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.

Intravenous administration of IL-12 encoding self-replicating RNA-lipid nanoparticle complex leads to safe and effective antitumor responses.

Interleukin 12 (IL-12) is a potent immunostimulatory cytokine mainly produced by antigen-presenting cells (e.g., dendritic cells, macrophages) and plays an important role in innate and adaptive immunity against cancers. Therapies that can synergistically modulate innate immunity and stimulate adaptive anti-tumor responses are of great interest for cancer immunotherapy. Here we investigated the lipid nanoparticle-encapsulated self-replicating RNA (srRNA) encoding IL-12 (referred to as JCXH-211) for the treatment of cancers. Both local (intratumoral) and systemic (intravenous) administration of JCXH-211 in tumor-bearing mice induced a high-level expression of IL-12 in tumor tissues, leading to modulation of tumor microenvironment and systemic activation of antitumor immunity. Particularly, JCXH-211 can inhibit the tumor-infiltration of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). When combined with anti-PD1 antibody, it was able to enhance the recruitment of T cells and NK cells into tumors. In multiple mouse solid tumor models, intravenous injection of JCXH-211 not only eradicated large preestablished tumors, but also induced protective immune memory that prevented the growth of rechallenged tumors. Finally, intravenous injection of JCXH-211 did not cause noticeable systemic toxicity in tumor-bearing mice and non-human primates. Thus, our study demonstrated the feasibility of intravenous administration of JCXH-211 for the treatment of advanced cancers.

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.

Deep analysis of skin molecular heterogeneities and their significance on the precise treatment of patients with psoriasis.

Background: Psoriasis is a highly heterogeneous autoinflammatory disease. At present, heterogeneity in disease has not been adequately translated into concrete treatment options. Our aim was to develop and verify a new stratification scheme that identifies the heterogeneity of psoriasis by the integration of large-scale transcriptomic profiles, thereby identifying patient subtypes and providing personalized treatment options whenever possible. Methods: We performed functional enrichment and network analysis of upregulated differentially expressed genes using microarray datasets of lesional and non-lesional skin samples from 250 psoriatic patients. Unsupervised clustering methods were used to identify the skin subtypes. Finally, an Xgboost classifier was utilized to predict the effects of methotrexate and commonly prescribed biologics on skin subtypes. Results: Based on the 163 upregulated differentially expressed genes, psoriasis patients were categorized into three subtypes (subtypes A-C). Immune cells and proinflammatory-related pathways were markedly activated in subtype A, named immune activation. Contrastingly, subtype C, named stroma proliferation, was enriched in integrated stroma cells and tissue proliferation-related signaling pathways. Subtype B was modestly activated in all the signaling pathways. Notably, subtypes A and B presented good responses to methotrexate and interleukin-12/23 inhibitors (ustekinumab) but inadequate responses to tumor necrosis factor-α inhibitors and interleukin-17A receptor inhibitors. Contrastly, subtype C exhibited excellent responses to tumor necrosis factor-α inhibitors (etanercept) and interleukin-17A receptor inhibitors (brodalumab) but not methotrexate and interleukin-12/23 inhibitors. Conclusions: Psoriasis patients can be assorted into three subtypes with different molecular and cellular characteristics based on the heterogeneity of the skin's immune cells and the stroma, determining the clinical responses of conventional therapies.

Single-cell ATAC sequencing identifies sleepy macrophages during reciprocity of cytokines in L. major infection.

The hallmark characteristic of macrophages lies in their inherent plasticity, allowing them to adapt to dynamic microenvironments. Leishmania strategically modulates the phenotypic plasticity of macrophages, creating a favorable environment for intracellular survival and persistent infection through regulatory cytokine such as interleukin (IL)-10. Nevertheless, these effector cells can counteract infection by modulating crucial cytokines like IL-12 and key components involved in its production. Using sophisticated tool of single-cell assay for transposase accessible chromatin (ATAC) sequencing, we systematically examined the regulatory axis of IL-10 and IL-12 in a time-dependent manner during Leishmania major infection in macrophages Our analysis revealed the cellular heterogeneity post-infection with the regulators of IL-10 and IL-12, unveiling a reciprocal relationship between these cytokines. Notably, our significant findings highlighted the presence of sleepy macrophages and their pivotal role in mediating reciprocity between IL-10 and IL-12. To summarize, the roles of cytokine expression, transcription factors, cell cycle, and epigenetics of host cell machinery were vital in identification of sleepy macrophages, which is a transient state where transcription factors controlled the epigenetic remodeling and expression of genes involved in pro-inflammatory cytokine expression and recruitment of immune cells.IMPORTANCELeishmaniasis is an endemic affecting 99 countries and territories globally, as outlined in the 2022 World Health Organization report. The disease's severity is compounded by compromised host immune systems, emphasizing the pivotal role of the interplay between parasite and host immune factors in disease regulation. In instances of cutaneous leishmaniasis induced by L. major, macrophages function as sentinel cells. Our findings indicate that the plasticity and phenotype of macrophages can be modulated to express a cytokine profile involving IL-10 and IL-12, mediated by the regulation of transcription factors and their target genes post-L. major infection in macrophages. Employing sophisticated methodologies such as single-cell ATAC sequencing and computational genomics, we have identified a distinctive subset of macrophages termed "sleepy macrophages." These macrophages exhibit downregulated housekeeping genes while expressing a unique set of variable features. This data set constitutes a valuable resource for comprehending the intricate host-parasite interplay during L. major infection.

IL-12 reprograms CAR-expressing natural killer T cells to long-lived Th1-polarized cells with potent antitumor activity.

Human natural killer T cells (NKTs) are innate-like T lymphocytes increasingly used for cancer immunotherapy. Here we show that human NKTs expressing the pro-inflammatory cytokine interleukin-12 (IL-12) undergo extensive and sustained molecular and functional reprogramming. Specifically, IL-12 instructs and maintains a Th1-polarization program in NKTs in vivo without causing their functional exhaustion. Furthermore, using CD62L as a marker of memory cells in human NKTs, we observe that IL-12 maintains long-term CD62L-expressing memory NKTs in vivo. Notably, IL-12 initiates a de novo programming of memory NKTs in CD62L-negative NKTs indicating that human NKTs circulating in the peripheral blood possess an intrinsic differentiation hierarchy, and that IL-12 plays a role in promoting their differentiation to long-lived Th1-polarized memory cells. Human NKTs engineered to co-express a Chimeric Antigen Receptor (CAR) coupled with the expression of IL-12 show enhanced antitumor activity in leukemia and neuroblastoma tumor models, persist long-term in vivo and conserve the molecular signature driven by the IL-12 expression. Thus IL-12 reveals an intrinsic plasticity of peripheral human NKTs that may play a crucial role in the development of cell therapeutics.

Consanguineous marriages increase the incidence of recurrent tuberculosis: Evidence from whole exome sequencing.

BACKGROUND: In this study, we have identified multiple mutations in the IL-12R1 gene among Pakistani patients who have inherited them through consanguineous marriages. These patients have experienced severe Bacille-Calmette-Guérin (BCG) infection as well as recurrent tuberculosis. We will demonstrate the pivotal role of interleukin (IL)-12/interferon (IFN)-γ axis in the regulation of mycobacterial diseases. METHODOLOGY: First, we checked the patients' medical records, and then afterward, we assessed interferon-gamma (IFN-γ) production through ELISA. Following that, DNA was extracted to investigate IL-12/IFN- abnormalities. Whole exome sequencing was conducted through Sanger sequencing. Secretory cytokine levels were compared from healthy control of the same age groups and they were found to be considerably less in the disease cohort. To evaluate the probable functional impact of these alterations, an in silico study was performed. RESULTS: The study found that the patients' PBMCs produced considerably less IFN-γ than expected. Analysis using flow cytometry showed that activated T cells lacked surface expression of IL-12Rß1. Exon 7 of the IL-12Rß1 gene, which encodes a portion of the cytokine binding region (CBR), and exon 10, which encodes the fibronectin-type III (FNIII) domain, were found to have the mutations c.641 A > G; p.Q214R and c.1094 T > C; p.M365T, respectively. In silico analysis showed that these mutations likely to have a deleterious effect on protein function. CONCLUSION: Our findings indicate the significant contribution of the IL-12/IFN-γ is in combating infections due to mycobacterium. Among Pakistani patients born to consanguineous marriages, the identified mutations in the IL-12Rß-1 gene provide insights into the genetic basis of severe BCG infections and recurrent tuberculosis. The study highlights the potential utility of newborn screening in regions with mandatory BCG vaccination, enabling early detection and intervention for primary immunodeficiencies associated with mycobacterial infections. Moreover, the study suggests at the potential role of other related genes such as IL-23Rß1, TYK2, or JAK2 in IFN-γ production, warranting further investigation.

Inhalable extracellular vesicle delivery of IL-12 mRNA to treat lung cancer and promote systemic immunity.

Lung carcinoma is one of the most common cancers and has one of the lowest survival rates in the world. Cytokines such as interleukin-12 (IL-12) have demonstrated considerable potential as robust tumour suppressors. However, their applications are limited due to off-target toxicity. Here we report on a strategy involving the inhalation of IL-12 messenger RNA, encapsulated within extracellular vesicles. Inhalation and preferential uptake by cancer cells results in targeted delivery and fewer systemic side effects. The IL-12 messenger RNA generates interferon-γ production in both innate and adaptive immune-cell populations. This activation consequently incites an intense activation state in the tumour microenvironment and augments its immunogenicity. The increased immune response results in the expansion of tumour cytotoxic immune effector cells, the formation of immune memory, improved antigen presentation and tumour-specific T cell priming. The strategy is demonstrated against primary neoplastic lesions and provides profound protection against subsequent tumour rechallenge. This shows the potential for locally delivered cytokine-based immunotherapies to address orthotopic and metastatic lung tumours.

Association between several immune response-related genes and the effectiveness of biological treatments in patients with moderate-to-severe psoriasis.

Biological therapies are safer and more effective against psoriasis than conventional treatments. Even so, 30-50% of psoriatic patients show an inadequate response, which is associated with individual genetic heterogeneity. Pharmacogenetic studies have identified several single nucleotide polymorphisms (SNPs) as possible predictive and prognostic biomarkers for psoriasis treatment response. The objective of this study was to determine the link between several SNPs and the clinical response to biological therapies in patients with moderate-severe psoriasis. A set of 21 SNPs related to psoriasis and/or other immunological diseases were selected and analysed from salivary samples of patients (n = 88). Treatment effectiveness and patient improvement was assessed clinically through Relative Psoriasis Area and Severity Index (PASI), also called 'PASI response', as well as absolute PASI. Associations between SNPs and PASI factors were assessed at 3 and 12 months for every treatment category of IL-17, IL-23, IL-12&23 and TNF-α inhibitors. Multivariate correlation analysis and Fisher's exact test were used to analyse the relationship between SNPs and therapy outcomes. Several SNPs located in the TLR2, TLR5, TIRAP, HLA-C, IL12B, SLC12A8, TNFAIP3 and PGLYRP4 genes demonstrated association with increased short and long-term therapy-effectiveness rates. Most patients achieved values of PASI response ≥75 or absolute PASI<1, regardless of the biological treatment administered. In conclusion, we demonstrate a relationship between different SNPs and both short- and especially long-term effectiveness of biological treatment in terms of PASI. These polymorphisms may be used as predictive markers of treatment response in patients with moderate-to-severe psoriasis, providing personalized treatment.

Oncolytic virus Ad-TD-nsIL-12 inhibits glioma growth and reprograms the tumor immune microenvironment.

AIMS: Gliomas are the most common central nervous system malignancies, with limited therapeutic options and poor prognosis, which are primarily attributed to the "immune desert" microenvironment. Previously, we constructed a three-gene-deleted oncolytic adenovirus (Ad-TD) loaded with non-secreting interleukin-12 (nsIL-12), which could be amplified in tumor cells and induce immunity to suppress tumors. However, the effects of this oncolytic virus on gliomas and their immune microenvironment remain unclear. There is an urgent need for further research. MATERIALS AND METHODS: We constructed a Syrian hamster brain tumor model and demonstrated the efficacy and mechanism of the novel oncolytic virus in treating brain tumors through a series of in vitro and in vivo experiments. We investigated the efficacy and safety (the number of hamsters in each group is either 5 or 10) of the oncolytic virus treatment in Syrian hamsters using a virus-treated group, a control virus-treated group, and a blank control group. KEY FINDINGS: In vitro assays showed that Ad-TD-nsIL-12 could specifically proliferate in brain tumor cells which induce tumor cell apoptosis and intracellular expression of interleukin (IL)-12. Moreover, in vivo experiments demonstrated that Ad-TD-nsIL-12 could effectively inhibit the progression of brain tumors and prolong survival. Ad-TD-nsIL-12 significantly enhanced T-cell infiltration in the brain tumor microenvironment. SIGNIFICANCE: Ad-TD-nsIL-12 can inhibit glioma progression and increase T-cell infiltration in the tumor tissue, particularly infiltration by cytotoxic T cells (CD8+). Ad-TD-nsIL-12 can amplify and produce IL-12, inducing anti-glioma immune responses to inhibit tumor progression.

Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory.

Current immunotherapies have proven effective in strengthening antitumor immune responses, but constant opposing signals from tumor cells and the surrounding microenvironment eventually lead to immune escape. We hypothesized that in situ release of antigens and regulation of both the innate and adaptive arms of the immune system would provide a robust and long-term antitumor effect by creating immunologic memory against tumors. To achieve this, we developed CARG-2020, a genetically modified virus-like vesicle (VLV) that is a self-amplifying RNA with oncolytic capacity and encodes immune regulatory genes. CARG-2020 carries three immune modulators: (i) the pleiotropic antitumor cytokine IL12, in which the subunits (p35 and p40) are tethered together; (ii) the extracellular domain (ECD) of the protumor IL17RA, which serves as a dominant-negative antagonist; and (iii) a shRNA targeting PD-L1. Using a mouse model of ovarian cancer, we demonstrated the oncolytic effect and immune-modulatory capacities of CARG-2020. By enhancing IL12 and blocking IL17 and PD-L1, CARG-2020 successfully reactivated immune surveillance by promoting M1, instead of M2, macrophage differentiation, inhibiting MDSC expansion and establishing a potent CD8+ T cell-mediated antitumoral response. Furthermore, we demonstrated that this therapeutic approach provided tumor-specific and long-term protection against the establishment of new tumors. Our results provide a rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients to enhance antitumor responses and prevent a recurrence.

IFN-γ inhibitory molecules derived from Eimeria maxima inhibit IL-12 secretion by modulating MAPK pathways in chicken macrophages.

IFN-γ plays a crucial role in resisting intracellular parasitic protozoa, such as Eimeria species. In our previous study, we identified 4 molecules derived from Eimeria maxima (E. maxima) that significantly inhibited IFN-γ production. However, the mechanism underlying this inhibitory effect remains unknown. In this study, we first investigated the effects of these 4 IFN-γ inhibitory molecules on the expression levels of chicken Toll-like receptors (chTLRs), IL-12, IL-10, TGF-ß, and TNF-α in chicken macrophage HD11 and bone marrow-derived dendritic cells (BMDCs). The results demonstrated that these 4 inhibitory molecules significantly downregulated the mRNA levels of chTLR-2, chTLR-4, chTLR-21, and both mRNA and protein levels of IL-12. Subsequently, to clarify the effects of these 4 inhibitory molecules on the IL-12 secretion-related signaling pathways in chicken macrophages, qRT-PCR and Western blot were used to detect the changes of key molecules involved in the signaling pathways of IL-12 secretion (NF-κB, ERK1/2, p38, JNK, STAT3) following coincubation with these inhibitory molecules. Finally, RNAi was employed to verify the function of key molecules in the signaling pathway. The results revealed a significant upregulation in the expression of ERK1/2 phosphorylated protein induced by the 4 inhibitory molecules. Knockdown of the ERK1/2 gene significantly reduced the inhibitory effect of the 4 E. maxima inhibitory molecules on IL-12. These findings indicate that the 4 inhibitory molecules can inhibit the secretion of IL-12 by upregulating the expression of ERK1/2 phosphorylated protein, which is a key molecule in the ERK-MAPK pathway. Our study may contribute to elucidating the mechanisms underlying immune evasion during E. maxima infections, thereby providing new insights for the control of chicken coccidiosis.

Effect of interleukin-12 gene expression on insulin resistance in patients with acne vulgaris.

BACKGROUND: Insulin resistance (IR) is a condition where cells become resistant to insulin, causing impaired glucose uptake and increased blood glucose levels. Interleukin-12 (IL-12), a cytokine, regulates the immune system. High levels of IL-12 can lead to chronic inflammation, exacerbate resistance to insulin, and contribute to type 2 diabetes. Also, link IR to acne vulgaris (AV), as it reduces tissue sensitivity to insulin, causing increased insulin levels and sebum production, which can contribute to acne development. AIM: To explore the role of IL-12 gene expression on IR in AV patients and to study the role of IL-12 gene in the development of AV. SUBJECTS AND METHODS: A case-control study was performed on 68 AV patients and 68 healthy controls. The biochemical analysis included fasting glucose, fasting insulin, (HOMA-IR), and serum IL-12 level. IL-12 gene expression was performed by quantitative real-time PCR for both two groups. In addition, folding change was calculated by using the standard 2-(∆∆Ct) method. RESULT: IL-12 level, IL-12 folding change, fasting insulin, and IR were all increased in acne patients. A highly significant linear correlation was found between IL-12 folding change and both IL-12 levels and IR. There is a substantial positive significant simple linear association between IL-12 level and IL-12 folding change, as well as IR and IL-12 folding change, in moderate and severe acne. CONCLUSION: IL-12 gene has an important role in IR and the development of acne in AV patients.

Intraperitoneal administration of a modified vaccinia virus Ankara confers single-chain interleukin-12 expression to the omentum and achieves immune-mediated efficacy against peritoneal carcinomatosis.

BACKGROUND: Peritoneal carcinomatosis is an advanced stage of cancer in which the disease has spread to the peritoneal cavity. In order to restore antitumor immunity subverted by tumor cells in this location, we evaluated intraperitoneal administrations of modified vaccinia virus Ankara (MVA) engineered to express single-chain interleukin 12 (scIL-12) to increase antitumor immune responses. METHODS: MVA encoding scIL-12 (MVA.scIL-12) was evaluated against peritoneal carcinomatosis models based on intraperitoneal engraftment of tumor cells. CD8-mediated immune responses, elucidated antitumor efficacy, and safety were evaluated following intravenous, intratumoral, or intraperitoneal administration of the viral vector. The immune response was measured by ELISpot (enzyme-linked immunosorbent spot), RNA sequencing, flow cytometry, intravital microscopy, and depletion of lymphocyte subsets with monoclonal antibodies. Safety was assessed by body-weight follow-up and blood testing. Tissue tropism on intravenous or intraperitoneal administration was assessed by bioluminescence analysis using a reporter MVA encoding luciferase. RESULTS: Intraperitoneal or locoregional administration, but not other routes of administration, resulted in a potent immune response characterized by increased levels of tumor-specific CD8+ T lymphocytes with the ability to produce both interferon-γ and tumor necrosis factor-α. The antitumor immune response was detectable not only in the peritoneal cavity but also systemically. As a result of intraperitoneal treatment, a single administration of MVA.scIL-12 encoding scIL-12 completely eradicated MC38 tumors implanted in the peritoneal cavity and also protected cured mice from subsequent subcutaneous rechallenges. Bioluminescence imaging using an MVA encoding luciferase revealed that intraperitoneal administration targets transgene to the omentum. The omentum is considered a key tissue in immune protection of the peritoneal cavity. The safety profile of intraperitoneal administration was also better than that following intravenous administration since no weight loss or hematological toxicity was observed when the vector was locally delivered into the peritoneal cavity. CONCLUSION: Intraperitoneal administration of MVA vectors encoding scIL-12 targets the omentum, which is the tissue where peritoneal carcinomatosis usually begins. MVA.scIL-12 induces a potent tumor-specific immune response that often leads to the eradication of experimental tumors disseminated to the peritoneal cavity.

Engineered IL-7 synergizes with IL-12 immunotherapy to prevent T cell exhaustion and promote memory without exacerbating toxicity.

Cancer immunotherapy is moving toward combination regimens with agents of complementary mechanisms of action to achieve more frequent and robust efficacy. However, compared with single-agent therapies, combination immunotherapies are associated with increased overall toxicity because the very same mechanisms also work in concert to enhance systemic inflammation and promote off-tumor toxicity. Therefore, rational design of combination regimens that achieve improved antitumor control without exacerbated toxicity is a main objective in combination immunotherapy. Here, we show that the combination of engineered, tumor matrix-binding interleukin-7 (IL-7) and IL-12 achieves remarkable anticancer effects by activating complementary pathways without inducing any additive immunotoxicity. Mechanistically, engineered IL-12 provided effector properties to T cells, while IL-7 prevented their exhaustion and boosted memory formation as assessed by tumor rechallenge experiments. The dual combination also rendered checkpoint inhibitor (CPI)-resistant genetically engineered melanoma model responsive to CPI. Thus, our approach provides a framework of evaluation of rationally designed combinations in immuno-oncology and yields a promising therapy.

Assembly-dependent Structure Formation Shapes Human Interleukin-23 versus Interleukin-12 Secretion.

Interleukin 12 (IL-12) family cytokines connect the innate and adaptive branches of the immune system and regulate immune responses. A unique characteristic of this family is that each member is anα:ßheterodimer. For human αsubunits it has been shown that they depend on theirßsubunit for structure formation and secretion from cells. Since subunits are shared within the family and IL-12 as well as IL-23 use the same ßsubunit, subunit competition may influence cytokine secretion and thus downstream immunological functions. Here, we rationally design a folding-competent human IL-23α subunit that does not depend on itsßsubunit for structure formation. This engineered variant still forms a functional heterodimeric cytokine but shows less chaperone dependency and stronger affinity in assembly with its ßsubunit. It forms IL-23 more efficiently than its natural counterpart, skewing the balance of IL-12 and IL-23 towards more IL-23 formation. Together, our study shows that folding-competent human IL-12 familyαsubunits are obtainable by only few mutations and compatible with assembly and function of the cytokine. These findings might suggest that human α subunits have evolved for assembly-dependent folding to maintain and regulate correct IL-12 family member ratios in the light of subunit competition.

Evaluation of SOCS5 mRNA and its association with serum IL-12 levels and rs41379147 SNP in various subsets of allergic disorders: A case control study.

BACKGROUND: The SOCS proteins act as suppressors of cytokine signaling by impeding certain signaling pathways. SOCS5, a constituent of the SOCS family, has been associated with the management of allergic reactions, primarily by impeding the signaling of interleukin-4 (IL-4), which is known to have a cardinal function in accelerating the development of an allergic reaction. The key goal of our research was to explore the probable ramifications of the SOCS5 single nucleotide polymorphism (SNP) namely rs41379147 on the expression of SOCS5 mRNA and serum IL-12 levels, as well as to analyze the interaction between SOCS5 genotypes and various clinicopathological parameters in atopic diseases. METHODS: The study involved the enrollment of 314 subjects comprising 154 atopic individuals and 160 healthy controls. PCR-RFLP was employed to conduct SNP analysis. Real-Time PCR was employed to quantify SOCS5 mRNA. The enzyme-linked immunosorbent assay (ELISA) technique was used for the quantification of interleukin-12 and total IgE levels in the serum while as chemiluminescence was used to determine Vitamin D levels. RESULTS: The PCR-RFLP analysis indicated a lack of statistically significant variation in genotypic and allelic frequencies between the cases and controls (p > 0.05) for - 9147 C/T SNP either in total atopy (OR-0.70, 95% CI=0.43-1.12, p =0.15), and on subgroup stratifications of chronic urticaria (OR-0.81, 95 % CI = 0.42-1.59, p = 0.61), allergic rhinitis (OR-0.63, 95 % CI = 0.33-1.19, p = 0.16) and bronchial asthma (OR-0.66,95% CI = 0.29-1.4, p=0.32). There was reduced mRNA expression of SOCS5 in total atopic cases, allergic rhinitis, bronchial asthma and chronic urticaria in comparison to controls which advocates the fact that SOCS5 has a protective role in allergic disease development. Despite the reduced amounts of IL-12 in total atopic cases and different allergic disorders in comparison to controls, IL-12 showed significant positive correlation with SOCS5 mRNA expression (p < 0.05). CONCLUSION: SOCS5 SNP rs41379147(C/T) does not pose any significant risk towards the development of any allergic disorder and has no impact on the expression of SOCS5 and IL-12. Our study has shown the reduced mRNA expression of SOCS5 among individuals diagnosed with chronic urticaria, allergic rhinitis and bronchial asthma and the expression of SOCS5 showed complete dependence on the cytokine milieu of IL12. The modulation of SOCS5 and IL-12 may represent potential curative targets for treating the menace of allergic diseases and present promising avenues for future investigation.

Overcoming lung cancer immunotherapy resistance by combining nontoxic variants of IL-12 and IL-2.

Engineered cytokine-based approaches for immunotherapy of cancer are poised to enter the clinic, with IL-12 being at the forefront. However, little is known about potential mechanisms of resistance to cytokine therapies. We found that orthotopic murine lung tumors were resistant to systemically delivered IL-12 fused to murine serum albumin (MSA, IL12-MSA) because of low IL-12 receptor (IL-12R) expression on tumor-reactive CD8+ T cells. IL2-MSA increased binding of IL12-MSA by tumor-reactive CD8+ T cells, and combined administration of IL12-MSA and IL2-MSA led to enhanced tumor-reactive CD8+ T cell effector differentiation, decreased numbers of tumor-infiltrating CD4+ regulatory T cells, and increased survival of lung tumor-bearing mice. Predictably, the combination of IL-2 and IL-12 at therapeutic doses led to significant dose-limiting toxicity. Administering IL-12 and IL-2 analogs with preferential binding to cells expressing Il12rb1 and CD25, respectively, led to a significant extension of survival in mice with lung tumors while abrogating dose-limiting toxicity. These findings suggest that IL-12 and IL-2 represent a rational approach to combination cytokine therapy whose dose-limiting toxicity can be overcome with engineered cytokine variants.

A phase I clinical trial of oncolytic adenovirus mediated suicide and interleukin-12 gene therapy in patients with recurrent localized prostate adenocarcinoma.

In a phase I dose escalation and safety study (NCT02555397), a replication-competent oncolytic adenovirus expressing yCD, TK and hIL-12 (Ad5-yCD/mutTKSR39rep-hIL-12) was administered in 15 subjects with localized recurrent prostate cancer (T1c-T2) at increasing doses (1 × 1010, to 1 × 1012 viral particles) followed by 7-day treatment of 5-fluorocytosine (5-FC) and valganciclovir (vGCV). The primary endpoint was toxicity through day 30 while the secondary and exploratory endpoints were quantitation of IL-12, IFNγ, CXCL10 and peripheral blood mononuclear cells (PBMC). The study maximum tolerated dose (MTD) was not reached indicating 1012 viral particles was safe. Total 115 adverse events were observed, most of which (92%) were grade 1/2 that did not require any treatment. Adenoviral DNA was detected only in two patients. Increase in IL-12, IFNγ, and CXCL10 was observed in 57%, 93%, and 79% patients, respectively. Serum cytokines demonstrated viral dose dependency, especially apparent in the highest-dose cohorts. PBMC analysis revealed immune system activation after gene therapy in cohort 5. The PSA doubling time (PSADT) pre and post treatment has a median of 1.55 years vs 1.18 years. This trial confirmed that replication-competent Ad5-IL-12 adenovirus (Ad5-yCD/mutTKSR39rep-hIL-12) was well tolerated when administered locally to prostate tumors.

Genetically engineered human umbilical cord-derived mesenchymal stem cells expressing human interleukin-12 and in vitro growth inhibition against lung adenocarcinoma cells.

INTRODUCTION: Mesenchymal stromal cells (MSCs) are promising vehicles for cancer therapy due to their homing ability and potency to be genetically manipulated through either viral or non-viral methods. Interleukin-12 (IL-12) is one of the key immunomodulatory cytokines which has anti-tumour effect. However, systemic administration of the cytokine at therapeutic dosage can cause serious toxicity in the host system due to the high systemic level of interferon-γ (IFN-γ) induced. OBJECTIVES: This study aimed to investigate the in vitro growth inhibition of genetically engineered human umbilical cord-derived mesenchymal stromal cells (hUCMSC) expressing IL-12 on H1975 human lung adenocarcinoma cells. MATERIALS AND METHODS: Both adenoviral method and electroporation which used to generate hUCMSC-IL12 were compared. The method with better outcome was selected to generate hUCMSC-IL12 for the co-culture experiment with H1975 or MRC-5 cells. Characterisation of hUCMSC and hUCMSC-IL12 was performed. RESULTS: Adenoviral method showed superior results in transfection efficiency (63.6%), post-transfection cell viability (82.6%) and hIL-12 protein expression (1.2 x 107 pg/ml) and thus was selected for the downstream experiments. Subsequently, hUCMSC-IL12 showed significant inhibition effect on H1975 cells after 5 days of co-culture. No significant difference was observed for all other co-culture groups, indicating that the inhibition effect was because of hIL-12. Lastly, the integrity of hUCMSC-IL12 remained unaffected by the transduction through examination of their surface markers and differentiation properties. CONCLUSION: This study provided proof of concept that hUCMSC can be genetically engineered to express hIL-12 which exerts direct growth inhibition effect on human lung adenocarcinoma cells.