IL-12 Gene Therapy Research Articles

An initial investigation of transcutaneous delivery of plasmid DNA encoding interleukin-10 for the treatment of psoriatic skin conditions

Psoriasis is a chronic immune-mediated skin disorder characterized by intense local inflammation, epidermal hyperplasia, and leukocyte infiltration. Current treatment approaches for psoriasis aim to alleviate symptoms and prevent disease progression, including systemically administered drugs with whole body side effects. Despite some advances in psoriasis treatment, success has been quite limited. To begin to address this challenge, we undertook an initial investigation of whether transcutaneous delivery of an endogenous anti-inflammatory cytokine could provide an effective, local treatment of psoriatic-like skin conditions. To do this, we utilized a previously documented rodent model of psoriasis, induced via a single topical application of Imiquimod (IMQ) to the shaved back of rats. The therapeutic approach used for this initial investigation was delivery of plasmid DNA encoding rat interleukin-10 (pDNA-rIL10), a non-viral gene therapy approach previously shown to be effective in suppressing neuroinflammatory disorders after localized delivery either intracerebrally or intrathecally. Translation of this CNS therapeutic for use in psoriatic-like skin disorders required reformulation to enable transcutaneous delivery. Toward that end, pDNA-rIL10 was topically applied in Lipoderm HMW, a base explicitly designed to deliver higher molecular weight compounds into skin. Here we show that a single topical application of pDNA-rIL10 in Lipoderm HMW was effective in decreasing mRNA levels of pro-inflammatory cytokines as well as reducing the recruitment of T-cells to IMQ-treated skin. Furthermore, this transcutaneous IL-10 gene therapy decreased signs of skin inflammation, reflected by reduced erythema. Moreover, the results provide an initial indication that IL10 may stimulate hair regrowth in psoriatic-like skin.

Degradable Cationic Polycarbamates Designed for Robust IL-12 Gene Therapy against Metastatic Lung Cancer

Degradable Cationic Polycarbamates Designed for Robust IL-12 Gene Therapy against Metastatic Lung Cancer

Abstract 3289: PD-1 peptide/IL-12 combination gene therapy for the treatment of melanoma

Abstract Immune checkpoint inhibitors (ICIs) such as PD-1 targeting antibodies nivolumab and pembrolizumab are FDA approved as therapies for many cancers. These ICIs inhibit PD-1 ligand binding, removing interference with T cell function, allowing T cell proliferation, and restoring the anti-tumor activity. Potential factors predicting checkpoint blockade success include PD-L1 expression, the presence of tumor infiltrating lymphocytes, microsatellite instability, and tumor mutational burden. ICIs are most effective clinically in combination therapies. Here, we devised a simple ICI gene therapy as a possible alternative to traditional and expensive monoclonal antibodies. We designed plasmids encoding the mouse sequences homologous to the nivolumab and pembrolizumab binding regions, which overlap the PD-1 ligand binding site. We hypothesized that, when produced intratumorally, each PD-1 peptide should bind PD-L1 and block T cell binding. Since B16F10 mouse melanoma cells and tumors express PD-L1, we tested this theory in this model. We directly demonstrated peptide-PD-L1 binding histologically on B16F10 mouse melanoma tumors. Systemic expression of the encoded peptides did not induce changes in general demeanor, discomfort or weight changes in mice, implying that intratumor expression would also be non-toxic. We then tested these peptide plasmids therapeutically. Intratumor electroporation delivery of the peptide plasmids to palpable B16F10 mouse melanoma tumors significantly increased tumor doubling time and induced approximately 10% long-term tumor regression. Delivery of a plasmid encoding IL-12 induced long-term tumor regression in approximately 40% of mice as well as a reduction myeloid-derived suppressor cells and T regulatory cells and an increase infiltration of CD8+ T cells, creating an inflamed tumor microenvironment. This modulation of the tumor microenvironment suggested the potentiation of a combination therapy. This combination synergistically increased long-term regression to 80-100%, depending on the peptide plasmid delivered, over either PD-1 peptide or IL-12 gene therapy alone. These results demonstrated that the delivery of immune regulators with differing activities may be a promising and potentially inexpensive alternative to monoclonal antibody therapies. Citation Format: Loree Heller, Guilan Shi, Jody Synowiec, Richard Heller. PD-1 peptide/IL-12 combination gene therapy for the treatment of melanoma [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 3289.

Intratumoral DNA-based delivery of checkpoint-inhibiting antibodies and interleukin 12 triggers T cell infiltration and anti-tumor response.

To improve the anti-tumor efficacy of immune checkpoint inhibitors, numerous combination therapies are under clinical evaluation, including with IL-12 gene therapy. The current study evaluated the simultaneous delivery of the cytokine and checkpoint-inhibiting antibodies by intratumoral DNA electroporation in mice. In the MC38 tumor model, combined administration of plasmids encoding IL-12 and an anti-PD-1 antibody induced significant anti-tumor responses, yet similar to the monotherapies. When treatment was expanded with a DNA-based anti-CTLA-4 antibody, this triple combination significantly delayed tumor growth compared to IL-12 alone and the combination of anti-PD-1 and anti-CTLA-4 antibodies. Despite low drug plasma concentrations, the triple combination enabled significant abscopal effects in contralateral tumors, which was not the case for the other treatments. The DNA-based immunotherapies increased T cell infiltration in electroporated tumors, especially of CD8+ T cells, and upregulated the expression of CD8+ effector markers. No general immune activation was detected in spleens following either intratumoral treatment. In B16F10 tumors, evaluation of the triple combination was hampered by a high sensitivity to control plasmids. In conclusion, intratumoral gene electrotransfer allowed effective combined delivery of multiple immunotherapeutics. This approach induced responses in treated and contralateral tumors, while limiting systemic drug exposure and potentially detrimental systemic immunological effects.

Abstract LB-396: Amplification of the CXCR3/CXCL9 axis via intratumoral electroporation of CXCL9 synergizes with IL-12 gene therapy (TAVO) to elicit robust anti-tumor immunity

Abstract Clinical and preclinical studies have demonstrated that plasmid IL-12 (tavokinogene telseplasmid; TAVO) delivered intratumorally via electroporation drives IFN-γ expression and recruits T cells to the tumor microenvironment, ultimately yielding durable systemic T cell responses. Interrogation of longitudinal biomarker data from our IL-12/anti-PD-1 clinical trial has identified that clinical responses are closely tied to intratumoral CXCR3 levels. While all patients had a similar frequency of activated CD8+ T cells in the periphery, responding patients had a significant increase of intratumoral CXCR3 transcripts post-treatment (p=0.03) compared to nonresponding patients (p=0.4), underscoring the clinical relevance of tumor-infiltrating CXCR3+ immune cells. Since the IFN-γ/CXCL9/CXCR3 axis is known to increase sensitivity to anti-PD-1 therapies, we hypothesized that combining intratumoral TAVO with a DNA-encoded locally secreted CXCL9 (cognate ligand for CXCR3) would further augment/restore this axis and ‘license' a robust anti-PD-1 response beyond the treated lesion. An appropriate CXCL9 gradient can productively modulate frequencies of tumor infiltrating tumor-reactive CXCR3+ T cells. We have previously demonstrated that intratumoral electroporation of plasmid IL-12 and CXCL9 elicits a robust antitumor immune response evidenced by increased systemic antigen-specific CD8+ T cells and improved regression of both treated and contralateral CT26 tumors. In the current study, we demonstrate that an enhanced CXCL9 gradient via intratumoral electroporation leads to efficient trafficking of CXCR3+ CD8+ T cells into CT26 tumors. We further explored how DNA-encodable IL-12/CXCL9 can work together to improve checkpoint inhibitor response. We demonstrated that intratumoral TAVO rapidly drives a population of CXCR3+ CD8+ T cells in the lymph node and importantly, depletion of these CXCR3+ immune cells abrogated an IL-12-mediated anti-tumor response. Furthermore, upon electroporation of IL-12 and CXCL9, transcriptomic analysis of the tumor microenvironment revealed an enrichment of genes associated with immune-related pathways (IFN-γ signaling, interleukin signaling, GPCR signaling), antigen presentation machinery, and TCR signaling, indicating that this combination therapy augments anti-tumor immunity. Lastly, leveraging the partially responsive anti-PD-1 CT26 tumor model, we demonstrated that intratumoral electroporation of plasmid IL-12 with CXCL9 significantly improved anti-PD1 response, providing a strong rationale for filing an Investigational New Drug application based on this intratumoral DNA-encodable combination approach.Intratumoral CXCL9 with IL12 Citation Format: Anandaroop Mukhopadhyay, Bianca Nguyen, Jack Y. Lee, Mia Han, Jon Salazar, Reneta Hermiz, Lauren Svenson, Erica Browning, Daniel J. O'Connor, David A. Canton, Daniel Fisher, Joseph Skitzki, Christopher G. Twitty. Amplification of the CXCR3/CXCL9 axis via intratumoral electroporation of CXCL9 synergizes with IL-12 gene therapy (TAVO) to elicit robust anti-tumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-396.

CRS free T cell therapy: T cell membrane anchored and tumor targeted IL12 (ATTIL12)-T cell therapy eliminates large tumors, overcomes heterogeneity, and avoids adverse effects

Abstract Adoptive transfer of genetic engineered T cells becomes an effective approach in treating hematologic malignancies, but shows poor responses against solid tumors due to multifactorial reasons. Interleukin-12 exhibits robust T cell activating and anti-tumor efficacy. However, its clinical application was impeded due to the lethal adverse effects. We demonstrated previously that tumor targeted IL-12 (ttIL12) gene therapy avoids the toxicity of wildtype IL12 and effectively eliminate tumor metastasis. In this study, we developed a second generation of ttIL12— membrane-anchored ttIL-12 (attIL12)—to modify naive T cells, CD19CAR-T cells and OT1 T cells. This attIL12-modified T cells silent the toxicity of both IL12 and CAR-T cells associated cytokine release syndrome (CRS). Surprisingly, this ATTIL12-T cell therapy is able to eliminate multiple types of tumors with either homo- or hetero-generous environment or large size. We are in full speed to prepare a phase I clinical trial.

Relaxin-FOLFOX-IL-12 triple combination therapy engages memory response and achieves long-term survival in colorectal cancer liver metastasis

Induction of memory T cell response is inefficient in colorectal cancer (CRC) liver metastasis following existing therapies due to abundant stroma and immunosuppressive environment within the metastatic liver, which leads to fast disease progression, high recurrence rate, and short survival. Two fundamental steps are involved to elicit extensive memory T cell response: stimulation of naive T cells with robust and persistent antigen signals; and maintenance of differentiated memory T cells with survival factors. Here, we demonstrate a rational design of triple combination regimen, including relaxin (RLN), FOLFOX (combination of 5-fluorouracil, leucovorin, and oxaliplatin), and IL-12, successfully stimulates central memory T cells and achieves long-term survival in an aggressive experimental CRC liver metastasis model. Sequential administration of FOLFOX and IL-12 gene therapy following stromal deactivation by RLN gene therapy completely cured established CRC liver metastases in ~50% of mice and provided long-lasting protection against tumor recurrence. The study here may highlight the potential of evoking memory response as a curative therapy for the treatment of CRC liver metastasis.

Cholesterol-conjugated PEGylated PAMAM as an efficient nanocarrier for plasmid encoding interleukin-12 immunogene delivery toward colon cancer cells.

IL-12 is a pleiotropic cytokine, which shows an ideal applicant for tumor immunotherapy, because of its features of creating an interconnection between innate (NK cells) and adaptive (cytotoxic T lymphocyte) immunity. IL-12 gene therapy is a useful technique to deliver an immune-modulatory gene directly into tumor site thereby limiting the adverse effects of systemic administration of IL-12 proteins. One of the most largely investigated non-viral gene carriers is polyamidoamine (PAMAM). In the current research, 5 and 3% of PAMAM primary amines were substituted to transmit the plasmid encoding IL-12 gene to cells by cholesteryl chloroformate and alkyl-PEG, respectively. The features of modified PAMAMs containing size and surface charge density, cytotoxicity, and transfection efficiency were investigated in colon cancer cells. in vitro experiment showed that this modified carrier with average size of about 160 nm and zeta potential of 30 mV was able to increase the level of IL-12 production up to two folds as compared to that of the unmodified PAMAM. Improvement of the polymer hydrophobic balance along with of the modulation of the surface positive charge could provide an efficient and safe non-viral IL-12 gene for colon cancer immunogene therapy.

Intratumoral IL-12 Gene Therapy Inhibits Tumor Growth In A HCC-Hu-PBL-NOD/SCID Murine Model.

PurposeThis study aimed to evaluate the efficacy and safety of intratumoral IL-12 gene therapy in an HCC-hu-PBL-NOD/SCID mouse model.Materials and methodsThe HCC murine model was generated in NOD/SCID mice, and mice with grafted tumors were injected intraperitoneally with 2 × 107 human peripheral blood lymphocytes 14 days after modeling. After 4 days, mice were randomly divided into the 9597/IL-12 group, the 9597/plasmid group and the PBS group. The changes of tumor volume were measured and mouse peripheral blood was sampled post-treatment for ELISA and CBA analyses, and the grafted tumors were collected 28 days post-treatment for immunohistochemistry, ELISA, CBA and detection of cell cycle and apoptosis.ResultsThe tumor volume was smaller in the 9597/IL-12 group than in the 9597/plasmid and PBS groups on days 7, 14, 21, and 28 post-treatment (P < 0.05). Higher IL-12 levels were detected in the peripheral blood and the supernatants of grafted tumor homogenates in the 9597/IL-12 group than in the 9597/plasmid and PBS groups 7, 14, 21 and 28 days post-treatment (P < 0.05). IHC revealed higher counts of CD3+T cells, CD4+T helper cells, IFN-γ Th1 cells+ and S-100 protein positive dentric cells and lower MVD in the 9597/IL-12 group than in the 9597/plasmid and PBS groups (P < 0.05). Flow cytometry showed a significantly higher proportion of HCC cells at the G0/G1 phase and a significantly lower proportion of HCC cells at the S phase in the 9597/IL-12 group than in the PBS group (P < 0.05) and a greater apoptotic rate of HCC cells in the 9597/IL-12 group than in the 9597/plasmid and PBS groups (P < 0.05).ConclusionIntratumoral IL-12 gene therapy may inhibit tumorigenesis with mild adverse effects in a HCC-hu-PBL-NOD/SCID murine model through inhibiting angiogenesis, arresting cells in G0/G1 phase and inducing apoptosis.

Spectrum of chronic lung allograft pathology in a mouse minor-mismatched orthotopic lung transplant model.

Chronic lung allograft dysfunction (CLAD) is a fatal condition that limits survival after lung transplantation (LTx). The pathological hallmark of CLAD is obliterative bronchiolitis (OB). A subset of patients present with a more aggressive CLAD phenotype, called restrictive allograft syndrome (RAS), characterized by lung parenchymal fibrosis (PF). The mouse orthotopic single LTx model has proven relevant to the mechanistic study of allograft injury. The minor-alloantigen-mismatched strain combination using C57BL/10(B10) donors and C57BL/6(B6) recipients reportedly leads to OB. Recognizing that OB severity is a spectrum that may coexist with other pathologies, including PF, we aimed to characterize and quantify pathologic features of CLAD in this model. Left LTx was performed in the following combinations: B10→B6, B6→B10, B6→B6. Four weeks posttransplant, blinded pathologic semi-quantitative assessment showed that OB was present in 66% of B10→B6 and 30% of B6→B10 grafts. Most mice with OB also had PF with a pattern of pleuroparenchymal fibroelastosis, reminiscent of human RAS-related pathology. Grading of pathologic changes demonstrated variable severity of airway fibrosis, PF, acute rejection, vascular fibrosis, and epithelial changes, similar to those seen in human CLAD. These assessments can make the murine LTx model a more useful tool for further mechanistic studies of CLAD pathogenesis.

Characterization of abscopal effects of intratumoral electroporation-mediated IL-12 gene therapy

Intratumoral electroporation-mediated IL-12 gene therapy (IT-pIL12/EP) has been shown to be safe and effective in clinical trials, demonstrating systemic antitumor effects with local delivery of this potent cytokine. We recently optimized our IL-12 gene delivery platform to increase transgene expression and efficacy in preclinical models. Here we analyze the immunological changes induced with the new IT-pIL12/EP platform in both electroporated and distant, non-electroporated lesions. IT-pIL12/EP-treated tumors demonstrated rapid induction of IL-12-regulated pathways, as well as other cytokines and chemokines pathways, and upregulation of antigen presentation machinery. The distant tumors showed an increase in infiltrating lymphocytes and gene expression changes indicative of a de novo immune response in these untreated lesions. Flow cytometric analyses revealed a KLRG1hi CD8+ effector T-cell population uniquely present in mice treated with IT-pIL12/EP. Despite being highly activated, this population expressed diminished levels of PD-1 when re-exposed to antigen in the PD-L1-rich tumor. Other T-cell exhaustion markers appeared to be downregulated in concert, suggesting an orchestrated “armoring” of these effector T cells against T-cell checkpoints when primed in the presence of IL-12 in situ. These cells may represent an important mechanism by which local IL-12 gene therapy can induce a systemic antitumor immune response without the associated toxicity of systemic IL-12 exposure.

Abstract 2623: CHP-IL12 combined with surgery suppresses tumor metastasis and prolongs survival

Abstract Interleukin 12 (IL-12) is the most promising cytokine for treating tumors by itself or arming immunogenic cells but its toxicity and moderate response limit its clinical use. To avoid or minimize the toxicity issue and boost the anti-tumor immune response, our lab has discovered a comprehensive tumor-targeting carcinoma homing peptide (CHP). Fusion of this peptide encoding DNA sequence to IL12 encoding sequence in frame yields the proprietary product—CHP-IL12 (US patent 9,657,077 B2), or tumor-targeting IL12, which increased the accumulation of IL12 in tumor environment and decreased liver toxicity compared to wild-type IL12 (wt-IL12) gene therapy. Here, we report the therapeutic efficacy of this CHP-IL12 on metastasis and to comprehend the underlying mechanism description between CHP-IL12 and wildtype (wt)-IL12 for boosting anti-tumor response, two orthotopic and spontaneous tumor metastasis models (one adult and one pediatric tumor) were utilized. CHP-IL12 gene therapy showed greater efficacy in controlling growth of established tumors compared to wt-IL12. CHP-IL12 gene therapy, in combination with surgery, reduced number of metastatic nodules in lungs, livers and bones. CHP-IL12, compared to wt-IL12, conferred a potential immunostimulatory effect involving 1) reduction of T-reg cells, myeloid-derived suppressor cells (MDSC) and tumor associated macrophages (TAM); 2) recruitment of activated CD8+ T cells to the tumor. Moreover, CHP-IL12 increased IL12, IFNγ level in tumor environment and decreased IL12, IFNγ level in serum, which is the major cause of toxicity. Furthermore, in combination with surgery, CHP-IL12 gene therapy led to long-term survival and enhanced immunological memory development of tumor bearing mice compared to wt-IL12 gene therapy. These results collectively indicate that CHP-IL12 fusion gene therapy coupled with surgery has a powerful potency in inhibition of tumor metastasis and prolonging survival and a low potency in inducing toxicity. Therefore, CHP-IL12 fusion gene therapy in combination with surgery may translate into clinic as a promising anti-tumor strategy for both adult and pediatric tumors. Citation Format: Qingnan Zhao, Jiemiao Hu, Jeffry J. Cutrera, Abhisek Mitra, Xueqing Xia, Shulin Li. CHP-IL12 combined with surgery suppresses tumor metastasis and prolongs survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2623.

Interleukin 4 modulates microglia homeostasis and attenuates the early slowly progressive phase of amyotrophic lateral sclerosis

Microglia activation is a commonly pathological hallmark of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), a devastating disorder characterized by a selective motor neurons degeneration. Whether such activation might represent a causal event rather than a secondary epiphenomenon remains elusive. Here, we show that CNS-delivery of IL-4—via a lentiviral-mediated gene therapy strategy—skews microglia to proliferate, inducing these cells to adopt the phenotype of slowly proliferating cells. Transcriptome analysis revealed that IL-4-treated microglia express a broad number of genes normally encoded by embryonic microglia. Since embryonic microglia sustain CNS development, we then hypothesized that turning adult microglia to acquire such phenotype via IL-4 might be an efficient in vivo strategy to sustain motor neuron survival in ALS. IL-4 gene therapy in SOD1G93A mice resulted in a general amelioration of clinical outcomes during the early slowly progressive phase of the disease. However, such approach did not revert neurodegenerative processes occurring in the late and fast progressing phase of the disease.

Abstract OT1-01-02: Pilot trial of a type I polarized autologous dendritic cell vaccine incorporating tumor blood vessel antigen-derived peptides in patients with metastatic breast cancer

Abstract BACKGROUND. Cancer vaccines based on tumor-associated antigens are rarely curative in advanced cancer. This limitation relates to the heterogeneity of cancer due to defects in antigen presentation and altered immunophenotypes. Therefore, another method to promote anti-tumor immunity is to prime T cells against tumor-associated stromal cells. We have reported that IL-12 gene-therapy of established HLA-A2neg B16 melanomas in HLA-A2 transgenic (Tg) mice resulted in CD8+ T cell-mediated immunity against the host HLA-A2+ stromal cells within the tumor microenvironment (TME). We have also shown that vaccines based on a subset of tumor blood vessel antigen (TBVA)-derived peptides (DLK1310-318, EphA2883-891, HBB31-39, NRP1433-441, RGS55-13 and TEM1691-700) prevented HLA-A2neg MC38 tumor establishment and promoted the regression of melanomas in HLA-A2 Tg mice by CD8+ T cell targeting of HLA-A2+ pericytes and vascular endothelial cells in the TME. TRIAL DESIGN. Based on this pre-clinical data, we are undertaking a Susan G. Komen-funded (IIR13261822; IND 15722) IRB-approved clinical trial of chemo-immunotherapy using the immunomodulatory drug gemcitabine (GEM) to suppress tumor infiltrating suppressor cells such as myeloid-derived suppressor cells (MDSC) and regulatory T cells (Tregs) with a dendritic cell (DC) vaccine pulsed with the above six HLA-A2-presented TBVA-derived peptides (DC-TBVA) in 30 HLA-A2+ patients with metastatic breast cancer (MBC). Eligible patients will first undergo leukapheresis for the generation of the DC-TBVA vaccine. Patients will then receive 3 cycles of GEM, 1000 mg/m2 IV on days 1 and 8 of a 21-day cycle for 3 cycles. Patients will then receive the DC-TBVA vaccine administered twice intradermally 7 days apart. ELIGIBILITY CRITERIA. Patients must be HLA-A2+ and have radiologically measurable MBC, an ECOG performance status of 0-1 and not have any active immune disorders. Prior GEM therapy is acceptable as long as the last dose was ≥ 3 months from registration on this study. Patients may not be on steroids. SPECIFIC AIMS. The 4 specific aims are to 1) assess the safety of GEM + αDC1-TBVA vaccination, 2) assess the clinical response of MBC to GEM + αDC1-TBVA vaccination, 3) determine the clinical efficacy of GEM + αDC1-TBVA vaccination in generating Tc1 immunity, and 4) correlate changes in MDSC and Tregs with the generation of anti-TBVA Tc1-cell immunity STATISTICAL METHODS. Clinical response: if the response rate is less than 10%, then there is probability 0.05 or less of accepting the vaccine therapy; if the response rate is bigger than 32%, then the probability of rejecting the combination is less than 0.2. While the secondary goals of the study are exploratory, there is sufficient statistical power to identify moderate to large effects (i.e., there will be statistical power &amp;gt;.80 to detect changes from baseline in the different immune function parameters that are &amp;gt;0.6 standard deviations of the parameter.) TARGET ACCRUAL. We will enroll 30 patients over 3 years, with the first patient expected to be enrolled in July 2015. CONTACT INFORMATION. Joseph Baar, MD, PhD. Seidman Cancer Center. E-mail: joseph.baar@uhhospitals.org. Citation Format: Baar J, Storkus W, Finke J, Butterfield L, Lazarus H, Reese J, Brufsky A, Downes K, Budd GT, Fu P. Pilot trial of a type I polarized autologous dendritic cell vaccine incorporating tumor blood vessel antigen-derived peptides in patients with metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT1-01-02.

Abstract 298: Overcoming TGF-β-related immunosuppression for cancer immunotherapy by oncolytic adenovirus co-expressing Interleukin-12 and decorin

Abstract Interleukin (IL)-12 has been shown to be one of the most promising antitumor cytokines. The major antitumor activities of IL-12 rely on its ability to promote T helper type 1 (Th1) immune responses and CTL responses. However, immune suppression-dominated microenvironments in advanced tumors hinder the effect of IL-12 gene therapy for cancer. Most tumor cells produce large amounts of transforming growth factor-β (TGF-β), which plays crucial roles of tumor-induced immune suppression. The TGF-β inhibits proliferation of immune cells such as T, NK, and dendritic cells, resulting in suppression of antitumor immunity. Moreover, TGF-β is also known to regulate the maintenance and induction of regulatory T (Treg) cells. Thus, it has been hypothesized that inhibiting TGF-β might be an apt strategy for generating more effective cancer immunotherapy. Therefore, to enhance the efficiency of IL-12-mediated cancer immunotherapy, we generated oncolytic adenovirus co-expressing IL-12 and Decorin (hDCN) (RdB/IL12/hDCN) as suitable therapeutic adjuvant to overcome immune suppression by down-regulation of TGF-β through hDCN. Intratumoral administration of RdB/IL12/hDCN promoted enhanced antitumor effects compared with an oncolytic adenovirus expressing IL-12 or hDCN alone (RdB/IL12 or RdB/hDCN, respectively). We showed markedly elevated levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α and IFN-γ secreting cells in RdB/IL12/hDCN-treated tumors. Moreover, Treg in draining lymph nodes (DLNs) dramatically decreased in mice treated with RdB/IL12/hDCN, suggesting an association with the down-regulation of TGF-β in tumor treated with RdB/IL12/hDCN. Consistent with these data, tumor tissue injected with RdB/IL12/hDCN showed increased infiltration of CD4+ T and CD8+ T cells infiltration. Furthermore, hDCN enhanced viral distribution and tumor penetration, leading to improved virus-mediated cancer gene therapy by overcoming the extracellular matrix barrier within tumor masses. Together, these results provide new insight into the ability of hDCN to eliminate immune suppression and demonstrate that adenovirus co-expressing IL-12 and hDCN is a promising therapeutic tool for cancer treatment than adenovirus expressing IL-12 alone. Note: This abstract was not presented at the meeting. Citation Format: Eonju Oh, Il-Kyu Choi, June Kyu Hwang, Chae-Ok Yun. Overcoming TGF-β-related immunosuppression for cancer immunotherapy by oncolytic adenovirus co-expressing Interleukin-12 and decorin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 298. doi:10.1158/1538-7445.AM2015-298

(119) - A Novel Ex Vivo Intrabronchial Delivery of Ientivirus IL-10 Gene Therapy Attenuates Both Acute Allograft Rejection and Bronchiolitis Obliterans After Murine Orthotopic Lung Transplantation

Interleukin (IL)-10 is an important regulatory cytokine in transplantation. We hypothesized that IL-10 gene expression can attenuate both acute rejection (AR) and bronchiolitis obliterans (BO) after lung transplant (LTx). The objective of this study was to investigate the effects of a clinically-relevant IL-10 gene therapy in a minor-mismatch mouse orthotopic single LTx model.

Retinoic Acid promotes interleukin-4 plasmid-dimethylsulfoxide topical transdermal delivery for treatment of psoriasis.

BackgroundPsoriasis is an autoimmune disease that is caused by a shift in the Th1/Th2 balance toward Th1-dominant immunity. It has been established as an effective treatment to counteract psoriasis by subcutaneous injection of recombinant interleukin (IL)-4, and IL-4 gene therapy by topical transdermal penetration has shown its antipsoriatic effect in mice. Retinoic acid (RA) and dimethylsulfoxide can increase the efficiency of gene transfection in the topical transdermal delivery system.ObjectiveWe investigated whether RA could improve anti-psoriasis efficiency using IL-4 expression plasmid pORF-mIL-4 (pIL-4) via transdermal delivery system in K14-vascular endothelial growth (K14-VEGF) factor transgenic mice.MethodsAfter pretreatment with RA, plasmid pIL-4 in 10% dimethylsulfoxide was applied to the ear skin by topical transdermal penetration. Hematoxylin- eosin staining and immunohistochemistry were performed with ear samples to evaluate anti-psoriasis efficiency in mice.ResultsThe psoriasis pathological features were relieved and psoriasis-associated factors were significantly reduced.ConclusionOur results reveal that topical application of pIL-4 in dimethylsulfoxide by transdermal delivery with RA pretreatment can improve psoriasis significantly.

The relationship between the antitumor effect of the IL-12 gene therapy and the expression of Th1 cytokines in an HPV16-positive murine tumor model.

Objective. The goal of the present study was to investigate the effect of IL-12 expressed in plasmid on the Th1 cytokine profile in an experimental HPV16-positive murine tumor model and the association with the IL-12's antitumor effect. Methods. Mice were injected with BMK-16/myc cells to establish HPV16-positive tumor and then pNGVL3-mIL-12 plasmid; pcDNA3 plasmid or PBS was injected directly into tumor site. The antitumor effect of the treatment was evaluated and the cytokines expression profile in each tumor tissue was analyzed. Results. Treatment with pNGVL3-mIL-12 plasmid had a significant antitumor effect, and a Th2-Th3-type cytokines prolife was detected in the murine tumor model with expression of the cytokines IL-10, IL-4, and TGF-β1. However, after the tumor was treated with three intratumoral injections of plasmid containing IL-12 cDNA, it showed a cytokine profile associated with Th1 with expression of IL-2, IL-12, and IFN-γ cytokines and reduced expression of IL-10, IL-4, and TGF-β1. Conclusions. The treatment with the IL-12 gene in the experimental HPV16-positive tumor model promoted the activation of the cellular immune response via expression of a Th1-type cytokine profile and was associated with the inhibition of tumor growth. Thus, IL-12 treatment represents a novel approach for gene therapy against cervical cancer.

Lentivirus IL-10 Gene Therapy Down-Regulates IL-17 and Attenuates Mouse Orthotopic Lung Allograft Rejection

The purpose of the study was to examine the effect of lentivirus-mediated IL-10 gene therapy to target lung allograft rejection in a mouse orthotopic left lung transplantation model. IL-10 may regulate posttransplant immunity mediated by IL-17. Lentivirus-mediated trans-airway luciferase gene transfer to the donor lung resulted in persistent luciferase activity up to 6 months posttransplant in the isograft (B6 to B6); luciferase activity decreased in minor-mismatched allograft lungs (B10 to B6) in association with moderate rejection. Fully MHC-mismatched allograft transplantation (BALB/c to B6) resulted in severe rejection and complete loss of luciferase activity. In minor-mismatched allografts, IL-10-encoding lentivirus gene therapy reduced the acute rejection score compared with the lentivirus-luciferase control at posttransplant day 28 (3.0 ± 0.6 vs. 2.0 ± 0.6 (mean ± SD); p = 0.025; n = 6/group). IL-10 gene therapy also significantly reduced gene expression of IL-17, IL-23, and retinoic acid-related orphan receptor (ROR)-γt without affecting levels of IL-12 and interferon-γ (IFN-γ). Cells expressing IL-17 were dramatically reduced in the allograft lung. In conclusion, lentivirus-mediated IL-10 gene therapy significantly reduced expression of IL-17 and other associated genes in the transplanted allograft lung and attenuated posttransplant immune responses after orthotopic lung transplantation.

Abstract 4737: IL-4 and CpG therapy suppresses the outgrowth of tumors by activating tumor-specific Th1-type immune responses.

Abstract Although interleukin (IL) -4 is often regarded as a type-2 response inducer, recent studies have demonstrated that local IL-4 delivery at the site of vaccination activates local dendritic cells (DCs) and promote type-1 T cell responses. In particular, IL-4 supports DC maturation and enhances IL-12p70 secretion from DCs. Microbial molecules such as lipopolysaccharides, bacteriaderived RNA, and DNA are recognized by host cells through the TLR family, which belongs to the family of pattern-recognition receptors and stimulates immune responses. It has been reported that DNA vaccines and synthetic oligodeoxynucleotides (ODN) containing an unmethylated cytidine-phosphate-guanosine (CpG) motif promote Th1-type immune responses. CpG stimulates DCs through TLR9 and enhances DC maturation, which may improve the therapeutic effects on established tumors. In this study, we evaluated the antitumor effects of IL-4 gene therapy and CpG-ODN treatment in a poorly immunogenic murine cancer model. We used the IL-4 gene-overexpressing murine colorectal cancer MC38 cell line (MC38-IL4). Incubation with MC38-IL4 and CpG-ODN enhanced bone marrow-derived dendritic cell (DC) maturation in vitro. In addition, interferon (IFN)-γ production was significantly increased in naïve splenocytes after they were coincubated with MC38-IL4 and CpG-ODN. When mice bearing MC38 wild-type tumors were inoculated subcutaneously with MC38-IL4 cells and CpG-ODN, the outgrowth of established parental tumors was significantly suppressed compared to those in the MC38-IL4-treated group (IL-4 vs IL-4 + CpG-ODN, p= 0.015). A marked infiltration of CD8+ cells in the established parental tumors of mice treated with MC38-IL4 and CpG-ODN was confirmed by immunohistochemical analyses (MC38-IL4: 2.8 ± 1.9 cells/field vs MC38-IL4 + CpG-ODN: 20.7 ± 15.3 cells/field, p=0.027). Obvious tumor-specific cytolysis was detected when splenocytes of MC38-IL4 + CpG-ODN-treated mice were stimulated by γ-irradiated MC38-IL4 cells and CpG-ODN twice weekly in vitro and used as effector cells in a chromium-release assay (32.2% ± 3.5% for MC38 cells vs 3.2% ± 1.1% for YAC-1 cells; at an effector to target ratio of 40). These results suggest that IL-4 and CpG-ODN treatment promotes potent Th1-type antitumor immune responses. Therefore, the combination of IL-4 gene therapy and CpG-ODN treatment for cancer should be evaluated in clinical trials. Citation Format: Atsushi Kajiwara, Hiroyoshi Doi, Junichi Eguchi, Shigeaki Ishii, Ayako Sasagawa, Masaki Sakaki, Risa Omori, Eiichi Hayashi, Reiko Makino, Jin Yoshida, Kazumasa Hiroishi, Michio Imawari. IL-4 and CpG therapy suppresses the outgrowth of tumors by activating tumor-specific Th1-type immune responses. [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 4737. doi:10.1158/1538-7445.AM2013-4737