Interleukin-12 Gene Therapy Research Articles

IL-12 and PD-1 peptide combination gene therapy for the treatment of melanoma

Interleukin-12 (IL-12) gene electrotransfer (GET) delivery is highly effective in inducing long-term, complete regression in mouse and human melanoma and other solid tumors. Therapeutic efficacy is enhanced by immune checkpoint inhibitors, and the combination of IL-12 plasmid GET (pIL-12 GET) and anti-programmed cell death protein 1 (PD-1) monoclonal antibodies has reached clinical trials. In this study, we designed peptides and plasmids encoding the mouse homologs of the pembrolizumab and nivolumab programmed cell death 1 ligand 1 (PD-L1) binding regions. We hypothesized that intratumor autocrine/paracrine peptide expression would block PD-1/PD-L1 binding and provide cancer patients with an effective and cost-efficient treatment alternative. We demonstrated that the mouse homolog to pembrolizumab was effective at blocking PD-1/PD-L1 invitro. After intratumor plasmid delivery, both peptides bound PD-L1 on tumor cells. We established that plasmid DNA delivery to tumors invivo or to tumor cells invitro upregulated several immune modulators and PD-L1 mRNA and protein, potentiating this therapy. Finally, we tested the combination of pIL-12 GET therapy and peptide plasmids. We determined that pIL-12 GET therapeutic efficacy could be enhanced by combination with the plasmid encoding the pembrolizumab mouse homolog.

Long-Term Follow-Up of Phase I Trial of Oncolytic Adenovirus-Mediated Cytotoxic and Interleukin-12 Gene Therapy for Treatment of Metastatic Pancreatic Cancer.

The long-term follow-up findings of the phase I trial evaluating the efficacy of oncolytic adenovirus-mediated cytotoxic and interleukin-12 gene therapy in metastatic pancreatic cancer (mPC) seem very promising. The study employed a replication-competent Adenovector in combination with chemotherapy in a dose-escalation format. The trial demonstrated a clinically meaningful median overall survival (OS) benefit, with patients in the highest dose cohort exhibiting an impressive median OS of 18.4 months. This contrasts starkly with patients receiving lower doses who experienced a median OS of 4.8 and 3.5 months, respectively. Remarkably, subject number 10, who received the highest dose, demonstrated an extraordinary survival of 59.1 months, presenting a compelling case for further exploration. Additionally, this patient displayed complete responses in lung and liver metastases, a rare occurrence in mPC treatment. Statistical analyses supported the observed survival benefit. The unprecedented OS results emphasize the potential of this treatment strategy and pave the way for future investigations into this promising gene therapy approach.

Riboswitch-controlled IL-12 gene therapy reduces hepatocellular cancer in mice.

Hepatocellular carcinoma (HCC) and solid cancers with liver metastases are indications with high unmet medical need. Interleukin-12 (IL-12) is a proinflammatory cytokine with substantial anti-tumor properties, but its therapeutic potential has not been realized due to severe toxicity. Here, we show that orthotopic liver tumors in mice can be treated by targeting hepatocytes via systemic delivery of adeno-associated virus (AAV) vectors carrying the murine IL-12 gene. Controlled cytokine production was achieved in vivo by using the tetracycline-inducible K19 riboswitch. AAV-mediated expression of IL-12 led to STAT4 phosphorylation, interferon-γ (IFNγ) production, infiltration of T cells and, ultimately, tumor regression. By detailed analyses of efficacy and tolerability in healthy and tumor-bearing animals, we could define a safe and efficacious vector dose. As a potential clinical candidate, we characterized vectors carrying the human IL-12 (huIL-12) gene. In mice, bioactive human IL-12 was expressed in a vector dose-dependent manner and could be induced by tetracycline, suggesting tissue-specific AAV vectors with riboswitch-controlled expression of highly potent proinflammatory cytokines as an attractive approach for vector-based cancer immunotherapy.

Abstract CT196: A phase I clinical trial of oncolytic adenovirus mediated suicide and interleukin-12 gene therapy in patients with recurrent prostate cancer

Abstract Introduction: Men with locally recurrent prostate cancer, after definitive radiotherapy, have few therapeutic options. Oncolytic adenovirus-mediated cytotoxic gene therapy is an investigational cancer therapy. Delivery of the suicide gene to the tumor is by direct intratumoral or systemic injection of a viral vector containing the suicide gene. Our approach incorporates yeast cytosine deaminase (yCD) and herpes simplex virus thymidine kinase (HSV-1 TK), to confer sensitivity to 5-fluorocytosine (5-FC) and Valganciclovir (vGCV), respectively. The pro-drugs are converted into active drugs that inhibit DNA damage repair. Here we report the safety of oncolytic adenovirus-mediated suicide gene therapy that incorporates an interleukin-12 (IL12) gene for treatment of recurrent prostate cancer. Methods: In this phase I study, a replication-competent adenovirus (Ad5-yCD/mutTKSR39rep-hIL-12) expressing yCD/mutTKSR39 (yeast cytidine deaminase/mutant S39R HSV-1 thymidine kinase) and human IL-12 (IL12) was injected into tumors of 15 subjects with recurrent prostate cancer (T1c-T2) at escalating doses (1 × 1010, 3 × 1010, 1 × 1011, 3 × 1011, or 1 × 1012 viral particles). Subjects received 5-FC and vGCV for 7 days. The study endpoint was toxicity through day 30. Experimental endpoints included measurements of serum IL12, interferon gamma (IFNγ), and CXCL10 to assess immune system activation. Peripheral blood mononuclear cells (PBMC) and proliferation markers were analyzed by flow cytometry. Results and Conclusions: Fifteen patients received Ad5-yCD/mutTKSR39rep-hIL-12 and oral 5-FC and vGCV. Approximately 92% of the 115 adverse events observed were grade 1/2 requiring no medical intervention. Ad5-yCD/mutTKSR39rep-hIL-12 DNA was detected in the blood of only two patients. Elevated serum IL12, IFNγ, and CXCL10 levels were detected in 57%, 93%, and 79% of subjects, respectively. Serum cytokines demonstrated viral dose dependency, especially apparent in the highest-dose cohorts. Analysis of immune cell populations indicated activation after Ad5-yCD/mutTKSR39rep-hIL-12 administration in cohort 5. The study did not detect a significant difference in the PSA doubling time (PSADT) between pre and post treatment by paired Wilcoxon rank test (p=0.17). There was no correlation between adenoviral dose and PSADT in each cohort separately or pooled (cohorts1-3 and cohorts 4-5). The study maximum tolerated dose (MTD) was not reached indicating 1012 viral particles was safe. This trial confirmed that replication-competent Ad5-IL-12 adenovirus (Ad5-yCD/mutTKSR39 rep-hIL-12) was well tolerated when administered locally to prostate tumors. Citation Format: Shyam Nyati, Hans Stricker, Kenneth L. Barton, Piln Li, Mohamed Elshaikh, Haythem Ali, Stephen L. Brown, Clara Hwang, James Peabody, Svend O. Freytag, Benjamin Movsas, Farzan Siddiqui. A phase I clinical trial of oncolytic adenovirus mediated suicide and interleukin-12 gene therapy in patients with recurrent prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT196.

Localized Nano-mediated Interleukin-12 Gene Therapy: Promising Candidate for Cancer Immunotherapeutics.

Interleukin-12 (IL-12) has a pleiotropic nature that allows it to induce immune responses while reversing tumour-induced immunosuppression. Therefore, this paper discusses the application and potential of IL-12 as an antitumor immunotherapeutic agent, emphasizing its advantages and limitations and the need for and the development of localized IL-12 nano-delivery strategies in cancer immunotherapy. Several databases from the National Centre for Biotechnology Information, WorldCat.org and the National Library of Medicine were searched for peer-reviewed studies to assess the potential of localized nano-mediated interleukin-12 gene therapy for cancer treatment. The literature search showed that IL-12 is a promising cancer immunotherapeutic agent. However, the systemic delivery of IL-12 was compromised by severe dose-limiting side effects, prompting the need for localized gene therapy to express the interleukin within the tumour microenvironment while minimizing systematic exposure. Although viral and non-viral gene therapy have demonstrated some efficacy in preclinical trials, the era of nanomedicine has opened novel avenues to improve therapeutic indices with minimal side effects. IL-12 activity can be further potentiated with other anticancer molecules that display immunostimulatory, autoantigenic and cytotoxic properties. Combination therapy has gained significant interest in the last decade as it increases gene therapy's therapeutic properties by decreasing the threshold for IL-12 efficacy and preventing systematic toxicity. The findings of this article will provide researchers with the knowledge to create immunotherapeutic nanovectors which work synergistically with their therapeutic payload to enhance the therapeutic effect of the IL-12 gene to eliminate cancer cells.

Integrating disulfides into a polyethylenimine gene carrier selectively boosts significant transfection activity in lung tissue enabling robust IL-12 gene therapy against metastatic lung cancers

Bioreducible polyethylenimines (SSPEIs) are promising non-viral carriers for cancer gene therapy. However, the availability of significant gene transfection activity by SSPEIs remains a challenge. Herein, an essential step was taken to ascertain whether or not the disulfide bonds of SSPEIs play a critical role in promoting significant gene transfection activity in different tissues. Initially, a disulfide-linked linear polyethylenimine (denoted as SSLPEI) consisting of one 5.0 kDa LPEI main chain and three disulfide-linked 5.7 kDa LPEI grafts was designed and prepared to possess similar molecular weight with commercialized 25 kDa LPEI as a positive control. The SSLPEI could induce superior in vitro transfection activity in different cells to the LPEI control as well as low cytotoxicity. Notably, such enhanced in vitro transfection effect by the SSLPEI was more marked in type-II alveolar epithelial cells compared to different cancer cells. In a Balb/c nude mouse model bearing SKOV-3 tumor, the SSLPEI caused parallel level of transgene expression with the LPEI control in the tumor but significantly higher level in the mouse lung. Furthermore, the SSLPEI and LPEI groups afforded an identical antitumor efficacy against the SKOV-3 tumor via intravenous delivery of a shRNA for silencing VEGF expression in the tumor. However, via intravenous delivery of an interleukin-12 (IL-12) gene into metastatic lung cancers in a C57BL/6 mouse model, the SSLPEI group exerted markedly higher IL-12 expression level in the mouse lung and peripheral blood as compared to the LPEI group, thereby boosting IL-12 immunotherapy against the lung metastasis with longer medium survival time. The results of this work elicit that the disulfide bonds of SSPEIs play a pivotal role in enhancing gene transfection activity selectively in the lung tissue rather than solid tumor, enabling high translational potential of SSPEIs for non-viral gene therapy against metastatic lung cancers.

Phase I trial of oncolytic adenovirus-mediated cytotoxic and interleukin-12 gene therapy for the treatment of metastatic pancreatic cancer

The safety of oncolytic adenovirus-mediated suicide and interleukin-12 (IL12) gene therapy was evaluated in metastatic pancreatic cancer patients. In this phase I study, a replication-competent adenovirus (Ad5-yCD/mutTKSR39rep-hIL-12) expressing yCD/mutTKSR39 (yeast cytidine deaminase/mutant S39R HSV-1 thymidine kinase) and human IL-12 (IL12) was injected into tumors of 12 subjects with metastatic pancreatic cancer (T2N0M1-T4N1M1) at escalating doses (1 × 1011, 3 × 1011, or 1 × 1012 viral particles). Subjects received 5-fluorocytosine (5-FC) therapy for 7 days followed by chemotherapy (FOLFIRINOX or gemcitabine/albumin-bound paclitaxel) starting 21 days after adenovirus injection. The study endpoint was toxicity through day 21. Experimental endpoints included measurements of serum IL12, interferon gamma (IFNG), and CXCL10 to assess immune system activation. Peripheral blood mononuclear cells and proliferation markers were analyzed by flow cytometry. Twelve patients received Ad5-yCD/mutTKSR39rep-hIL-12 and oral 5-FC. Approximately 94% of the 121 adverse events observed were grade 1/2 requiring no medical intervention. Ad5-yCD/mutTKSR39rep-hIL-12 DNA was detected in the blood of two patients. Elevated serum IL12, IFNG, and CXCL10 levels were detected in 42%, 75%, and 92% of subjects, respectively. Analysis of immune cell populations indicated activation after Ad5-yCD/mutTKSR39rep-hIL-12 administration. The median survival of patients in the third cohort is 18.1 (range, 3.5–20.0) months. The study maximum tolerated dose (MTD) was not reached.

CTIM-05. COMBINATION OF CONTROLLED INTERLEUKIN-12 GENE THERAPY WITH IMMUNE CHECKPOINT BLOCKADE IN RECURRENT GLIOBLASTOMA: UPDATED RESULTS OF A MULTI-INSTITUTIONAL, OPEN LABEL PHASE 1 TRIAL

Abstract A published clinical trial of veledimex (V)-regulatable interleukin-12 (IL-12) gene therapy (“Controlled IL-12”) under the control of a transcriptional switch (RheoSwitch Therapeutic Systemâ, RTSâ) as monotherapy in recurrent glioblastoma (rGBM) showed sustained infiltration of activated T cells within the tumor months after treatment (Sci Transl Med. 2019;11(505)). These T cells demonstrated up-regulation of immune checkpoint signaling, providing a rationale for combination therapy with the PD-1 inhibitor, nivolumab (nivo). We report interim findings following completion of enrollment (with follow-up ongoing) for a multi-institutional, open label, dose-escalation phase 1 trial (NCT03636477) evaluating safety and tolerability of loco-regional Controlled IL-12 in combination with nivo in adults with rGBM. Replication-incompetent adenovirus coding for RTS-IL-12 (Ad) was administered during surgery by free-hand injection into the tumor and periphery (2 x 1011 viral particles, Day 0) accompanied by V (10 or 20 mg) PO QD x 15 (Days 0 to 14) in combination with nivo (1 or 3 mg/kg) IV on Days -7, 15, then Q2W. Twenty-one subjects were treated (Cohort 1: V 10 mg, nivo 1 mg/kg, n=3; Cohort 2: V 10 mg, nivo 3 mg/kg, n=3; and Cohort 3: V 20 mg, nivo 3 mg/kg, n=3 & 12 in expansion). Safety data were comparable to Ad+V monotherapy. Adverse reactions (ARs) during follow-on nivo dosing were consistent with nivo labeling. ARs were manageable and generally reversible with no synergistic toxicities. Updated overall survival findings will be presented. Baseline and post-treatment histochemical staining and multiplex immunofluorescence analyses for a subgroup of subjects will be discussed. The safety of this combination immunotherapy has been established, leading to a currently accruing phase 2 clinical trial of loco-regional Controlled IL-12 gene therapy in combination with the PD-1 inhibitor cemiplimab-rwlc (NCT 04006119).

Pre-treatment with IL2 gene therapy alleviates Staphylococcus aureus arthritis in mice

BackgroundStaphylococcus aureus (S. aureus) arthritis is one of the most detrimental joint diseases known and leads to severe joint destruction within days. We hypothesized that the provision of auxiliary immunoregulation via an expanded compartment of T regulatory cells (Tregs) could dampen detrimental aspects of the host immune response whilst preserving its protective nature. Administration of low-dose interleukin 2 (IL2) preferentially expands Tregs, and is being studied as a treatment choice in several autoimmune conditions. We aimed to evaluate the role of IL2 and Tregs in septic arthritis using a well-established mouse model of haematogenously spred S. aureus arthritis.MethodsC57BL/6 or NMRI mice we intravenously (iv) injected with a defined dose of S. aureus LS-1 or Newman and the role of IL2 and Tregs were assessed by the following approaches: IL2 was endogenously delivered by intraperitoneal injection of a recombinant adeno-associated virus vector (rAAV) before iv S. aureus inoculation; Tregs were depleted before and during S. aureus arthritis using antiCD25 antibodies; Tregs were adoptively transferred before induction of S. aureus arthritis and finally, recombinant IL2 was used as a treatment starting day 3 after S. aureus injection. Studied outcomes included survival, weight change, bacterial clearance, and joint damage.ResultsExpansion of Tregs induced by IL2 gene therapy prior to disease onset does not compromise host resistance to S. aureus infection, as the increased proportions of Tregs reduced the arthritis severity as well as the systemic inflammatory response, while simultaneously preserving the host’s ability to clear the infection.ConclusionsPre-treatment with IL2 gene therapy dampens detrimental immune responses but preserves appropriate host defense, which alleviates S. aureus septic arthritis in a mouse model.

TAC Chemotherapy and Pembrolizumab Plus Interleukin-12 Gene Therapy and L-NMMA in Triple Negative Breast Cancer

TAC Chemotherapy and Pembrolizumab Plus Interleukin-12 Gene Therapy and L-NMMA in Triple Negative Breast Cancer

Gene therapy based on interleukin-12 loaded chitosan nanoparticles in a mouse model of fibrosarcoma.

Interleukin-12 (IL-12) as a cytokine has been proved to have a critical role in stimulating the immune system and has been used as immunotherapeutic agents in cancer gene therapy. Chitosan as a polymer, with high ability of binding to nucleic acids is a good candidate for gene delivery since it is biodegradable, biocompatible and non-allergenic polysaccharide. The objective of the present study was to investigate the effects of cells transfected with IL-12 loaded chitosan nanoparticles on the regression of fibrosarcoma tumor cells (WEHI-164) in vivo. WEHI-164 tumor cells were transfected with IL-12 loaded chitosan nanoparticles and then were injected subcutaneously to inoculate tumor in BALB/c mice. Tumor volumes were determined and subsequently extracted after mice sacrifice. The immunohistochemistry staining was performed for analysis of Ki-67 expression (a tumor proliferation marker) in tumor masses. The expression of IL-12 and IFN-γ were studied using real-time polymerase chain reaction and immunoblotting. The group treated with IL-12 loaded chitosan nanoparticles indicated decreasing of tumor mass[r1] volume (P<0.001). The results of western blotting and real-time PCR showed that the IL-12 expression was increased in the group. Immunohistochemistry staining indicated that the Ki-67expression was reduced in the group treated with IL-12 loaded chitosan nanoparticles. IL-12 gene therapy using chitosan nanoparticles has therapeutic effects on the regression of tumor masses in fibrosarcoma mouse model.

Abstract 2078: Interleukin-12 gene therapy in combination with bevacizumab and pegylated liposomal doxorubicin for treatment of disseminated ovarian cancer

Abstract Despite recent improvements in treatment options for ovarian cancer patients, notably, the approval of using bevacizumab in combination with chemotherapies including pegylated liposomal doxorubicin (PLD), this disease is still the most deadly of all gynecological malignancies requiring new and novel therapeutics. Interleukin-12 (IL-12) is a highly active cytokine that can induce a potent anti-cancer immunity mediated through activation of cytotoxic T-lymphocytes, natural killer cell proliferation, and secretion of interferon-γ. We are developing an IL-12 based gene therapy for the treatment of gynecological malignancies that have spread into the peritoneal cavity. Our approach utilizes IL-12 plasmid (pIL-12) formulated with the PPC delivery system, which is comprised of a low molecular weight polyethylenimine covalently linked to polyethyleneglycol and cholesterol. Previously we have shown in a mouse model of disseminated ovarian cancer efficacy of a treatment regimen of pIL-12/PPC used in combination with paclitaxel and carboplatin. The combination treatment significantly improved survival compared to either pIL-12/PPC alone or chemotherapy alone and demonstrated the feasibility of using an IL-12 immunotherapy in combination with cytotoxic chemotherapies to achieve additive therapeutic effects. Results from a Phase I clinical trial in platinum resistant patients have shown that intraperitoneal delivery of pIL-12/PPC in combination with PLD produced an overall clinical benefit of 57.1% (PR = 21.4%; SD = 35.7%) in patients with measurable disease. The highest percentage of PRs were found at the highest dose level (28.6%) along with highest percentage of patients achieving SD (57.1%). Here we describe studies evaluating the combination of pIL-12/PPC with bevacizumab and PLD. For these studies 5,000,000 human SKOV3 cells were implanted into the peritoneal cavity of immunocompromised Hsd:Athymic Nude-Foxn1nu mice. Treatment with pIL-12/PPC alone and bevacizumab alone resulted in a 50% and a 39% reduction of animals with visible tumors at the end of the study. Combining pIL-12/PPC + bevacizumab improved the response to 78% of animals with no visible tumors. Further, combining pIL-12/PPC + bevacizumab + PLD resulted in a &amp;gt;98% decrease in tumor burden in animals compared to controls and ∼92% decrease in tumor burden compared to animals treated only with bevacizumab + PLD. All treatments were well tolerated and analysis of serum chemistries and hematology showed normal ranges of all parameters examined for all groups. There were no significant differences in animal weights between groups during the experiment. Together these results suggest synergistic efficacies can be achieved by combining a novel pIL-12/PPC immunotherapy with anti-angiogenesis therapies and cytotoxic chemotherapies in disseminated ovarian cancer. Citation Format: Jason G. Fewell, Majed M. Matar, Jennifer Rice, Diane McClure, Elaine Brunhoeber, Jeff Sparks, Stefanie Greenleaf, Kelley Smith, Khursheed Anwer. Interleukin-12 gene therapy in combination with bevacizumab and pegylated liposomal doxorubicin for treatment of disseminated ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2078.

Gene therapy with IL-12 induced enhanced anti-tumor activity in fibrosarcoma mouse model

Context Immunotherapy is among the most promising modalities for treatment of cancer. Recently, interleukin 12 (IL-12) has been used as an immunotherapeutic agent in cancer gene therapy. IL-12 can activate dendritic cells (DCs) and boost anti-tumor immune responses. Objective In the current study, we have investigated if IL-12 gene therapy can lead to the regression of tumor mass in a mouse model of fibrosarcoma. Material and methods To investigate the therapeutic efficacy of IL-12, WEHI-164 tumor cells were transfected with murine-IL12 plasmids using Lipofectamine. Enzyme linked immunosorbent assay (ELISA) was used to confirm IL-12 expression in transfected cells. The fibrosarcoma mouse model was established by subcutaneous injection of transfected cells to Balb/C mice. Mice were sacrificed and the tumors were extracted. Tumor sizes were measured by caliper. The expression of IL-12 and IFN-γ was studied with real-time PCR and western blotting. The expression of Ki-67(a tumor proliferation marker) in tumor mass was studied by immunohistochemistry staining. Results and discussion The group treated with IL-12 showed a significant decrease in tumor mass volume (P: 0.000). The results of real-time PCR and western blotting showed that IL-12 and IFN-γ expression increased in the group treated with IL-12 (relative expression of IL-12: 1.9 and relative expression of IFN-γ: 1.766). Immunohistochemistry staining showed that Ki-67 expression was reduced in the group treated with IL-12. Conclusion IL-12 gene therapy successfully led to regress of tumor mass in the fibrosarcoma mouse model. This may serve as a candidate therapeutic approach for treatment of cancer.

IL-12 immunotherapy of BrafV600E-induced papillary thyroid cancer in a mouse model

Papillary thyroid carcinoma (PTC) accounts for >80% thyroid malignancies, and BRAFV600E mutation is frequently found in >40% PTC. Interleukin-12 (IL-12) is a proinflammatory heterodimeric cytokine with strong antitumor activity. It is not known whether IL-12 immunotherapy is effective against BrafV600E-induced PTC. In the present study, we investigated the effectiveness of IL-12 immunotherapy against BrafV600E-induced PTC in LSL-BrafV600E/TPO-Cre mice. LSL-BrafV600E/TPO-Cre mice were created for thyroid-specific expression of BrafV600E under the endogenous Braf promoter, and spontaneous PTC developed at about 5 weeks of age. The mice were subjected to two treatment regimens: (1) weekly intramuscular injection of 50 μg plasmid DNA expressing a single-chain IL-12 fusion protein (scIL-12/CMVpDNA), (2) daily intraperitoneal injection of mouse recombinant IL-12 protein (mrIL-12, 100 ng per day). The role of T cells, natural killer (NK) cells, and transforming growth factor-β (TGF-β) in IL-12-mediated antitumor effects was determined by a 51Cr-release cytotoxicity assay. Tumor size and weight were significantly reduced by either weekly intramuscular injection of scIL-12/CMVpDNA or daily intraperitoneal injection of mrIL-12, and tumor became more localized. Survival was significantly increased when treatment started at 1 week of age as compared with that at the 6 weeks of age. Both NK and CD8+ T cells were involved in the cytotoxicity against tumor cells and their antitumor activity was significantly reduced in tumor-bearing mice. TGF-β also inhibited the antitumor activity of NK and CD8+ T cells. The immune suppression was completely reversed by IL-12 treatment and partially recovered by anti-TGF-β antibody. We conclude that both IL-12 gene therapy and recombinant protein therapy are effective against PTC. Given that the immune response is significantly suppressed in tumor-bearing mice and can be restored by IL-12, the current study raises a possibility of the application of IL-12 as an adjuvant therapy for thyroid cancer.

Abstract 292: Interleukin-12 gene therapy combined with local ablative technique electrochemotherapy for treatment of canine mastocytoma

Abstract Electrochemotherapy is now well established ablative local tumor treatment for veterinary as well as human cancer. Electrochemotherapy combines the use of permeabilizing electric pulses applied directly at the tumor site with the injection of chemotherapeutic drugs, cisplatin or bleomycin. Increased membrane permeability enables enhanced entry of chemotherapeutic drug into the cells and resulted in higher tumor cell kill. Up to 80% complete responses are obtained after single electrochemotherapy treatment of cutaneous tumors of different histology (Yarmush et al. Annu Rev Biomed Eng. 2014; 16:295-320). To add a systemic component to this very effective local treatment, in preclinical studies it was combined with several different immune therapies, including gene therapy with plasmid DNA encoding interleukin-12 (IL-12) (Radiol Oncol. 2012;46:302-11). In our on-going clinical research, the effect of IL-12 gene therapy combined with electrochemotherapy was evaluated in 18 client- owned dogs with mastocytomas. Written consent for participation was obtained from the owners and the study was approved by the competent authorities (Veterinary administration and Administration for biotechnology of the Ministry of agriculture and environment; Republic Ethical Committee for the experiments involving animals). Electrochemotherapy using intratumoral injection of bleomycin and application of electric pulses was preformed prior to gene therapy. Plasmid encoding IL-12 was injected peritumorally into the skin and immediately thereafter electric pulses were applied. Local response of tumors was evaluated by measurements of tumors’ size. Systemic response was assessed by determination of IL-12 and IFN-γ in patients’ sera. Safety of gene therapy was evaluated by qRT-PCR measurement for the presence of plasmid DNA at the site of application and possible horizontal gene transfer by in vitro transformation of plasmid DNA encoding IL-12 into the bacteria isolated from the dog's (patient’s) skin. One month after the therapy, 11 dogs (61%) had a complete response, 4 (22%) partial response, and 3 (16%) stable disease. At the end of observation period (median 25 months) 16 dogs (89%) had complete response and 2 had to be euthanized due to the progression of disease. In all dogs, except two, serum IFN-γ or IL-12 levels were detected, without systemic toxicity. At the site of DNA plasmid application, except for one patient, the plasmid was not detected at 1 week post-treatment. No horizontal gene transfer was detected; plasmid was not detected in any of the residential bacteria and in vitro transformation of plasmid into isolated strains was negative. Collectively, the results of our on-going clinical trial demonstrate that IL-12 gene therapy combined with electrochemotherapy is safe, feasible and effective treatment for canine mastocytoma. Citation Format: Maja Cemazar, Jerneja Ambrozic Avgustin, Gregor Sersa, Darja Pavlin, Ana Krhac Levacic, Natasa Tesic, Mitja Rak, Ursa Lampreht, Natasa Tozon. Interleukin-12 gene therapy combined with local ablative technique electrochemotherapy for treatment of canine mastocytoma. [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 292. doi:10.1158/1538-7445.AM2015-292

Abstract 4047: CD8+T cell-mediated tumor-specific induction of the NKG2D ligands to trigger NKG2D-dependent tumor cell death in vivo

Abstract Introduction: High profile studies have shown that restoring NKG2D ligand expression greatly promotes antitumor immune surveillance and tumor regression, but malignant tumor cells often down-regulate NKG2D ligands to escape from immune surveillance. Previous studies showed that engineering tumor cells with NKG2D ligand resulted in inhibition of tumor progression. However, it is not feasible to engineer each tumor cell with NKG2D ligand in clinical treatment. Therefore, a therapeutic treatment that can restore NKG2D ligand on tumor cells is required to promote tumor immune surveillance. In this regard, many therapeutic approaches were discovered to induce NKG2D ligands on tumor cells in vitro. Unfortunately, no effective in vivo approaches are available to restore this ligand across different types of tumors because the classic in vitro stress signal-dependent NKG2D ligand induction has rarely been duplicated in solid tumors in vivo. Experimental procedure: This study reveals a unique approach_two independent co-administration of immune stimulatory signal interleukin-12 and doxorubicin_restores the NKG2D ligands in multiple murine and human tumor types. The induction of NKG2D ligands was determined using immunoblotting, flow cytometry and immunohistochemistry staining. The comparison of Rae-1 induction in immune competent mice versus immune deficient mice (nude, Rag2 -/- or Pfp -/-) implied that mature CD8+T cells are the most critical immune subpopulation. The concentration of interferon γ in tumors was measured using ELISA assay. The accumulation of NKG2D positive immune cells in tumor sections was examined using immunofluorescence straining, followed by TUNEL assay to determine tumor cell death. Results: We showed for the first time that a chemoimmunotherapy led to the tumor specific restoration of NKG2D ligands in a variety of tumor models in vivo. Systemic delivery of interleukin-12 gene therapy plus doxorubicin effectively restored murine NKG2D ligand Rae-1 in vivo in two subcutaneous tumor models (LLC and CT26), one orthotopic tumor model (K7M3) and one spontaneous breast carcinoma model. Significantly, such therapeutic approach can also stimulate human NKG2D ligands in xenograft tumor models. Intriguingly, this tumor-specific Rae-1 induction in vivo requires CD8+T cells. This NKG2D ligand-restoring therapy itself can also attract endogenous NKG2D-positive cells to attack tumor cells. Along with the tumor-specific NKG2D ligand induction, the promoted immune surveillance leads to massive tumor cell death. Conclusion: This study provides the first therapeutic treatment for inducing immune surveillance ligand in tumors in vivo. Further, it reveals a CD8+T cell-dependent novel mechanism for the restoration of NKG2D ligand. The revealed in vivo NKG2D ligand restoring approach could be translated for cancer prevention and treatment. Citation Format: Jiemiao Hu, Xueqing Xia, Eugenie S. Kleinerman, Shulin Li. CD8+T cell-mediated tumor-specific induction of the NKG2D ligands to trigger NKG2D-dependent tumor cell death in vivo. [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 4047. doi:10.1158/1538-7445.AM2015-4047

Gene Electrotransfer of Canine Interleukin 12 into Canine Melanoma Cell Lines.

A gene electrotransfer (GET) of interleukin 12 (IL-12) had already given good results when treating tumors in human and veterinary clinical trials. So far, plasmids used in veterinary clinical studies encoded a human or a feline IL-12 and an ampicillin resistance gene, which is not recommended by the regulatory agencies to be used in clinical trials. Therefore, the aim of the current study was to construct the plasmid encoding a canine IL-12 with kanamycin antibiotic resistance gene that could be used in veterinary clinical oncology. The validation of the newly constructed plasmid was carried out on canine malignant melanoma cells, which have not been used in GET studies so far, and on human malignant melanoma cells. Canine and human malignant melanoma cell lines were transfected with plasmid encoding enhanced green fluorescence protein at different pulse parameter conditions to determine the transfection efficiency and cell survival. The IL-12 expression of the most suitable conditions for GET of the plasmid encoding canine IL-12 was determined at mRNA level by the qRT-PCR and at protein level with the ELISpot assay. The obtained results showed that the newly constructed plasmid encoding canine IL-12 had similar or even higher expression capacity than the plasmid encoding human IL-12. Therefore, it represents a promising therapeutic plasmid for further IL-12 gene therapy in clinical studies for spontaneous canine tumors. Additionally, it also meets the main regulatory agencies' (FDA and EMA) criteria.

Combined Tumor Cell-Based Vaccination and Interleukin-12 Gene Therapy Polarizes the Tumor Microenvironment in Mice.

Tumor progression depends on tumor milieu, which influences neovasculature formation and immunosuppression. Combining immunotherapy with antiangiogenic/antivascular therapy might be an effective therapeutic approach. The aim of our study was to elaborate an anticancer therapeutic strategy based on the induction of immune response which leads to polarization of tumor milieu. To achieve this, we developed a tumor cell-based vaccine. CAMEL peptide was used as a B16-F10 cell death-inducing agent. The lysates were used as a vaccine to immunize mice bearing B16-F10 melanoma tumors. To further improve the therapeutic effect of the vaccine, we combined it with interleukin (IL)-12 gene therapy. IL-12, a cytokine with antiangiogenic properties, activates nonspecific and specific immune responses. We observed that combined therapy is significantly more effective (as compared with monotherapies) in inhibiting tumor growth. Furthermore, the tested combination polarizes the tumor microenvironment, which results in a switch from a proangiogenic/immunosuppressive to an antiangiogenic/immunostimulatory one. The switch manifests itself as a decreased number of tumor blood vessels, increased levels of tumor-infiltrating CD4+, CD8+ and NK cells, as well as lower level of suppressor lymphocytes (Treg). Our results suggest that polarizing tumor milieu by such combined therapy does inhibit tumor growth and seems to be a promising therapeutic strategy.

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.

Preclinical toxicology of oncolytic adenovirus-mediated cytotoxic and interleukin-12 gene therapy for prostate cancer

The purpose of this study was to examine the toxicity of combining oncolytic adenovirus-mediated cytotoxic and interleukin 12 (IL-12) gene therapy in a preclinical model to support future phase 1 trials. One hundred and twenty C57BL/6 male mice received an intraprostatic injection of saline (n = 24) or an oncolytic adenovirus (Ad5-yCD/mutTKSR39rep-mIL12) expressing two suicide genes and mouse IL-12 (n = 96). The adenovirus was administered at three dose levels (1.3 × 106, 1.3 × 107, 1.3 × 108 vp/kg) followed by 2 weeks of 5-flurocytosine (5-FC) and gancliclovir (GCV) prodrug therapy. There were no premature deaths. Daily observations of animals did not reveal any obvious clinical problems throughout the 78-day in-life phase of the study. Animals in the highest adenovirus dose group exhibited lymphopenia and transaminitis on day 3, both of which resolved by day 17. Except for mild inflammation of the prostate and seminal vesicles, histopathology of major organs was largely unremarkable. IL-12 and interferon-gamma levels in prostate and serum peaked on day 3 and were either undetectable or returned to baseline levels by day 17. No adenoviral DNA was detected in serum in any group at any time point. The results demonstrate that local administration of an oncolytic adenovirus expressing two suicide genes and IL-12 is well tolerated and support moving this investigational approach into human trials.