect of tumor-antigen-primed MHC-haploidentical lymphocytes in a TC-1 (H-2b) murine lung cancer model. The haploidentical effector cells prepared from ...splenocytes stimulated with the tumor cell lysates pulsed-DCs from F1 (C57BL/6 × Balb/C, H-2bd) mice, or immunized from F1 mice vaccinated with inactivated tumor cells. Haploidentical lymphocytes immunized with TC-1 could specifically kill the TC-1 cells, as well as up-regulate the NK activity. The effector cells inactivated with Mitomycin C in vitro kept the killing capacity, and migrated to tumor tissue. Compared with the cells from non-inactivated, or the syngeneic mice, the cells from Mitomycin C inactivated mice with haploidentical showed enhanced tumor inhibition. The survival time increased in tumor bearing mice and some showed tumor free, which developed no tumor after rechallenged with TC-1.Th1 cytokines including IL-2, IFN-γwere significantly increased. These studies demonstrate that the tumor antigen immunized MHC-haploidentical lymphocytes could specificity target to the tumor and kill them. MHC-mismatched alloantigens could be an effective adjuvant to break the immune tolerance and to activate innate and adoptive immunity in the tumor bearing host. The infusion of the inactivated MHC-haploidentical lymphocytes immunized with tumor antigen could be a safe and effective adoptive immunotherapy against tumor.
Abstract
Pediatric-onset Systemic Lupus Erythematosus (SLE) is linked to a deficiency of the endonuclease Dnase1L3. Dnase1L3 can function extracellularly as a barrier to transfection or localize to ...the nucleus of macrophages to cleave DNA during apoptosis, though the mechanism by which Dnase1L3 protects from pediatric-onset SLE is unknown. Two polygenic lupus murine models, MRL-lpr and NZB/W F1, bear the activity-reducing T89I mutation in Dnase1L3. Dnase1L3 T89I has an eight-fold decrease in barrier-to-transfection activity, while the endonuclease activity is only decreased two-fold compared to the wildtype, providing a tool with which to dissect Dnase1L3 trafficking in SLE. We hypothesized that Dnase1L3 T89I localizes to the nucleus, which decreases barrier-to-transfection activity and enhances SLE onset. We transfected HEK cells with wild type or T89I Dnase1L3, and determined the subcellular localization of Dnase1L3. We found that the majority of wild type Dnase1L3 is secreted from the cell, while Dnase1L3 T89I showed increased nuclear localization. Since Dnase1L3 alters inflammasome function, we tested whether inflammatory stimuli alter Dnase1L3 trafficking. LPS treatment of cells transfected with Dnase1L3 T89I reduced its nuclear localization. Altered Dnase1L3 trafficking may impair inflammasome function, since macrophages from NZB/W F1 mice secreted less IL-1β than wild type mice. Overall, the increase in nuclear localization of Dnase1L3 T89I suggests that barrier to transfection or extracellular nuclease activity may promote IL-1β release and protect against pediatric-onset SLE.
Highlights • Haploidentical lymphocytes could induce tumor specific CTL against recipient tumor by MHC identical molecules. • Haploidentical lymphocyte mismatched molecules can play a role of ...adjuvant. • Inactivated haploidentical CTL can be accumulated to the tumor tissue to take effect. • Host innate, adoptive and memory immune responses to tumor could be induced by transfer of inactivated haploidentical CTL.
Lysosome-associated protein transmembrane 4β (LAPTM4B) is a gene that has been indicated to be involved in cancer. It is located at chromosome 8q22 and is composed of seven exons and six introns. ...LAPTM4B encodes two protein isoforms: LAPTM4B-35 and LAPTM4B-24. LAPTM4B-35 is markedly upregulated and LAPTM4B-24 is downregulated in several types of cancer. LAPTM4B-35 is 91 amino acids (N91) longer than LAPTM4B-24 at the N-terminus. In the present study, western blotting, enzyme-linked immunosorbent spot analysis and the B16F10-N91 tumor bearing-mice experiments were used to evaluate whether the overexpression of N91 indicates its potential as a candidate tumor-associated antigen. The results revealed that N91 was expressed in a wide range of normal mouse tissues and human peripheral blood mononuclear cells, with varying expression levels. The weak immunogenicity of N91 protein suggested it was a weak candidate antigen; however, the N91 protein was associated with cell proliferation.
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