Interleukin-12 Plasmid Research Articles

Neoadjuvant intratumoral plasmid interleukin-12 electro-gene-transfer and nivolumab in patients with operable locoregionally advanced melanoma.

Intratumoral (IT) TAVO-EP (tavokinogene telseplasmid delivered by electroporation) results in localized expression of interleukin-12 (IL-12) within the tumor microenvironment (TME). This study evaluated neoadjuvant TAVO-EP combined with intravenous (IV) nivolumab followed by surgery and adjuvant nivolumab in patients with operable locoregionally advanced melanoma. The neoadjuvant phase comprised up to 3 Χ 4-week cycles where TAVO-EP was given IT on days 1, 8, and 15 (optional) concurrently with 480 mg nivolumab IV on day 8 of each 4-week cycle. Surgery followed, and adjuvant nivolumab was initiated after surgery. The primary endpoint was pathologic complete response (pCR). Secondary endpoints included major pathological response (MPR; pCR or near pCR). Sixteen patients were enrolled and the preoperative radiological response rate was 63%. One patient declined surgery after experiencing a significant clinical response. Among the remaining 15 patients, pCR rate was 60% and MPR was 80%. No patient with MPR has had disease recurrence with a median follow-up from the date of surgery of 15.4 months. At baseline, most patients exhibited low CD8+ TIL, PD-L1 and IFN-γ gene expression signature. There was enhanced immune activation following treatment in the TME and blood including increased immune-related gene expression, CD8+ TIL and proliferating immune cell subsets. The clinical efficacy of neoadjuvant IT TAVO-EP + nivolumab is promising with 80% of patients achieving an MPR. Evidence of potent immune activation both systemically and within the TME along with a favorable safety profile supports the activity of local IL-12 and anti-PD1 based regimens.

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.

Genetically engineered electrospinning contributes to spinal cord injury repair by regulating the immune microenvironment.

Introduction: Spinal cord injury (SCI) is associated with microenvironment imbalance, thereby resulting in poor regeneration and recovery of the spinal cord. Gene therapy can be used to balance the inflammatory response, however target genes cannot exist in localized injured areas. Methods: A genetically engineered electrospun scaffold (GEES) to achieve long-term immunoregulation and nerve repair was constructed. By combining the microfluidic and electrospinning techniques, interleukin-10 plasmid (pIL10) was loaded into lipid nanoparticles (LNPs) (pIL10-LNP), which was encapsulated to the nerve growth factor (NGF). Immunofluorescence staining, qRT-PCR, ELISA, flow cytometry, and other tests were employed to comprehensively assess the role of GEES in modulating macrophage polarization and facilitating neural repair. Results: The results showed that the scaffold released >70% of the pIL10-LNP within 10 d and continued slow release within 30 d. In vitro cell experiments have demonstrated that GEES effectively stimulates macrophages to secrete anti-inflammatory cytokines and facilitates the differentiation of neural stem cells into neuronal cells. In rat T9 SCI model, the GEES significantly inhibited the inflammatory response in the acute and chronic phases of SCI by transfecting local tissues with slow-release pIL10-LNP to promote the release of the anti-inflammatory factor IL10, thereby creating a favorable microenvironment. With the addition of NGF, the repair and regeneration of nerve tissues was effectively promoted, and the post-SCI motor function of rats improved. Discussion: GEES can regulate post-SCI immune responses through continuous and effective gene delivery, providing a new strategy for the construction of electrospun scaffolds for nerve repair in gene therapy.

Targeted delivery of interleukin-12 plasmid into HepG2 cells through folic acid conjugated graphene oxide nanocarrier.

Successful gene therapy relies on carriers to transfer genetic materials with high efficiency and low toxicity in a targeted manner. To enhance targeted cell binding and uptake, we developed and synthesized a new gene delivery vector based on graphene oxide (GO) modified by branched polyethyleneimine (BPEI) and folic acid (FA). The GO-PEI-FA nanocarriers exhibit lower toxicity compared to unmodified PEI, as well as having the potential to efficiently condense and protect pDNA. Interestingly, increasing the polymer content in the polyplex formulation improved plasmid transfer ability. Substituting graphene oxide for PEI at an N/P ratio of 10 in the HepG2 and THP1 cell lines improved hIL-12 expression by up to approximately eightfold compared to simple PEI, which is twice as high as GO-PEI-FA in Hek293 at the same N/P ratio. Therefore, the GO-PEI-FA described in this study may serve as a targeting nanocarrier for the delivery of the hIL-12 plasmid into cells overexpressing folic acid receptors, such as those found in hepatocellular carcinoma.

Surface decoration of low molecular weight polyethylenimine (LMW PEI) by phthalated dextrin for improved delivery of interleukin-12 plasmid.

In this investigation, low molecular weight polyethyleneimine (LMW PEI; 1.8 kDa branched PEI) was conjugated to phathalated dextrin. The aim of this chemical modification was to decorate PEI molecules with a hydrophilic layer to improve its biophysical properties while the phthalic moiety may improve the hydrophilic-hydrophobic balance of the final structure. The polymers were prepared at various conjugation degrees ranging from 6.5% to 16.5% and characterized in terms of biophysical characteristics as well as their gene transfer ability and cell-induced toxicity. The results showed that dextrin-phthalated-PEI (DPHPEI) polymer was able to form nanoparticles with the size range of around 118-170 nm, with the zeta potential of 6.2-9.5 mV. DPHPEI polymers could increase the level of desired protein expression in the cells by up to three folds compared with unmodified LMW PEI while the cell viability of the modified polymers was around 80%. The result of this study shows a promising approach to improve the transfection efficiency of LMW PEI while maintaining its low toxic effects.

Safety and Efficacy of IL-12 Plasmid DNA Transfection into Pig Skin: Supportive Data for Human Clinical Trials on Gene Therapy and Vaccination.

Gene electrotransfer (GET) of plasmids encoding interleukin 12 (IL-12) has already been used for the treatment of various types of tumors in human oncology and as an adjuvant in DNA vaccines. In recent years, we have developed a plasmid encoding human IL-12 (phIL12) that is currently in a phase I clinical study. The aim was to confirm the results of a non-clinical study in mice on pharmacokinetic characteristics and safety in a porcine model that better resembled human skin. The GET of phIL12 in the skin was performed on nine pigs using different concentrations of plasmid phIL12 and invasive (needle) or noninvasive (plate) types of electrodes. The results of our study demonstrate that the GET of phIL-12 with needle electrodes induced the highest expression of IL-12 at the protein level on day 7 after the procedure. The plasmid was distributed to all tested organs; however, its amount decreased over time and was at a minimum 28 days after GET. Based on plasmid copy number and expression results, together with blood analysis, we showed that IL-12 GET is safe in a porcine animal model. Furthermore, we demonstrated that pigs are a valuable model for human gene therapy safety studies.

Macrophage-hitchhiking interleukin-10 plasmid DNA delivery system modulates rheumatoid arthritis microenvironment via the re-polarization of macrophages

Rheumatoid arthritis (RA) is a common chronic inflammatory disease and the current treatments for RA are unsatisfactory due to the buffer barrier from the RA microenvironment. Interleukin-10 (IL-10) was found to be able to modulate rheumatoid arthritis microenvironment (RAM) by the metabolic reprogramming of macrophages. However, its clinical application has proven challenging mainly due to its short half-life, non-ideal targeting efficiency via systemic administration and poor tissue penetration. Taking advantage of the recruitment of macrophages, we constructed an innovative macrophage-hitchhiking IL-10 pDNA delivery system using the peptides bearing nuclear localization signal (NLS) and tuftsin-modified disulfide-crosslinked polymers (bPEI-SS-PEG-T) which was responsive to the intracellular glutathione concentration. Firstly, we formed NLS/DNA complexes via electrostatic interactions to enhance the nuclear entry efficiency. Then NLS/DNA complexes were combined with bPEI-SS-PEG-T to construct bPEI-SS-PEG-T/NLS/DNA NPs with sizes of 168.4 nm and zeta potentials of + 10.9 mV. As the bPEI-SS-PEG-T was biodegradable and the tuftsin peptides enhanced macrophage-mediated phagocytosis, the nanoparticles exhibited lower cytotoxicity and better cellular uptake in addition to higher nuclear entry efficiency, leading to better IL-10 plasmid transfection. In addition, these macrophage-hitchhiking nanoparticles could effectively accumulate at inflammatory sites of RA via intra-peritoneal injection due to the macrophage migration to the inflammatory joints and alleviate symptoms of inflammation in vivo by inducing the re-polarization of macrophages, which might be mediated by the inhibition of mammalian target of rapamycin (mTOR) and the induction of arginase-2 (Arg2). As a result, this macrophage-hitchhiking gene delivery system demonstrated excellent inflammation targeting ability, good nuclear entry ability, high IL-10 plasmid transfection efficiency and great therapeutic efficacy in vivo, which is promising for the treatment of rheumatoid arthritis. Besides, this delivery system also provides a novel strategy for the gene therapy and the gene delivery system design for rheumatoid arthritis and other similar inflammatory diseases from the perspective of the inflammatory microenvironment.

Self-Assembled Nanoparticle-Forming Derivatives of Dextrin-Conjugated Polyethylenimine Containing Urethane Bonds for Enhanced Delivery of Interleukin-12 Plasmid

Background and Objective: In the present investigation, low molecular weight polyethylenimine (LMW PEI, 1.8 kDa PEI) was conjugated to dextrin via urethane units and tested to transfer plasmid encoding interleukin-12 (IL-12) plasmid. Although high molecular weight PEI (HMW PEI, 25 kDa PEI) has shown substantial transfection efficiency, its wide application has been hampered due to considerable cytotoxicity. Therefore, LMW PEI with low toxic effects was used as the core of our gene transfer construct. Methods: LMW PEI was conjugated to dextrin via urethane units to improve its biophysical characteristics as well as cytotoxic effects. The conjugates were characterized in terms of buffering capacity, plasmid DNA condensation ability, particle size, and zeta potential as well as protection against enzymatic degradation. In Vitro experiments were carried out to evaluate the ability of these LMW PEI conjugates to transfer plasmid encoding human interleukin-12 (hIL- 12) to the cells. The MTT assay was performed to measure the cell-induced toxicity of the conjugates. Results: The results of our study demonstrated that the PEI derivatives with higher amounts of amine content (i.e. higher conjugation degrees) have considerable buffering capacity and plasmid condensation ability. These conjugates could condense plasmid DNA at Carrier to Plasmid ratios (C/P) ≥2 and form polyplexes at the size range of 120-165 nm while their zeta potential was around 5.5-8.5 mV. The results of transfection efficiency demonstrated that the level of IL- 12 production increased by 2-3 folds compared with unmodified LMW PEI while the level of cytotoxicity was not higher than 20%. Conclusion: The strategy used in this study shows a promising way to prepare gene carriers with high transfection efficiency and low toxicity.

Interleukin-10 plasmid delivery by polymeric nanocarrier shows efficient and safe tissue repair in acute muscle damage models in mice

We established a custom-designed nanocarrier for in vivo delivery of an immunomodulatory plasmid capable of facilitating tissue repair in acute muscle damage models in mice. The nanocarrier was constructed by selecting from versatile cationic designs that were used to complex with a negatively charged plasmid, while the excessive positive charge was shielded by the poly (glutamic acid)-polyethylene glycol co-polymer. The best-performing formulation was identified by high throughput screening in macrophages, in which we obtained the hierarchical ranking in terms of reporter gene expression (GFP plasmid, pGFP) and macrophage polarization status (interleukin 10 plasmid, pIL-10). For proof-of-principle, we performed intramuscular (IM) injection of pGFP nanocarrier in a cardiotoxin-induced muscle injury model. After confirming the in vivo GFP transfection, we demonstrated protected delivery of pIL-10 to the injured muscle site, leading to an accelerated tissue repair outcome. Immunophenotyping analysis revealed the increased number of M2 macrophages and regulatory T cells, as well as increased IL-10 production at muscle site. The effectiveness of the pIL-10 carrier was interfered by pharmaceutical macrophage depletion using pre-injected clodronate liposome. When intravenously (IV) injected, our pIL-10 nanocarrier preferentially biodistributed in the injured muscle (owing to transiently decreased blood vessel integrity) and improved tissue repair. Unlike IL-10 protein (that was clinically abandoned), sustained IL-10 production by our nanocarrier dramatically reduced the degree of spleen enlargement and kidney inflammation, suggesting a favorable safety characteristic in vivo.

Abstract 4210: Gene therapy approach to suppress metastasis in a mouse melanoma model

Abstract Immune checkpoint inhibitors (ICIs) have been demonstrated to have significant clinical benefits. Effectiveness of ICIs have been limited by both primary and acquired resistance. In addition, absence of T cell infiltration within the tumor site is one of the major obstacles limiting ICIs efficacy against solid tumors. Interleukin-12 (IL-12) is a potent immune stimulator that has been shown to stimulate growth and survival of T cells as well as NK cells. Utilizing a B16.F10 melanoma model in C57Bl/6 mice, we observed a significant reduction in T regulatory cells and myeloid derived suppressor cells while T effector cells were significantly increased within the tumor microenvironment (TME) following intratumor delivery of a plasmid encoding IL-12 using gene electrotransfer. Monotherapy with plasmid IL-12 delivered with gene electrotransfer (pIL-12 GET) resulted in prolonged disease-free survival as well as long term immune memory. To determine the robustness of this therapeutic approach and to evaluate the responsiveness in combination with anti-PD1, we used a two-tumor model consisting of a subcutaneous B16F10 tumor and B16F10 cells expressing luciferase injected via the intraperitoneal route in a C57Bl/6 mouse. Intratumor delivery of pIL-12 GET as a monotherapy resulted in reduction or elimination of the subcutaneous tumor. However, the monotherapy approach was only successful in reducing the peritoneal spread in about 50% of the mice. When pIL-12 GET was combined with anti-PD1 administered via an intraperitoneal injection not only was there an elimination of the subcutaneous tumor, but it resulted in the elimination of intraperitoneal metastatic growth. The elimination of peritoneal spread was confirmed utilizing an In Vivo Imaging System. Observations on day 60 revealed background levels of luminescence in 90% of mice treated with the combination therapy thus confirming long-term disease-free survival of these mice. This level of response was not seen in mice treated with anti-PD-1 alone or with simple injection of pIL-12. In addition, the combination therapy resulted in an enhanced memory response which protected against growth of new tumors following challenge. In addition, exposure of tumor cells to IL-12 resulted in upregulation of MHC class 1 and PDL1 expression. The results have this study suggest that the combination of pIL-12 GET combined with ICIs could be utilized as an effective combination therapy for melanoma. Citation Format: Richard Heller, Megan Scott, Cathryn Mangiamele, Jody Synowiec, Guilan Shi. Gene therapy approach to suppress metastasis in a mouse melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4210.

Interleukin-12 Plasmid DNA Delivery by N-[(2-Hydroxy-3-trimethylammonium)propyl]chitosan-Based Nanoparticles.

Cationic polysaccharides are capable of forming polyplexes with nucleic acids and are considered promising polymeric gene carriers. The objective of this study was to evaluate the transfection efficiency and cytotoxicity of N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan salt (HTCS), a quaternary ammonium derivative of chitosan (CS), which benefits from non-ionizable positive charges. In this work, HTCS with a full quaternization of amino groups and a molar mass of 130,000 g·mol−1 was synthesized to use for delivery of a plasmid encoding the interleukin-12 (IL-12) gene. Thus, a polyplex based on HTCS and the IL-12 plasmid was prepared and then was characterized in terms of particle size, zeta potential, plasmid condensation ability, and protection of the plasmid against enzymatic degradation. We showed that HTCS was able to condense the IL-12 plasmid by the formation of polyplexes in the range of 74.5 ± 0.75 nm. The level of hIL-12 production following the transfection of the cells with HTCS polyplexes at a C/P ratio of 8:1 was around 4.8- and 2.2-fold higher than with CS and polyethylenimine polyplexes, respectively. These findings highlight the role of HTCS in the formation of polyplexes for the efficient delivery of plasmid DNA.

Evaluation of the long-term anti-Spike immune response following a novel coronavirus vaccine (CORVax): electroporation of SARS-CoV-2 Spike plasmid DNA plus IL12 plasmid DNA

Abstract The global SARS-CoV-2 (CoV2) pandemic has focused efforts to generate effective vaccines that induce potent and persistent immunity. Recently, we described a novel vaccine approach using electroporation (EP) of a plasmid encoding a prefusion stabilized CoV2 spike protein (CORVax) plus plasmid Interleukin-12 (IL-12). IL-12 is an effective vaccine adjuvant that increases Th1 and Th2 antibodies in the serum. Here we examine the persistence of anti-Spike antibodies present in the serum of mice one year following CORVax vaccination. C57BL/6 and BALB/c were vaccinated intramuscular (IM) and/or intradermal (ID) with a plasmid encoding the CoV2 spike protein with or without plasmid-encoded murine IL-12. Mice received plasmid EP immediately following injection. Splenocytes and serum were harvested at various time points to interrogate virus-specific cellular responses as well anti-spike antibody titers. Anti-Spike IgG antibodies were elicited by EP of CORVax (IC50 = 1/2112), as well as EP of CORVax combined with IL-12 (IC50 = 1/4214) approximately 40 days after the booster vaccination. These anti-Spike IgG titers decayed over time but were still present 1 year after vaccination: CORVax (IC50 = 1/351 day 146, IC50 = 1/208 day 383); CORVax + IL12 (IC50 = 1/590 day 146, IC50 = 1/266 day 383). Our data shows that EP of CORVax induces IgG responses to CoV2 Spike and the CoV2 Spike receptor binding domain. At one year following vaccination the anti-Spike IgG titers were higher in mice that received CORVax plus IL-12, however the rate at which the titers waned from their initial peak was comparable whether the mice received IL-12 or not. Additional studies are ongoing to determine whether the addition of IL-12 will enhance an anti-Spike memory response. Generous support from The Chiles Foundation, Nancy Lematta, and the Providence Medical Foundation.

Abstract P2-14-06: Durable responses with intratumoral electroporation of plasmid interleukin-12 plus pembrolizumab in patients with advanced triple-negative breast cancer: Cohort 1 update from KEYNOTE-890

Abstract BACKGROUND: Electroporated plasmid interleukin-12 (tavokinogene telseplasmid; TAVO-EP) delivered to accessible tumors by intratumoral injection induces sustained local expression of IL-12. IL-12 is a potent immunoregulatory cytokine that plays a key role in the crosstalk between innate (dendritic, macrophage, and natural killer) and adaptive (T and B) cells, promoting anti-tumor immune responses. TAVO-EP has been shown to induce activation of innate and adaptive tumor-infiltrating and peripheral immune cells, regression of treated and distant untreated lesions (abscopal effect), and expression of PD-L1 in patients with melanoma or triple-negative breast cancer (TNBC), without the systemic toxicity that limited therapeutic use of IL-12 historically. The combination of TAVO-EP and pembrolizumab has demonstrated durable responses in melanoma patients with immunologically “cold” tumors or with prior progression on anti-PD1 therapy. Anti-PD1 monotherapy has just over 5% overall response rate (ORR) in the second-line or later (2L+) treatment setting for advanced TNBC. New antibody-directed conjugate (ADC) chemotherapy has increased rates of responses compared with prior standard chemotherapy in 2L+ advanced TNBC; however, short duration of response (DOR), and toxicity are issues of concern. Therapies that can induce durable responses with limited toxicity are needed. METHODS: Cohort 1 of this Phase 2, open-label, multicenter study assessed the safety and efficacy of TAVO-EP in combination with pembrolizumab as 2L+ treatment for advanced TNBC. Eligible patients had at least 1 line of prior systemic therapy for advanced or metastatic disease, measurable disease by RECIST v1.1, and ≥1 lesion accessible for TAVO-EP treatment. Patients received pembrolizumab (200 mg IV) every 3 weeks and TAVO-EP (0.5 mg/mL at dose volume of ~1/4 lesion volume) on Days 1, 5, and 8 every 6 weeks. Tumor assessments were performed every 12 weeks. The primary endpoint was RECIST v.1.1 ORR by investigator review. Secondary endpoints included safety and tolerability, DOR, progression-free survival (PFS), immune-related RECIST (iRECIST) ORR and PFS, disease control rate, and overall survival (OS). ClinicalTrials.gov: NCT03567720. RESULTS: Between 01Nov2018 and 30Jan2020, 26 patients were enrolled and received at least one dose of study treatment (median follow up of 11.1 months). Patients had a median of 2 prior lines of systemic therapy for advanced disease (range 1-5). Among 23 patients evaluable for response, the ORR was 17.4% (4 with partial response [PR]). One responder with centrally confirmed PD-L1-negative disease and chest wall and bulky liver metastases had a sustained PR and an iRECIST complete response (CR). One responder had near complete regression of a large fungating chest wall skin lesion. The median DOR was 16.6 months. Median OS was 11.0 months (range 0.6-27.5+). The most common treatment-related adverse events (TRAEs) (all grades) were administration site pain and fatigue. Grade 3 TRAEs were reported in 6 patients (23%) including fatigue (11.5%); acute kidney injury, enterocolitis, and myocarditis (3.8% each). There were no Grade 4 or 5 TRAEs. CONCLUSIONS: The combination of TAVO-EP and pembrolizumab in pretreated patients with advanced TNBC resulted in durable RECIST v1.1 responses, including in PD-L1-negative disease, and was well tolerated. This novel immunotherapeutic regimen warrants further evaluation in 2L+ advanced TNBC. Cohort 2 exploring TAVO-EP + pembrolizumab + chemotherapy in frontline TNBC is currently enrolling. Citation Format: Melinda L. Telli, Irene Wapnir, Bianca Devitt, Katharine Cuff, Hatem Soliman, Shaveta Vinayak, David A. Canton, Christopher G. Twitty, Sunny Xie, Ying Lu, Donna Bannavong, Bridget O'Keeffe, Sandra Aung, Rohit Joshi. Durable responses with intratumoral electroporation of plasmid interleukin-12 plus pembrolizumab in patients with advanced triple-negative breast cancer: Cohort 1 update from KEYNOTE-890 [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-14-06.

Abstract OT2-01-03: Trial in progress: Phase 2 study of intratumoral plasmid interleukin-12 (tavokinogene telseplasmid; TAVO™) plus electroporation in combination with pembrolizumab with or without chemotherapy in patients with inoperable locally advanced or metastatic triple-negative breast cancer (KEYNOTE-890/OMS-I141)

Abstract BACKGROUND: Interleukin-12 (IL-12) is a potent immunoregulatory cytokine that plays a key role in the crosstalk between innate (dendritic, macrophage, and natural killer) and adaptive (T and B) cells promoting anti-tumor immune responses. DNA plasmid-based IL-12 delivered to accessible tumors by intratumoral injection and electroporation (TAVO-EP) has been shown to induce activation of innate and adaptive tumor-infiltrating and peripheral immune cells, regression of treated and distant untreated lesions (abscopal effect), and expression of PD-L1 in patients with melanoma or triple-negative breast cancer (TNBC), without the systemic toxicity that has limited therapeutic use of IL-12 historically. The combination of TAVO-EP and pembrolizumab has demonstrated durable responses in melanoma patients with immunologically “cold” tumors or with prior progression on anti-PD1 therapy. Early clinical data indicate similar potential for eliciting strengthened immunogenic responses in TNBC. KEYNOTE-890 (OMS-I141) is a Phase 2 study in patients with metastatic TNBC to evaluate the safety and efficacy of TAVO-EP + pembrolizumab in the second-line or later (2L+) treatment setting (Cohort 1) or TAVO-EP + pembrolizumab + chemotherapy in the first-line (1L) setting (Cohort 2). Cohort 1 data are presented in a separate abstract. Enrollment in Cohort 2 is ongoing. METHODS: Cohort 2 of this Phase 2, open-label, multicenter study will be assessing the safety and efficacy of TAVO-EP in combination with pembrolizumab and chemotherapy as a first-line treatment for metastatic TNBC. Eligible patients are adults with metastatic TNBC (ER and PR staining <10%, HER2 0 to 1+ or [F]ISH-negative), no prior systemic therapy for advanced disease (neo/adjuvant therapy allowed if at least 6-month disease-free interval from last treatment), measurable disease by RECIST v1.1, at least one lesion accessible for TAVO-EP treatment, and biopsy tissue available for post-hoc central determination of PD-L1 expression. Patients will receive pembrolizumab (200 mg IV) every 3 weeks, TAVO-EP (0.5 mg/mL at dose volume of ~1/4 lesion volume) on Days 1, 5, and 8 every 6 weeks, and nab-paclitaxel (100 mg/m2 IV) on Days 1, 8, and 15 every 4 weeks. Additional chemotherapy options may be introduced in future protocol amendments. Tumor assessments will be performed every 12 weeks. On-study biopsies will be collected approximately 3 weeks after start of treatment and at disease progression. The primary endpoint will be ORR assessed by blinded independent review per RECIST v1.1. Additional endpoints will include safety and tolerability, duration of response, immune ORR, progression-free survival (PFS), immune PFS, disease control rate, and overall survival. Planned enrollment in Cohort 2 is 40 patients. Based on positive efficacy data in Cohort 1, additional cohorts are being planned and will be presented. ClinicalTrials.gov: NCT03567720 Citation Format: Melinda Telli, Bianca Devitt, Katharine Cuff, Shaveta Vinayak, Rita Nanda, Alberto J. Montero, Rina Hui, David A. Canton, Christopher Twitty, Sunny Xie, Donna Bannavong, Bridget O'Keeffe, Sandra Aung, Rohit Joshi. Trial in progress: Phase 2 study of intratumoral plasmid interleukin-12 (tavokinogene telseplasmid; TAVO™) plus electroporation in combination with pembrolizumab with or without chemotherapy in patients with inoperable locally advanced or metastatic triple-negative breast cancer (KEYNOTE-890/OMS-I141) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-01-03.

Engineered Mesenchymal Stem Cells as a Biotherapy Platform for Targeted Photodynamic Immunotherapy of Breast Cancer.

Interleukin-12 (IL12) is a pleiotropic cytokine with promising prospects for cancer immunotherapy. Though IL12 gene-based therapy can overcome the fatal hurdle of severe systemic toxicity, targeted delivery and tumor-located expression of IL12 gene remain the challenging issues yet to be solved. Photo-immunotherapy emerging as a novel and precise therapeutic strategy, which elaborately combines immune-activating agents with light-triggered photosensitizers for potentiated anticancer efficacy. Herein, an engineered stem cell-based biotherapy platform (MB/IL12-MSCs) incorporating immune gene plasmid IL12 (pIL12) and photosensitizer methylene blue (MB) is developed to realize tumor-homing delivery of therapeutic agents and photo-immunotherapy efficacy enhancement. The biotherapy platform retained tumor-tropic migration and penetration functions, which improved the intratumoral distribution of therapeutic agents, thereby promoting photodynamic effects and reinforcing immune responses. Importantly, MB/IL12-MSCs restricted the expression and distribution of IL12 at tumor site, which minimized potential toxicity while eliciting sufficient anticancer immunity. In noteworthy, activation of immunity induced by MB/IL12-MSCs established long-term systemic immunologic memory to prevent tumor relapse. The MB/IL12-MSCs outperform their monotherapy counterparts in breast tumor models, and the growth of tumor significantly arrested as well as re-challenging abscopal tumor growth slowdown. Collectively, this work reveals that MSCs-based strategy may advance more efficient, durable, and safer cancer photo-immunotherapy.

Phase 1 study of safety, tolerability and immunogenicity of the human telomerase (hTERT)-encoded DNA plasmids INO-1400 and INO-1401 with or without IL-12 DNA plasmid INO-9012 in adult patients with solid tumors

BackgroundHuman telomerase reverse transcriptase (hTERT) is frequently classified as a ‘universal’ tumor associated antigen due to its expression in a vast number of cancers. We evaluated plasmid DNA-encoded hTERT as...

Results of Dynamic Contrast-Enhanced Ultrasound Correlate With Treatment Outcome in Canine Neoplasia Treated With Electrochemotherapy and Interleukin-12 Plasmid Electrotransfer.

Electrochemotherapy (ECT) and/or gene electrotransfer of plasmid DNA encoding interleukin-12 (GET pIL-12) are effective treatments for canine cutaneous, subcutaneous, and maxillofacial tumors. Despite the clinical efficacy of the combined treatments of ECT and GET, data on parameters that might predict the outcome of the treatments are still lacking. This study aimed to investigate whether dynamic contrast-enhanced ultrasound (DCE-US) results of subcutaneous tumors differ between tumors with complete response (CR) and tumors without complete response (non-CR) in dogs treated with ECT and GET pIL-12. Eight dogs with a total of 12 tumor nodules treated with ECT and GET pIL-12 were included. DCE-US examinations were performed in all animals before and immediately after therapy as well as 8 h and 1, 3, and 7 days later. Clinical follow-up examinations were performed 7 and 14 days, 1 and 6 months, and 1 year after treatment. Numerous significant differences in DCE-US parameters were noted between tumors with CR and non-CR tumors; perfusion and perfusion heterogeneity were lower in CR tumors than in non-CR tumors. Therefore, studies with larger numbers of patients are needed to investigate whether DCE-US results can be used to predict treatment outcomes and to make effective decisions about the need for repeated therapy or different treatment combinations in individual patients.

Targeted interleukin-10 plasmid DNA therapy in the treatment of osteoarthritis: Toxicology and pain efficacy assessments

Osteoarthritis results in chronic pain and loss of function. Proinflammatory cytokines create both osteoarthritis pathology and pain. Current treatments are poorly effective, have significant side effects, and have not targeted the cytokines central to osteoarthritis development and maintenance. Interleukin-10 is an anti-inflammatory cytokine that potently and broadly suppresses proinflammatory cytokine activity. However, interleukin-10 protein has a short half-life in vivo and poor joint permeability. For sustained IL-10 activity, we developed a plasmid DNA-based therapy that expresses a long-acting human interleukin-10 variant (hIL-10var). Here, we describe the 6-month GLP toxicology study of this therapy. Intra-articular injections of hIL-10var pDNA into canine stifle joints up to 1.5 mg bilaterally were well-tolerated and without pathologic findings. This represents the first long-term toxicologic assessment of intra-articular pDNA therapy. We also report results of a small double-blind, placebo-controlled study of the effect of intra-articular hIL-10var pDNA on pain measures in companion (pet) dogs with naturally occurring osteoarthritis. This human IL-10-based targeted therapy reduced pain measures in the dogs, based on veterinary and owner ratings, without any adverse findings. These results with hIL-10var pDNA therapy, well-tolerated and without toxicologic effects, establish the basis for clinical trials of a new class of safe and effective therapies for OA.

Enhancing immune responses by a novel multi-epitope ROP8 DNA vaccine plus interleukin-12 plasmid as a genetic adjuvant against acute Toxoplasma gondii infection in BALB/c mice

BackgroundToxoplasmosis is a widespread zoonotic infection, caused by an obligate intracellular protozoan. The infection is often asymptomatic in immunocompetent individuals, although in persons with impaired immune system may lead to severe and progressive complications. Constant attempts of scientists have made valuable findings in the development of Toxoplasma gondii (T. gondii) candidate vaccines. However, an effective vaccine has not been successfully developed yet. In the current study, we tested the co-delivery of a novel multi-epitope pcROP8 DNA vaccine with a plasmid encoding IL-12 (pcIL-12) to assess the immune responses in BALB/c mice against acute T. gondii infection. MethodsBALB/c mice were immunized on days 0, 21, and 42. The immune responses of both vaccinated and control groups were evaluated using cytokine and antibody measurements, lymphocyte proliferation assay, and survival time. ResultsThe findings demonstrated that immunization with multi-epitope pcROP8 significantly enhanced the level of anti-T. gondii antibodies, TH1-type cellular immune responses, lymphocyte proliferation, and prolonged survival time, compared to control groups (P < 0.05). Furthermore, the use of pcIL-12 as a genetic adjuvant led to enhancements of the above-mentioned immune responses in BALB/c mice (P < 0.05). ConclusionsThe co-administration of pcIL-12 with multi-epitope pcROP8 vaccine, could successfully enhance the level of protection. Thus, this immunization regimen may represent an effective vaccine strategy against acute T. gondii infection.

Intratumoral delivery of tavokinogene telseplasmid yields systemic immune responses in metastatic melanoma patients

Interleukin 12 (IL-12) is a pivotal regulator of innate and adaptive immunity. We conducted a prospective open-label, phase II clinical trial of electroporated plasmid IL-12 in advanced melanoma patients (NCT01502293). Patients with stage III/IV melanoma were treated intratumorally with plasmid encoding IL-12 (tavokinogene telseplasmid; tavo), 0.5 mg/ml followed by electroporation (six pulses, 1500 V/cm) on days 1, 5, and 8 every 90 days in the main study and additional patients were treated in two alternative schedule exploration cohorts. Correlative analyses for programmed death-ligand 1 (PD-L1), flow cytometry to assess changes in immune cell subsets, and analysis of immune-related gene expression were carried out on pre- and post-treatment samples from study patients, as well as from additional patients treated during exploration of additional dosing schedules beyond the pre-specified protocol dosing schedule. Response was measured by study-specific criteria to maximize detection of latent and potentially transient immune responses in patients with multiple skin lesions and toxicities were graded by the Common Terminology Criteria for Adverse Events version 4.0 (CTCAE v4.0). The objective overall response rate was 35.7% in the main study (29.8% in all cohorts), with a complete response rate of 17.9% (10.6% in all cohorts). The median progression-free survival in the main study was 3.7 months while the median overall survival was not reached at a median follow up of 29.7 months. A total of 46% of patients in all cohorts with uninjected lesions experienced regression of at least one of these lesions and 25% had a net regression of all untreated lesions. Transcriptomic and immunohistochemistry analysis showed that immune activation and co-stimulatory transcripts were up-regulated but there was also increased adaptive immune resistance. Intratumoral Tavo was well tolerated and led to systemic immune responses in advanced melanoma patients. While tumor regression and increased immune infiltration were observed in treated as well as untreated/distal lesions, adaptive immune resistance limited the response.