Systemic Administration Of IL-12 Research Articles

Target-Dependent Expression of IL12 by synNotch Receptor-Engineered NK92 Cells Increases the Antitumor Activities of CAR-T Cells.

IL12 is an immune-stimulatory cytokine for key immune cells including T cells and NK cells. However, systemic administration of IL12 has serious side effects that limit its clinical application in patients. Recently, synthetic Notch (synNotch) receptors have been developed that induce transcriptional activation and deliver therapeutic payloads in response to the reorganization of specific antigens. NK92 cell is a human natural killer (NK) cell line which has been developed as tools for adjuvant immunotherapy of cancer. Here, we explored the possibility of using synNotch receptor-engineered NK92 cells to selectively secrete IL12 at the tumor site and increase the antitumor activities of chimeric antigen receptor (CAR)-modified T cells. Compared with the nuclear factor of activated T-cells (NFATs) responsive promoter, which is another regulatory element, the synNotch receptor was better at controlling the expression of cytokines. NK92 cells transduced with the GPC3-specific synNotch receptor could produce the proinflammatory cytokine IL12 (GPC3-Syn-IL12-NK92) in response to GPC3 antigen expressed in cancer cells. In vivo GPC3-Syn-IL12-NK92 cells controlling IL12 production could enhance the antitumor ability of GPC3-redirected CAR T cells and increase the infiltration of T cells without inducing toxicity. Taken together, our results demonstrated that IL12 supplementation by synNotch-engineered NK92 cells could secrete IL12 in a target-dependent manner, and promote the antitumor efficiency of CAR-T cells. Local expression of IL12 by synNotch-engineered NK92 cells might be a safe approach to enhance the clinical outcome of CAR-T cell therapy.

Enhancement of Oncolytic Activity of oHSV Expressing IL-12 and Anti PD-1 Antibody by Concurrent Administration of Exosomes Carrying CTLA-4 miRNA

Systemic administration of checkpoint inhibitors alone and especially concurrent with intratumoral administration of oncolytic herpesviruses (oHSV) has a major impact on cancer therapy marred by rare failures of healthy organs. Furthermore, tumors vary with respect to susceptibility to oncolytic effects of oHSV. Here we report the construction and properties of 3 families of oncolytic herpes simplex viruses expressing no immunomodulatory genes (T1 series), murine IL-12 (T2 series) or murine or human IL-12 and anti PD-1 antibody (T3 series). We report that insertion of the gene encoding PD-1 Ab significantly augmented the oncolytic activity of oHSV bereft of immunostimulatory genes (T1 series) or expressing IL-12 alone (T2 series). The T3 oHSV expressed IL-12, PD-1 Ab were restricted to the tumor bed whereas the induced IFN-γ accumulated to high levels both in tumor bed and in blood. Furthermore, the T3 oHSV was superior to systemic administration of IL-12 and antibody to PD-1. This report also shows that the oncolytic activity of T3 oHSV in a relatively resistant tumor was enhanced by concurrent intratumoral administrationof genetically engineered exosomes carrying miRNA targeting CTLA-4 checkpoint.

Heterodimeric Fc-fused IL12 shows potent antitumor activity by generating memory CD8+ T cells

ABSTRACTInterleukin-12 (IL12) (p35/p40 complex) is a heterodimeric cytokine with potent anti-tumor activity. However, its short serum half-life and high dose-related toxicities limit its clinical efficacy. Here, we constructed heterodimeric immunoglobulin Fc-fused mouse IL12 (mIL12) in a monovalent binding format (mono-mIL12-Fc) to generate long-acting mIL12 in the naturally occurring heterodimeric form. Mono-mIL12-Fc exhibited a much longer plasma half-life than recombinant mIL12, enabling twice-weekly systemic injections to remove established tumors in syngeneic mouse models. Mono-mIL12-Fc was more potent than wild-type Fc-based bivalent-binding IL12-Fc (bi-mIL12-Fc) for eradicating large established immunogenic tumors without noticeable toxicities by enhancing interferon-γ production and the proliferation of immune effector cells in tumors. More importantly, mono-mIL12-Fc triggered weaker IL12 signaling than bi-mIL12-Fc, favoring the generation of functional and protective memory CD8+ T cells. Our results demonstrate that heterodimeric-Fc-fused IL12 is a suitable format for augmenting adaptive CD8+ T cell immune responses, providing a practical alternative to the systemic administration of IL12 for antitumor therapy.

Gene therapy approaches against cancer using in vivo and ex vivo gene transfer of interleukin-12.

IL-12 is an immunostimulatory cytokine with strong antitumor properties. Systemic administration of IL-12 in cancer patients led to severe toxic effects, prompting the development of gene therapy vectors able to express this cytokine locally in tumors. Both nonviral and viral vectors have demonstrated a high antitumor efficacy in preclinical tumor models. Some of these vectors, including DNA electroporation, adenovirus and ex vivo transduced dendritic cells, were tested in patients, showing low toxicity and moderate antitumor efficacy. IL-12 activity can be potentiated by molecules with immunostimulatory, antiangiogenic or cytotoxic activity. These combination therapies are of clinical interest because they could lower the threshold for IL-12 efficacy, increasing the therapeutic potential of gene therapy and preventing the toxicity mediated by this cytokine.

Copy number variations of HLA-I and activation of NKp30 pathway determine the sensitivity of gastric cancer cells to the cytotoxicity of natural killer cells.

Nude mice are important in vivo model for characterization of cell malignancy behavior; however, many cancer cells fail to form tumors in it. Understanding this defective mechanism may provide novel insights into tumorigenesis and how tumor cells escape innate immunity. Whole-genome sequencing was conducted on two gastric cancer (GC) cells, BGC823 and AGS, which do and do not form tumors in nude mice, to identify their genomic differences relevant to natural killer (NK) cells. We found that the tumorigenic capacity of human GC cell lines was dependent on the recruitment and activation of NK cells in xenograft tumors. We used whole-genome sequence (WGS) on GC cell lines to identify potential genes controlling susceptibility to NK-mediated killing. The tumorigenic cell line BGC823 expressed high levels of HLA-I because of copy gain and was resistant to NK cell killing. In contrast, another cell line AGS expressing low levels of HLA-I with activated NKp30/MAPK/IL-12 (interleukin-12) or IL-2 (interleukin-2) pathway was susceptible to NK lysis. Treatment of tumor bearing mice with systemic administration of IL-12 in combination with intratumor injection of anti-HLA-I antibody significantly increased NK cell recruitment into xenograft tumors, which became sensitive to NK killing, resulting in reduced tumor progression. In human GC specimens, decreased HLA-I expression and increased NK cells surrounding tumor cells were correlated with decreased metastasis potential and better prognosis of patients. Our results provide a mechanistic basis for GC cells to escape NK lysis and a promising prospect of NK immunotherapy for GC cells.

Defining the Pharmacodynamic Profile and Therapeutic Index of NHS-IL12 Immunocytokine in Dogs with Malignant Melanoma.

BackgroundInterleukin (IL)-12 is a pro-inflammatory cytokine that mediates T-helper type 1 responses and cytotoxic T-cell activation, contributing to its utility as anti-cancer agent. Systemic administration of IL-12 often results in unacceptable toxicity; therefore, strategies to direct delivery of IL-12 to tumors are under investigation. The objective of this study was to assist the preclinical development of NHS-IL12, an immunocytokine consisting of an antibody, which targets necrotic tumor regions, linked to IL-12. Specifically this study sought to evaluate the safety, serum pharmacokinetics, anti-tumor activity, and immune modulation of NHS-IL12 in dogs with naturally occurring cancers.Methodology/Principal FindingsA rapid dose-escalation study of NHS-IL12 administered subcutaneously to dogs with melanoma was conducted through the Comparative Oncology Trials Consortium (COTC). Eleven dogs were enrolled in four dose-escalation cohorts; thereafter, an additional seven dogs were treated at the defined tolerable dose of 0.8 mg/m2. The expanded cohort at this fixed dose (ten dogs in total) was accrued for further pharmacokinetics and pharmacodynamics assessment. NHS-IL12 levels, serum cytokine concentrations, and peripheral blood mononuclear cell characterization (post-treatment) and draining lymph node immune profiling, and tumor biopsies (pre- and post-treatment) were collected. Adverse events included thrombocytopenia, liver enzymopathies, fever, and vasculitis. Correlation between interferon (IFN)-γ induction, adverse events, and NHS-IL12 exposure (maximum concentration and area under the concentration-time curve) were dose-dependent. Serum IL-10 levels and intratumoral CD8+ populations increased after treatment. Partial responses, according to Response Evaluation Criteria in Solid Tumors (RECIST) criteria, were observed in two dogs treated with NHS-IL12 0.8 mg/m2 and 1.6 mg/m2.Conclusions/SignificanceNHS-IL12 was administered safely to dogs with melanoma and both immunologic and clinical activity was observed. This study successfully defined a narrow therapeutic window for systemic delivery of NHS-IL12 via the subcutaneous route. Results will inform the design and implementation of first-in-human clinical trials of NHS-IL12 in cancer patients.

413. Pre-Clinical Preparation and Validation of Tumor Cell-Based IL-12 Immunotherapy for Acute Myeloid Leukemia

Interleukin(IL)-12 is a potent pro-inflammatory cytokine that stimulates a variety of effector cells involved in anti-tumor immunity. Systemic administration of IL-12 has associated toxicities, however. Various strategies are being developed to reduce such toxicities by restricting IL-12 distribution. Options here include generating fusions with tumor-targeting molecules and directing gene delivery to specific cells. First – we used lentivirus vectors (LVs) to engineer expression of murine IL-12 in tumor cells ex vivo and subsequently infused such modified cells into recipient mice. This strategy restricts IL-12 to the local tumor microenvironment thereby promoting immune activation in the context of tumor-associated antigens (TAAs). Using mouse models of both leukemia and solid tumors, we found that this cell-based approach generated effective anti-tumor protection when as little as 1% of the tumor burden expressed IL-12 as long as threshold expression levels on a per cell basis were reached. Second – our groups showed that anti-tumor mechanisms here involve CD4+ killer T cells, dendritic cells, and direct cell-cell contact with effectors. Clinical translation of this cell-based IL-12 therapy is in progress in Toronto for AML using a novel LV to modify patients’ own blast cells. Patient AML cells collected to date (n = 21) were stratified based on in vivo growth kinetics and transduced with a near-GMP-grade bicistronic LV that encodes the human IL-12 cDNA as a p40-p70 fusion, as well as a mutant thymidylate kinase (tmpK) fused to the ectodomain of LNGFR (trLNGFR) as a suicide (cell-fate control) cassette. The trLNGFR/tmpK element allows selection and also selective ablation of transduced cells by administration of AZT. Furthermore, it also allows tracking of transduced cells and quantification of transduction frequencies/transgene expression levels. With our current protocol, functional transduction efficiencies of primary patient AML blasts ranged from 20% to 70% (n=17). Transduced AML cells displayed a strong correlation between vector copy number and trLNGFR/tmpK + IL-12 levels and dose-dependent sensitivity to AZT. In vitro immortalization (IVIM) assays determined that the near-GMP LV/IL-12 vector displayed minimal genotoxic risk in transduced lin- cells; insertion site analyses carried out on expanded clones displayed poly-to oligo-clonality patterns. Pre-clinical data on toxicity and scale-up considerations are being accumulated in preparation for a Clinical Trial Application to Health Canada targeting AML. This LV/IL-12 immunotherapy platform targeting tumor cells themselves thus holds potential to be effective against a wide variety of cancers.

Systemic Administration of a Novel Immune-Stimulatory Pseudovirion Suppresses Lung Metastatic Melanoma by Regionally Enhancing IFN-γ Production

Cancer immunotherapy has encountered many difficulties in the face of the expectation to eradicate cancer, and new breakthroughs are required. We have previously shown that UV-inactivated Sendai virus particles (hemagglutinating virus of Japan envelope; HVJ-E) induce immunity against multiple tumor types. In this study, a novel pseudovirion that stimulates more robust antitumor immunity was designed for cancer treatment. First, we found that culturing murine splenocytes with HVJ-E in combination with interleukin (IL)-12 resulted in a remarkable increase in IFN-γ production compared with that observed in splenocytes cultured with IL-12 alone. The synergistic effects of HVJ-E and IL-12 on IFN-γ production were caused by viral F proteins independently of HVJ-E fusion activity and not by hemagglutination from hemagglutinin-neuraminidase (HN) proteins. We next constructed HN-depleted HVJ-E expressing the Fc region of immunoglobulin G (IgG) on the envelope and single-chain IL-12 containing the ZZ domain of protein A to produce an IL-12-conjugated HVJ-E particle without hemagglutinating activity. IL-12-conjugated HVJ-E dramatically enhanced the amount of IFN-γ produced by immune cells. Intratumoral injection of IL-12-conjugated HVJ-E eradicated murine melanomas more effectively than injection of wild-type HVJ-E through increased production of melanoma-specific CTLs. IL-12-conjugated HVJ-E preferentially accumulated in the lungs after systemic administration. When small metastatic melanoma foci were formed in the lungs, systemic administration of IL-12-conjugated HVJ-E significantly reduced the number of metastatic foci by inducing local production of IFN-γ in the lungs and generating large numbers of melanoma-specific CTLs. IL-12-conjugated HVJ-E is a promising tool for the treatment of cancers, including lung metastasis.

First-in-human phase I trial of NHS-IL12 in advanced solid tumors.

TPS2617 Background: NHS-IL12 is an antibody-cytokine conjugate consisting of two heterodimers of interleukin (IL)-12, fused to a human monoclonal antibody specific for DNA-histone H1 complex exposed in tumor necrosis. IL12 is a proinflammatory cytokine, produced by activated phagocytes and dendritic cells, that plays a critical role in regulating the transition from innate to adaptive immunity. Early clinical trials of IL12 reported clinical responses in metastatic renal cell carcinoma, Kaposi sarcoma (50–71% response rate), T-cell lymphoma (56%), and non-Hodgkin’s lymphoma (21%). However, systemic toxicity limited further clinical development. NHS-IL12 is designed to reduce toxicity associated with systemic administration of IL12 by selectively targeting delivery to necrotic areas of tumors. A fluorescence imaging study of NHS-murine (mu)-IL12 in mice with syngeneic lung carcinoma demonstrated effective targeting by NHS-muIL12. Immunohistochemistry studies further confirmed effective targeting of tumors by NHS-muIL12. In another study in canines with spontaneously occurring solid tumors, 2 of 11 subjects achieved a partial response with a single dose. Methods: This is a phase I dose-escalation study designed to determine the maximum tolerated dose of NHS-IL12 and to define the biologically optimal dose and schedule based on immunologic (pharmacodynamic) analysis of cytokines such as interferon-g, and IL-10, at multiple times points from 4 hours up to 2 weeks following the administration of NHS-IL12. Eligible patients (pts) include those with evaluable or measurable solid tumors who have progressive disease on at least one prior line of therapy. Pts with a condition associated with significant necrosis of non-tumor bearing tissue, (e.g., ulcerative colitis), are excluded. Modified immune-related response criteria are used to assess response, the major features of these criteria being (a) imaging confirmation of both progression and response at least 4 weeks after initial imaging, and (b) no consideration of new lesions unless the total measurable tumor burden meets the criteria for progressive disease. This trial opened to accrual in Nov. 2011 and may enroll up to 78 pts.

IL-12p35 Subunit Contributes to Autoimmunity by Limiting IL-27–Driven Regulatory Responses

Contrasting results have emerged from studies performed using IL-12p35(-/-) mice. Animals lacking the IL-12p35 subunit can either be protected from or develop exacerbated autoimmune diseases, intracellular infections, and delayed-type hypersensitivity responses. In this study, we report that mice lacking the IL-12p35 subunit develop a significantly milder Ag-induced arthritis compared with wild-type (WT) mice. Lack of severe inflammation is accompanied by an increase in the mRNA levels of the Ebi-3 and p28 subunits and increased secretion of IL-27 and IL-10. This anti-inflammatory environment contributed to increased differentiation of regulatory T and B cells with intact suppressive function. Furthermore, IL-12p35(-/-) mice display reduced numbers of Th17 cells compared with WT arthritic mice. Neutralization of IL-27, but not the systemic administration of IL-12, restored inflammation and Th17 to levels seen in WT mice. The restoration of disease phenotype after anti-IL-27 administration indicates that the IL-12p35 subunit acts as negative regulator of the developing IL-27 response in this model of arthritis.

Enhanced paradoxical type 2 responses generated by interleukin-12 in patients with gastric and colorectal cancer

2599 Background: Interleukin-12 has been reported to induce type 1 helper T cells (Th1) that produce interleukin (IL)-2 and gamma-interferon (G-IFN), and recognized as one of the strongest immunotherapeutic agent. However, it was found that serum levels of type 2 cytokine such as IL-10 increased after systemic administration of IL-12. Methods: To study this paradoxical phenomenon, peripheral blood mononuclear cells (PBMC) were used collected from 7 healthy volunteer (HV) and 45 patients with gastric and colorectal cancer, and were used for this study. These PBMC were stimulated with IL-12 and the concentrations of E°-IFN, IL-6, IL-10 in the culture supernatant were measured as production of each cytokine after cultivation for 24 hours. Results: The production of IL-6 and IL-10 in patients were significantly higher than in HV. Those of IL-6 by patients with distant metastasis were higher than those by HV. Those of IL-10 were significantly increased in patients with nodal involvement and with distant metast...

Enhanced paradoxical type 2 responses generated by interleukin-12 in patients with gastric and colorectal cancer

2599 Background: Interleukin-12 has been reported to induce type 1 helper T cells (Th1) that produce interleukin (IL)-2 and gamma-interferon (G-IFN), and recognized as one of the strongest immunotherapeutic agent. However, it was found that serum levels of type 2 cytokine such as IL-10 increased after systemic administration of IL-12. Methods: To study this paradoxical phenomenon, peripheral blood mononuclear cells (PBMC) were used collected from 7 healthy volunteer (HV) and 45 patients with gastric and colorectal cancer, and were used for this study. These PBMC were stimulated with IL-12 and the concentrations of É°-IFN, IL-6, IL-10 in the culture supernatant were measured as production of each cytokine after cultivation for 24 hours. Results: The production of IL-6 and IL-10 in patients were significantly higher than in HV. Those of IL-6 by patients with distant metastasis were higher than those by HV. Those of IL-10 were significantly increased in patients with nodal involvement and with distant metastasis than in HV and patients without any metastasis, and these findings were clearer in gastric cancer. Those of IL-6 and IL-10 were significantly correlated each other. Those of G-IFN was increased in patients with nodal involvement and with distant metastasis. Conclusions: Thus IL-12 appeared to induce type 2 responses in patients with cancer and this phenomena may interfere an activation of cell-mediated immunity and may cause an unsuccessful immunotherapy against cancer. A new approach to alter this phenomena is required in order to realize an effective immunotherapy for malignant diseases. No significant financial relationships to disclose.

Induction of an antitumor immunological response by an intratumoral injection of dendritic cells pulsed with genetically engineered Semliki Forest virus to produce interleukin-18 combined with the systemic administration of interleukin-12.

The aim of this study was to investigate further immunogene treatment of malignant brain tumor to improve its therapeutic efficacy. Intratumoral dendritic cells pulsed with Semliki Forest virus (SFV)-interleukin-18 (IL-18) and/or systemic IL-12 were injected into mice bearing the B16 brain tumor. To study the immune mechanisms involved in tumor regression, we monitored the growth of implanted B16 brain tumor cells in T cell-depleted mice and IFNgamma-neutralized mice. To analyze the protective immunity created by tumor inoculation, B16 cells were injected into the left thighs of mice that had received an inoculation, and tumor growth was monitored. The local delivery of dendritic cells pulsed with IL-18 bound by SFV combined with the systemic administration of IL-12 enhanced the induction of the T helper type 1 response from tumor-specific CD4+ and CD8+ T cells and natural killer cells as well as antitumor immunity. Interferon-gamma is partly responsible for this IL-18-mediated antitumor immunity. Furthermore, the protective immunity is mediated mainly by CD8+ T cells. Immunogene therapy that combines the local administration of dendritic cells pulsed with IL-18 bound by SFV and the systemic administration of IL-12 may be an excellent candidate for the development of a new treatment protocol. A self-replicating SFV system may therefore open a novel approach for the treatment of malignant brain tumor.

Immunological prevention of spontaneous tumors: a new prospect?

Recent demonstrations of the specific immune prevention of mammary cancer in female BALB/c mice transgenic for the rat Her-2/neu oncogene (BALB-neuT) have resulted in reconsideration of the immune mechanisms that inhibit tumor growth. All the mammary glands of these mice progress asynchronously, but consistently, from hyperplasia to invasive carcinoma. Overexpression of the oncogene product p185 neu is first evident in the rudimentary glands of 3-week-old mice. Carcinogenesis is prevented by vaccination with plasmids coding for the extracellular and transmembrane domains of this p185 neu, or with allogeneic cells expressing p185 neu on their membrane, plus the systemic administration of IL-12. This inhibition is the outcome of a delayed-type hypersensitivity specific for p185 neu and the production of anti-p185 neu antibodies that restrain the proliferation of tumor cells by stripping p185 neu from their membrane, whereas cytotoxic T lymphocytes seem devoid of a major role.

IFN-γ and Fas/FasL are required for the antitumor and antiangiogenic effects of IL-12/pulse IL-2 therapy

Systemic administration of IL-12 and intermittent doses of IL-2 induce complete regression of metastatic murine renal carcinoma. Here, we show that overt tumor regression induced by IL-12/pulse IL-2 is preceded by recruitment of CD8+ T cells, vascular injury, disrupted tumor neovascularization, and apoptosis of both endothelial and tumor cells. The IL-12/IL-2 combination synergistically enhances cell surface FasL expression on CD8+ T lymphocytes in vitro and induces Fas and FasL expression within tumors via an IFN-γ–dependent mechanism in vivo. This therapy also inhibits tumor neovascularization and induces tumor regression by mechanisms that depend critically on endogenous IFN-γ production and an intact Fas/FasL pathway. The ability of IL-12/pulse IL-2 to induce rapid destruction of tumor-associated endothelial cells and regression of established metastatic tumors is ablated in mice with a dysregulated Fas/FasL pathway. The common, critical role for endogenous IFN-γ and the Fas/FasL pathway in early antiangiogenic effects and in antitumor responses suggests that early, cytokine-driven innate immune mechanisms and CD8+ T cell–mediated responses are interdependent. Definition of critical early molecular events engaged by IL-12/IL-2 may provide new perspective into optimal therapeutic engagement of a productive host-antitumor immune response.

IFN-gamma and Fas/FasL are required for the antitumor and antiangiogenic effects of IL-12/pulse IL-2 therapy.

Systemic administration of IL-12 and intermittent doses of IL-2 induce complete regression of metastatic murine renal carcinoma. Here, we show that overt tumor regression induced by IL-12/pulse IL-2 is preceded by recruitment of CD8(+) T cells, vascular injury, disrupted tumor neovascularization, and apoptosis of both endothelial and tumor cells. The IL-12/IL-2 combination synergistically enhances cell surface FasL expression on CD8(+) T lymphocytes in vitro and induces Fas and FasL expression within tumors via an IFN-gamma-dependent mechanism in vivo. This therapy also inhibits tumor neovascularization and induces tumor regression by mechanisms that depend critically on endogenous IFN-gamma production and an intact Fas/FasL pathway. The ability of IL-12/pulse IL-2 to induce rapid destruction of tumor-associated endothelial cells and regression of established metastatic tumors is ablated in mice with a dysregulated Fas/FasL pathway. The common, critical role for endogenous IFN-gamma and the Fas/FasL pathway in early antiangiogenic effects and in antitumor responses suggests that early, cytokine-driven innate immune mechanisms and CD8(+) T cell-mediated responses are interdependent. Definition of critical early molecular events engaged by IL-12/IL-2 may provide new perspective into optimal therapeutic engagement of a productive host-antitumor immune response.

Cytokine gene therapy of mesothelioma. Immune and antitumor effects of transfected interleukin-12.

Malignant mesothelioma (MM) is a solid tumor of the mesothelium for which there is no curative treatment. MM appears to be sensitive to immunotherapeutic approaches, and one of the most powerful immunomodulatory cytokines with antitumor effects is interleukin (IL)-12. We have previously shown in a murine model of MM that systemic administration of recombinant IL-12 induces a potent anti-MM immune response. The nature and accessibility of MM tumors means that they are suitable candidates for direct cytokine and gene-transfer therapeutic approaches. Therefore, we undertook a study to assess the antitumor effects induced by the local production of IL-12 within MM tumors by transfecting a murine MM line with the genes for IL-12. The IL-12 transfectant (AB1-IL-12) did not produce tumors in normal mice, but did so in athymic nude mice, implicating T cells in the prevention of MM tumor growth. In mixing experiments, paracrine IL-12 production inhibited growth of untransfected MM cells provided that cells producing IL-12 represented more than 50-80% of the inoculum. Furthermore, BALB/c mice previously challenged with AB1-IL-12 were protected against rechallenge with parental AB1 tumor, indicating that the transfectant induced long-term immunity. AB1-IL-12 induced systemic immunity that was effective at reducing the incidence of parental AB1 tumor at a distal site, but its effects were dose-dependent. Though both CD4(+) and CD8(+) cells infiltrated the rejecting tumor, CD8(+) effector cells were essential for protection against development of parental AB1 tumor. This study shows that paracrine secretion of IL-12, generated by gene transfer, can induce immunity against MM that can act locally and also at a distant site. In addition, there was no evidence of toxicity, which has been associated with the systemic administration of IL-12, indicating that this cytokine is a good candidate for experimental gene therapy in MM.

A Single-Chain IL-12 IgG3 Antibody Fusion Protein Retains Antibody Specificity and IL-12 Bioactivity and Demonstrates Antitumor Activity

IL-12 is a heterodimeric cytokine with many actions on innate and cellular immunity that may have antitumor and antimetastatic effects. However, systemic administration of IL-12 can be toxic. Tumor-specific Abs provide a means to selectively target a metastatic/residual nodule and deliver therapeutic quantities of an immunostimulatory molecule like IL-12 with lower systemic levels and ideally, toxicity. We report the construction and characterization of an Ab fusion protein in which single-chain murine IL-12 is fused to an anti-Her2/neu Ab at the amino terminus (mscIL-12.her2.IgG3). The use of single-chain IL-12 in the fusion protein simplifies vector construction, ensures equimolar concentrations of the two IL-12 subunits, and may confer greater stability to the fusion protein. SDS-PAGE analysis shows this 320-kDa protein is secreted and correctly assembled. FACS analysis demonstrates that this fusion protein binds to cells transfected with the Her2/neu Ag, thus retaining Ab specificity; this fusion protein also binds to a cell line and to PHA-activated PBMC that express the IL-12R, thus demonstrating cytokine receptor specificity. T cell proliferation assays and NK cytotoxicity assays demonstrate that this fusion protein exhibits IL-12 bioactivity comparable to recombinant murine IL-12. In vivo studies demonstrate that this fusion protein has antitumor activity. These results are significant and suggest that this IL-12 Ab fusion protein can effectively combine the therapeutic potential of IL-12 with the tumor-targeting ability of the Ab and may provide a viable alternative to systemic administration of IL-12.

Potent antitumor effects mediated by local expression of the mature form of the interferon-gamma inducing factor, interleukin-18 (IL-18).

IL-18 is produced during the acute immune response by macrophages and immature dendritic cells. IL-18 receptors are induced on T cells and NK cells by IL-12 and together they enhance a cellular immune response. We constructed retroviral and adenoviral vectors encoding the mature bioactive murine IL-18 in order to examine their immune and antitumor effects in murine tumor models. Secretion of bioactive IL-18 from murine tumor cells was facilitated by transfecting them with recombinant viral vectors carrying the prepro leader sequence of human parathyroid hormone fused to the 5' end of the mature murine IL-18 cDNA. Direct injection of the IL-18 recombinant adenoviral vector (Ad.PTH.IL-18) into an established MCA205 murine fibrosarcoma completely eradicated tumor in all animals with concomitant induction of protective systemic immunity. Co-administration of systemic IL-12 provided synergistic antitumor effects when combined with peritumoral injections of Ad.PTH.IL-18 without apparent side-effects as we observed with systemic administration of IL-18. Depletion of asialo GM-1+ cells completely abrogated the antitumor effects of Ad.PTH.IL-18, suggesting a major role for NK cells in mediating the anti-tumor effects of IL-18. Peritumoral injection of Ad.PTH.IL-18 was also associated with reduced numbers of CD8+ cells found within the tumor (HBSS versus Ad.PTH.IL-18, P < 0.0001). This suggests that IL-18 could be utilized as an alternative cancer gene therapy especially when combined with systemic administration of IL-12.

Local treatment with IL-12 is an effective inhibitor of airway hyperresponsiveness and lung eosinophilia after airway challenge in sensitized mice

Background: Systemic administration of IL-12 can prevent airway hyperresponsiveness (AHR) in mice after sensitization and repeated allergen challenge. However, systemic IL-12 has been associated with severe adverse effects. Objective: We determined whether IL-12 administration to the airways in a dose sufficiently low so as not to result in systemic effects can modify allergic inflammation and AHR after allergen challenge. Methods: Mice were sensitized to ovalbumin by intraperitoneal injection and challenged with ovalbumin aerosol on 3 consecutive days. During the period of challenge, IL-12 was administered intranasally following 2 regimens, designated high (500 ng) or low (50 ng). We monitored airway responsiveness to inhaled methacholine by barometric body plethysmography, lung inflammatory cells, local cytokine production, and, to assess systemic effects of IL-12 treatment, spleen weights and numbers of eosinophils in the bone marrow. Results: Allergen challenge resulted in increases in airway responsiveness and in numbers of lung eosinophils. These increases were prevented by both high- and low-dose IL-12. Additionally, IL-12 administration resulted in enhanced local interferon-γ production and prevented the increases in local IL-4 and IL-5 production after airway challenge. A high dose, but not a low dose, of IL-l2 resulted in increased spleen weights and prevented the increase in numbers of bone marrow eosinophils after allergen challenge. Conclusion: These data indicate that local administration of IL-12 can prevent AHR and reduce lung eosinophilia after allergen challenge in sensitized mice without eliciting systemic adverse effects. IL-12 exerts these effects by inducing local T H1-type responses in the airways in a setting that is normally dominated by T H2-type responses. (J Allergy Clin Immunol 1998;102:86-93.)