Tumor-targeted IL-12 combined with tumor resection yields a survival-favorable immune profile.

BACKGROUND: Although accumulated evidence provides a strong scientific premise for using immune profiles to predict survival in patients with cancer, a universal immune profile across tumor types is still lacking, and how to achieve a survival-associated immune profile remains to be evaluated. METHODS: We analyzed datasets from The Cancer Genome Atlas to identify an immune profile associated with prolonged overall survival in multiple tumor types and tested the efficacy of tumor cell-surface vimentin-targeted interleukin 12 (ttIL-12) in inducing that immune profile and prolonging survival in both mouse and patient-derived xenograft tumor models. RESULTS: We identified an immune profile (IFNγHiCD8HiFOXP3LowCD33Low) associated with prolonged overall survival across several human tumor types. ttIL-12 in combination with surgical resection of the primary tumor transformed tumors to this immune profile. Intriguingly, this immune profile transformation led to inhibition of metastasis and to prolonged survival in both mouse and patient-derived xenograft malignant models. Wild-type IL-12 combined with surgery was significantly less effective. In the IL-12-sensitive C3H mouse strain, in fact, wild-type IL-12 and surgery resulted in shorter overall survival than in mice treated with control pDNA; this surprising result is believed to be attributable to IL-12 toxicity, which was absent in the mice treated with ttIL-12. The ttIL-12-induced immune profile associated with longer overall survival was also associated with a greater accumulation of CD8+ T cells and reduced infiltration of regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages. The underlying mechanism for this transformation by ttIL-12 treatment was induction of expression of CXCL9 and reduction of expression of CXCL2 and CCL22 in tumors. CONCLUSIONS: ttIL-12 when combined with surgery led to conversion to the IFNγHiCD8HiFOXP3LowCD33Low immune profile, eliminated relapse and metastasis, and prolonged overall survival.

A practical guide for the safe implementation of early phase drug development and immunotherapy program in gynecologic oncology practice.

The success of targeted and immune therapies in other malignancies has led to an exponential increase in the number of active and pending clinical trials using these therapeutic approaches in patients with gynecologic cancers. These novel investigational agents are associated with unique and potentially life-threatening toxicities and many require special multidisciplinary logistical considerations. The objective of this review is to describe a practical approach for the safe implementation of targeted and immune therapies in academic gynecologic oncology practices based on our experience at M.D. Anderson Cancer Center.

IL27 controls skin tumorigenesis via accumulation of ETAR-positive CD11b cells in the pre-malignant skin.

Establishment of a permissive pre-malignant niche in concert with mutant stem are key triggers to initiate skin carcinogenesis. An understudied area of research is finding upstream regulators of both these triggers. IL27, a pleiotropic cytokine with both pro- and anti-inflammatory properties, was found to be a key regulator of both. Two step skin carcinogenesis model and K15-KRASG12D mouse model were used to understand the role of IL27 in skin tumors. CD11b-/- mice and small-molecule of ETAR signaling (ZD4054) inhibitor were used in vivo to understand mechanistically how IL27 promotes skin carcinogenesis. Interestingly, using in vivo studies, IL27 promoted papilloma incidence primarily through IL27 signaling in bone-marrow derived cells. Mechanistically, IL27 initiated the establishment of the pre-malignant niche and expansion of mutated stem cells in K15-KRASG12D mouse model by driving the accumulation of Endothelin A receptor (ETAR)-positive CD11b cells in the skin-a novel category of pro-tumor inflammatory identified in this study. These findings are clinically relevant, as the number of IL27RA-positive cells in the stroma is highly related to tumor de-differentiation in patients with squamous cell carcinomas.

Lack of Immunomodulatory Interleukin-27 Enhances Oncogenic Properties of Mutant p53 In Vivo.

PURPOSE: p53 is mutated in about 50% of human cancers, mostly through missense mutations. Expression of mutant p53 is associated with poor clinical outcomes or metastasis. Although mutant p53 is inherently instable, various stressors such as DNA damage or expression of the oncogenic Kras or c-myc affect the oncogenic properties of mutant p53. However, the effects of inflammation on mutant p53 are largely unknown. IL27 is an important immunomodulatory cytokine, but its impact on mutant p53-driven tumorigenesis has not been reported. EXPERIMENTAL DESIGN: IL27RA(-/-) mice were bred with mutant p53 heterozygous (p53(R172H/+)) mice to obtain IL27RA(-/-)p53(H/+) and IL27RA(-/-)p53(H/H) mice. Mouse survival and tumor spectra for the cohort were analyzed. Stability of p53 protein was analyzed via IHC and Western blot analysis. RESULTS: This study unraveled that lack of IL27 signaling significantly shortened the survival duration of mice with tumors expressing both copies of the mutant p53 gene (Li-Fraumeni mouse model). Interestingly, in mice that were heterozygous for mutant p53, lack of IL27 signaling not only significantly shortened survival time but also doubled the incidence of osteosarcomas. Furthermore, lack of IL27 signaling is closely associated with increased mutant p53 stability in vivo from early age. CONCLUSIONS: These results suggest that IL27 signaling modulates the oncogenic properties of mutant p53 in vivo Clin Cancer Res; 22(15); 3876-83. ©2016 AACR.

Mutant p53 in concert with an interleukin-27 receptor alpha deficiency causes spontaneous liver inflammation, fibrosis, and steatosis in mice.

UNLABELLED: The cellular and molecular etiology of unresolved chronic liver inflammation remains obscure. Whereas mutant p53 has gain-of-function properties in tumors, the role of this protein in liver inflammation is unknown. Herein, mutant p53(R172H) is mechanistically linked to spontaneous and sustained liver inflammation and steatosis when combined with the absence of interleukin-27 (IL27) signaling (IL27RA), resembling the phenotype observed in nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) patients. Indeed, these mice develop, with age, hepatocyte necrosis, immune cell infiltration, fibrosis, and micro- and macrosteatosis; however, these phenotypes are absent in mutant p53(R172H) or IL27RA(-/-) mice. Mechanistically, endothelin A receptor (ETAR)-positive macrophages are highly accumulated in the inflamed liver, and chemical inhibition of ETAR signaling reverses the observed phenotype and negatively regulates mutant p53 levels in macrophages. CONCLUSION: The combination of mutant p53 and IL27RA(-/-) causes spontaneous liver inflammation, steatosis, and fibrosis in vivo, whereas either gene alone in vivo has no effects on the liver.

Safe and effective treatment of spontaneous neoplasms with interleukin 12 electro-chemo-gene therapy.

Electroporation improves the anti-tumour efficacy of chemotherapeutic and gene therapies. Combining electroporation-mediated chemotherapeutics with interleukin 12 (IL-12) plasmid DNA produces a strong yet safe anti-tumour effect for treating primary and refractory tumours. A previously published report demonstrated the efficacy of a single cycle of IL-12 plasmid DNA and bleomycin in canines, and, similarly, this study further demonstrates the safety and efficacy of repeated cycles of chemotherapy plus IL-12 gene therapy for long-term management of aggressive tumours. Thirteen canine patients were enrolled in this study and received multiple cycles of electro-chemo-gene therapy (ECGT) with IL-12 pDNA and either bleomycin or gemcitabine. ECGT treatments are very effective for inducing tumour regression via an antitumour immune response in all tested histotypes except for sarcomas, and these treatments can quickly eradicate or debulk large squamous cell carcinomas. The versatility of ECGT allows for response-based modifications which can overcome treatment resistance for affecting refractory lesions. Importantly, not a single severe adverse event was noted even in animals receiving the highest doses of chemotherapeutics and IL12 pDNA over multiple treatment cycles. This report highlights the safety, efficacy and versatility of this treatment strategy. The data reveal the importance of inducing a strong anti-tumour response for successfully affecting not only the treated tumours, but also non-treated metastatic tumours. ECGT with IL12 pDNA plus chemotherapy is an effective strategy for treating multiple types of spontaneous cancers including large, refractory and multiple tumour burdens.

Safety and efficacy of tumor-targeted interleukin 12 gene therapy in treated and non-treated, metastatic lesions.

The ability to control the immune system to actively attack tumor tissues will be a marvelous weapon to combat the persistent attack of cancer. Unfortunately, safe and effective methods to gain this control are not yet available as cancer therapies. To overcome the impediments to this control, tumor-targeted (tt) Interleukin 12 (IL-12) plasmid DNA can be safely delivered to accessible tumors, and these treatments can induce antitumor immune responses in both the treated and untreated tumors. Here, electroporationmediated ttIL-12 pDNA treatments are shown to be safe and well tolerated in a dose escalation study in canines bearing naturally-occurring neoplasms. The final patient in the dose-escalation study received up to 3,800 µg pDNA distributed among five separate squamous cell carcinoma tumors in doses equivalent to those administered in a Phase I trial with wildtype IL-12 pDNA. Not a single severe adverse event occurred in any patient at any of the five dose levels, and only minor, transient changes were noted in any tested parameter. Clinical response analysis and immune marker mRNA detection of treated and non-treated lesions suggest that ttIL-12 pDNA treatments in only a few tumors can elicit antitumor immune responses in the treated lesions as well as distant metastatic lesions. These observations and results demonstrate that ttIL-12 pDNA can be safely administered at clinical levels, and these treatments can affect both treated and nontreated, metastatic lesions.

Managing local swelling following intratumoral electro-chemo-gene therapy.

Delivering genes and other materials directly into the tumor tissue causes specifically localized and powerfully enhanced efficacy of treatments; however, these specific effects can cause rapid, drastic changes in the appearance, texture, and consistency of the tumor. These changes complicate clinical response measurements which can confound the results and render recurring treatments difficult to perform and clinical response measurements nearly impossible to obtain accurately. One of the complicating issues is local swelling. Here, we demonstrate how swelling caused by intratumoral gene treatments can confound the clinical results and impede further treatments, and we demonstrate an easy technique to help overcome this potential hurdle.

The impact of non-electrical factors on electrical gene transfer.

Electrical pulses directly and effectively boost both in vitro and in vivo gene transfer, but this process is greatly affected by non-electrical factors that exist during electroporation. These factors include, but are not limited to, the types of cells or tissues used, property of DNA, DNA formulation, and expressed protein. In this mini-review, we only describe and discuss a summary of DNA properties and selected DNA formulations on gene transfer via electroporation. The properties of DNA were selected for review because a substantial amount of remarkable work has been performed during the past few years but has received less notice than other works, although DNA properties appear to be critical for boosting electroporation delivery. The selected formulations will be covered in this mini-review because we are only interested in the simple formulations that could be used for cell or gene therapy via electroporation. Plus, there was an extensive review of DNA formulations in the first edition of this book. The formulations discussed in this mini-review represent novel developments in recent years and may impact electroporation significantly. These advancements in DNA formulations could prove to be important for gene delivery and disease treatment.

Universal marker and detection tool for human sarcoma circulating tumor cells.

To date, no specific marker exists for the detection of circulating tumor cells (CTC) from different types of sarcomas, though tools are available for detection of CTCs in peripheral blood of patients with cancer for epithelial cancers. Here, we report cell-surface vimentin (CSV) as an exclusive marker on sarcoma CTC regardless of the tissue origin of the sarcoma as detected by a novel monoclonal antibody. Utilizing CSV as a probe, we isolated and enumerated sarcoma CTCs with high sensitivity and specificity from the blood of patients bearing different types of sarcoma, validating their phenotype by single cell genomic amplification, mutation detection, and FISH. Our results establish the first universal and specific CTC marker described for enumerating CTCs from different types of sarcoma, thereby providing a key prognosis tool to monitor cancer metastasis and relapse.

Intricacies for posttranslational tumor-targeted cytokine gene therapy.

The safest and most effective cytokine therapies require the favorable accumulation of the cytokine in the tumor environment. While direct treatment into the neoplasm is ideal, systemic tumor-targeted therapies will be more feasible. Electroporation-mediated transfection of cytokine plasmid DNA including a tumor-targeting peptide-encoding sequence is one method for obtaining a tumor-targeted cytokine produced by the tumor-bearing patient's tissues. Here, the impact on efficacy of the location of targeting peptide, choice of targeting peptide, tumor histotype, and cytokine utilization are studied in multiple syngeneic murine tumor models. Within the same tumor model, the location of the targeting peptide could either improve or reduce the antitumor effect of interleukin (IL)12 gene treatments, yet in other tumor models the tumor-targeted IL12 plasmid DNAs were equally effective regardless of the peptide location. Similarly, the same targeting peptide that enhances IL12 therapies in one model fails to improve the effect of either IL15 or PF4 for inhibiting tumor growth in the same model. These interesting and sometimes contrasting results highlight both the efficacy and personalization of tumor-targeted cytokine gene therapies while exposing important aspects of these same therapies which must be considered before progressing into approved treatment options.

Intraosseous inoculation of tumor cells into bone marrow promotes distant metastatic tumor development: A novel tool for mechanistic and therapeutic studies.

Bone marrow-derived cells have a potent impact on the formation and progression of tumor metastasis. This study demonstrates that bone marrow directly promotes metastasis to distant sites from tumor cells residing in the bone marrow in multiple types of tumors and multiple mouse strains. The bone marrow environment requires less tumor cells for inducing distant metastasis and overcomes the inhibition of metastasis resulting from engineering the tumor cells with reporter genes. This discovery provides an effective approach to generate spontaneous-like metastatic tumor models which will satisfy the urgent need for studying metastasis biology and discovering novel therapeutics.

Coordination between TLR9 signaling in macrophages and CD3 signaling in T cells induces robust expression of IL-30.

IL-30, the p28 subunit of IL-27, interacts with EBV-induced gene 3 to form IL-27, which modulates both proinflammatory and anti-inflammatory responses during autoimmune or infectious disease. It also acts as a natural antagonist of gp130, thereby attenuating the signals of other gp130-associated cytokines. IL-30 regulation via LPS has been reported by others, but the intercellular communication that induces IL-30 expression is unknown. In this study, we show that treatment with anti-CD3/CD28 Abs plus CpG oligodeoxynucleotides induces robust expression of IL-30, whereas either treatment alone induces only low expression of IL-30. This observation in vitro mirrors the murine model in which administration of CpG under inflammatory conditions in vivo induces IL-30 expression. This robust induction of IL-30 occurs through the coordination of helper CD4(+) T cells and innate immune cells (e.g., macrophages) and, to a lesser degree, B cells via the CD40/CD154 signaling pathway. These findings reveal a previously unrecognized mechanism that integrates signaling pathways from T cells and macrophages at the cellular level to induce IL-30 expression.

Interleukin-30: a novel antiinflammatory cytokine candidate for prevention and treatment of inflammatory cytokine-induced liver injury.

UNLABELLED: The liver is the major metabolic organ and is subjected to constant attacks from chronic viral infection, uptake of therapeutic drugs, life behavior (alcoholic), and environmental contaminants, all of which result in chronic inflammation, fibrosis, and, ultimately, cancer. Therefore, there is an urgent need to discover effective therapeutic agents for the prevention and treatment of liver injury, the ideal drug being a naturally occurring biological inhibitor. Here we establish the role of IL30 as a potent antiinflammatory cytokine that can inhibit inflammation-induced liver injury. In contrast, interleukin (IL)27, which contains IL30 as a subunit, is not hepatoprotective. Interestingly, IL30 is induced by the proinflammatory signal such as IL12 through interferon-gamma (IFN-γ)/signal transducer and activator of transcription 1 signaling. In animal models, administration of IL30 by way of a gene therapy approach prevents and treats both IL12-, IFN-γ-, and concanavalin A-induced liver toxicity. Likewise, immunohistochemistry analysis of human tissue samples revealed that IL30 is highly expressed in hepatocytes, yet barely expressed in inflammation-induced tissue such as fibrous/connective tissue. CONCLUSION: These novel observations reveal a novel role of IL30 as a therapeutic cytokine that suppresses proinflammatory cytokine-associated liver toxicity.

WSX1 expression in tumors induces immune tolerance via suppression of effector immune cells.

Crosstalk between tumor cells and the cognate microenvironment plays a crucial role in tumor initiation and progression. However, only a few genes are known to affect such a crosstalk. This study reveals that WSX1 plays such a role when highly expressed in tumor cells. The expression of WSX1 in Lewis Lung Carcinoma (LLC) and the melanoma cell line AGS induces the death of T cells and inhibits the production of the effector cytokine IFNγ from NK and T cells, resulting in the promotion of tumor growth. These pro-tumorigenic properties of WSX1 are independent of IL27. This key observation reveals a new pathway of tumor-host interaction, which will ultimately lead to better strategies in immune therapy to reverse tumor tolerance.

Discovery of a linear peptide for improving tumor targeting of gene products and treatment of distal tumors by IL-12 gene therapy.

Like many effective therapeutics, interleukin-12 (IL-12) therapy often causes side effects. Tumor targeted delivery may improve the efficacy and decrease the toxicity of systemic IL-12 treatments. In this study, a novel targeting approach was investigated. A secreted alkaline phosphatase (SEAP) reporter gene-based screening process was used to identify a mini-peptide which can be produced in vivo to target gene products to tumors. The coding region for the best peptide was inserted into an IL-12 gene to determine the antitumor efficacy. Affinity chromatography, mass spectrometry analysis, and binding studies were used to identify a receptor for this peptide. We discovered that the linear peptide VNTANST increased the tumor accumulation of the reporter gene products in five independent tumor models including one human xenogeneic model. The product from VNTANST-IL-12 fusion gene therapy increased accumulation of IL-12 in the tumor environment, and in three tumor models, VNTANST-IL-12 gene therapy inhibited distal tumor growth. In a spontaneous lung metastasis model, inhibition of metastatic tumor growth was improved compared to wild-type IL-12 gene therapy, and in a squamous cell carcinoma model, toxic liver lesions were reduced. The receptor for VNTANST was identified as vimentin. These results show the promise of using VNTANST to improve IL-12 treatments.

Expression of WSX1 in tumors sensitizes IL-27 signaling-independent natural killer cell surveillance.

It is well known that the interleukin (IL)-27 receptor WSX1 is expressed in immune cells and induces an IL-27-dependent immune response. Opposing this conventional dogma, this study reveals a much higher level of WSX1 expression in multiple types of epithelial tumor cells when compared with normal epithelial cells. Expression of exogenous WSX1 in epithelial tumor cells suppresses tumorigenicity in vitro and inhibits tumor growth in vivo. Different from the role of WSX1 in immune cells, the antitumor activity of WSX1 in epithelial tumor cells is independent of IL-27 signaling but is mainly dependent on natural killer (NK) cell surveillance. Deficiency of either the IL-27 subunit EBV-induced gene 3 or the IL-27 receptor WSX1 in the host animals had no effect on tumor growth inhibition induced by WSX1 expression in tumor cells. Expression of WSX1 in epithelial tumor cells enhances NK cell cytolytic activity against tumor cells, whereas the absence of functional NK cells impairs the WSX1-mediated inhibition of epithelial tumor growth. The underlying mechanism by which WSX1 expression in tumor cells enhances NK cytolytic activity is dependent on up-regulation of NKG2D ligand expression. Our results reveal an IL-27-independent function of WSX1: sensitizing NK cell-mediated antitumor surveillance via a NKG2D-dependent mechanism.

Administering plasmid DNA encoding tumor vessel-anchored IFN-alpha for localizing gene product within or into tumors.

Tumor-targeted gene delivery has been intensively studied in the field of gene therapy, but no attention has been given to targeting the therapeutic gene products, which are transcribed and translated from the injected genes, into tumors. Targeting immune stimulatory gene products into tumors is the key to triggering tumor-specific CD8(+) T-cell responses and reducing systemic toxicity. To target the gene products generated from the injected genes into tumors, genes encoding the tumor-targeted fusion gene product were generated and administered locally and systemically via electroporation. As anticipated, administration of a therapeutic gene encoding IFN-alpha and the tumor vessel-targeted peptide CDGRC fusion gene product minimizes the leakage of immunostimulatory cytokine from tumors into the blood circulation, increases the infiltration of CD8(+) T cells into tumors, induces a high magnitude of cytotoxic T-cell lysis (CTL) activity, and reduces tumor vessel density. As a result, tumor growth was more significantly inhibited by administering the IFN-alpha-CDGRC gene than by administering the wild-type IFN-alpha gene. The same result was obtained with the systemic administration of the tumor-targeted IFN-alpha gene. This gene product-based tumor-targeted gene therapy approach could complement any other tumor-targeted gene delivery method for improving tumor-targeting efficiency.

Intratumoral bleomycin and IL-12 electrochemogenetherapy for treating head and neck tumors in dogs.

Bleomycin and Interleukin 12 have been used clinically to treat tumors; however, the co-administration of Bleomycin and Interleukin 12 followed by electroporation has not been tested clinically. In this study, dogs with spontaneous head and neck tumors were treated with one co-administration of Bleomycin and Interleukin 12 plasmid DNA followed by electroporation. The regression of the recurrent papillary tumor and the adjacent metastatic bone tumor was analyzed by multiple CT scans. The papillary tumor was completely eradicated in less than 2 weeks, and the bone tumor was not visible 23 weeks after the administration.

Administering Plasmid DNA Encoding Tumor Vessel-anchored IFN-α for Localizing Gene Product Within or Into Tumors.

Tumor-targeted gene delivery has been intensively studied in the field of gene therapy, but no attention has been given to targeting the therapeutic gene products, which are transcribed and translated from the injected genes, into tumors. Targeting immune stimulatory gene products into tumors is the key to triggering tumor-specific CD8+ T-cell responses and reducing systemic toxicity. To target the gene products generated from the injected genes into tumors, genes encoding the tumor-targeted fusion gene product were generated and administered locally and systemically via electroporation. As anticipated, administration of a therapeutic gene encoding IFN-α and the tumor vessel-targeted peptide CDGRC fusion gene product minimizes the leakage of immunostimulatory cytokine from tumors into the blood circulation, increases the infiltration of CD8+ T cells into tumors, induces a high magnitude of cytotoxic T-cell lysis (CTL) activity, and reduces tumor vessel density. As a result, tumor growth was more significantly inhibited by administering the IFN-α-CDGRC gene than by administering the wild-type IFN-α gene. The same result was obtained with the systemic administration of the tumor-targeted IFN-α gene. This gene product-based tumor-targeted gene therapy approach could complement any other tumor-targeted gene delivery method for improving tumor-targeting efficiency.