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.

Armored modified vaccinia Ankara in cancer immunotherapy.

Cancer immunotherapy relies on unleashing the patient´s immune system against tumor cells. Cancer vaccines aim to stimulate both the innate and adaptive arms of immunity to achieve durable clinical responses. Some roadblocks for a successful cancer vaccine in the clinic include the tumor antigen of choice, the adjuvants employed to strengthen antitumor-specific immune responses, and the risks associated with enhancing immune-related adverse effects in patients. Modified vaccinia Ankara (MVA) belongs to the family of poxviruses and is a versatile vaccine platform that combines several attributes crucial for cancer therapy. First, MVA is an excellent inducer of innate immune responses leading to type I interferon secretion and induction of T helper cell type 1 (Th1) immune responses. Second, it elicits robust and durable humoral and cellular immunity against vector-encoded heterologous antigens. Third, MVA has enormous genomic flexibility, which allows for the expression of multiple antigenic and costimulatory entities. And fourth, its replication deficit in human cells ensures a excellent safety profile. In this review, we summarize the current understanding of how MVA induces innate and adaptive immune responses. Furthermore, we will give an overview of the tumor-associated antigens and immunomodulatory molecules that have been used to armor MVA and describe their clinical use. Finally, the route of MVA immunization and its impact on therapeutic efficacy depending on the immunomodulatory molecules expressed will be discussed.

A novel imidazole-based azo molecule: synthesis, characterization, quantum chemical calculations, molecular docking, molecular dynamics simulations and ADMET properties.

CONTEXT: Today, the treatment or prevention of cancer, which is one of the most important causes of death, has a very important place. On the other hand, the discovery of new antimicrobial agents is also important because of antibiotic resistance that can occur in humans. For these reasons, in this study, the synthesis, quantum chemical calculations, and in silico studies of a novel azo molecule with high bioactive potential were carried out. In the first step of the synthesis part, (3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)aniline compound, which is the raw material of the drug used in cancer treatments, was synthesized. In the second step, a novel product 2-hydroxy-5-((3-(4-methyl-1H-imidazol-1-yl)-5-trifluoromethyl)phenyl)diazenyl)benzaldehyde (HTB) was obtained as a result of the reaction of salicylaldehyde coupling to this compound. Then, as it was being spectroscopically described, its geometry was optimized. In order to perform quantum chemical calculations, the molecular structure, vibrational spectroscopic data, electronic transition absorption wavelengths, HOMO and LUMO analyses, molecular electrostatic potential (MEP) and potential energy surface (PES) of the molecule were all taken into consideration. Using molecular docking simulations, in silico interactions of the HTB molecule with some anticancer and antibacterial-related proteins were studied. In addition, the ADMET parameters of the HTB were also predicted. METHODS: The structure of the synthesized compound was elucidated using 1H-NMR, 13C-NMR (APT), 19F-NMR, FT-IR and UV-vis spectroscopic methods. The optimized geometry, molecular electrostatic potential diagram and vibrational frequencies of the HTB molecule were calculated at the DFT/B3LYP/6-311G(d,p) level. The TD-DFT method was used to calculate HOMOs-LUMOs and electronic transitions, and the GIAO method was used to calculate chemical shift values. It was observed that the experimental spectral data were in good agreement with the theoretical ones. Molecular docking simulations of the HTB molecule using 4 different proteins were investigated. Two of these proteins were involved in simulating anticancer activity and the other two in simulating antibacterial activity. According to molecular docking studies, the binding energies of the complexes formed by the HTB compound with the 4 selected proteins were between -9.6 and -8.7 kcal/mol. HTB showed the best affinity with VEGFR2 protein (PDB ID: 2XIR) and the binding energy of this interaction was found to be -9.6 kcal/mol. The HTB-2XIR interaction was examined with molecular dynamics simulation for 25 ns and it was determined that this complex was stable during this time. In addition, the ADMET parameters of the HTB were also calculated, and from these values, it was determined that the compound has very low toxicity and high oral bioavailability.

Preclinical development of a first-in-class vaccine encoding HER2, Brachyury and CD40L for antibody enhanced tumor eradication.

The induction of antiviral innate immunity by systemic immunization with live virus can be employed to positively impact the response to therapeutic vaccination. We previously demonstrated that systemic immunization with a non-replicating MVA encoding CD40 ligand (CD40L) enhances innate immune cell activation and function, and triggers potent antitumor CD8+ T cell responses in different murine tumor models. Antitumor efficacy was increased when combined with tumor targeting antibodies. Here we report the development of TAEK-VAC-HerBy (TVH), a first-in-class human tumor antibody enhanced killing (TAEK) vaccine based on the non-replicating MVA-BN viral vector. It encodes the membrane bound form of human CD40L, HER2 and the transcription factor Brachyury. TVH is designed for therapeutic use in HER2- or Brachyury-expressing cancer patients in combination with tumor targeting antibodies. To preclude possible oncogenic activities in infected cells and to prevent binding of vaccine-encoded HER2 by monoclonal antibodies trastuzumab and pertuzumab, genetic modifications of HER2 were introduced in the vaccine. Brachyury was genetically modified to prevent nuclear localization of the protein thereby inhibiting its transcriptional activity. CD40L encoded in TVH enhanced human leukocyte activation and cytokine secretion in vitro. Lastly, TVH intravenous administration to non-human primates was proven immunogenic and safe in a repeat-dose toxicity study. Nonclinical data presented here highlight TVH as a first-in-class immunotherapeutic vaccine platform currently under clinical investigation.

Comparative in vitro and DFT antioxidant studies of phenolic group substituted pyridine-based azo derivatives.

Two azo compounds 2-(3-pyridylazo)-3,5-dihydroxybenzoic acid (PAB) and 4-(3-pyridylazo)resorcinol (PAR) thought to have the potential to be used as antioxidants were designed, synthesized and antioxidant activities were investigated both in vitro and in silico. The synthesized compounds were characterized by 1H-NMR, 13C-NMR, FT-IR, UV-Vis and mass spectra. The molecular geometry and vibrational frequency calculations of the synthesized compounds in ground state were performed by the density functional theory (DFT) employing B3LYP level with the 6-311 G(d,p) basis set. The gauge independent atomic orbital (GIAO) method was used to determine the chemical shift values of 1H-NMR and 13C-NMR. HOMO-LUMO calculations were carried out by time-dependent DFT (TD-DFT) approach. Computational spectroscopic data of the synthesized compounds are fully compatible with experimental ones. The antioxidant activities of the PAB and PAR were investigated by using DPPH assay. It was determined that the PAB molecule showed better antioxidant activity than PAR and, butylated hydroxytoluene (BHT) which is the standard antioxidant. In addition, the thermodynamic stability parameters obtained with the help of DFT calculations were found to be quite compatible with the antioxidant capacity sequence derived from the DPPH assay. HighlightsTwo pyridine derivative azo compounds were synthesized and evaluated for its antioxidant activityMolecular geometry and spectroscopic properties of the molecules were calculatedin vitro and in silico antioxidant activities were investigated by DPPH free radical scavenging assayThe PAB molecule showed better antioxidant activity than BHTCommunicated by Ramaswamy H. Sarma.

Functional analysis of peripheral and intratumoral neoantigen-specific TCRs identified in a patient with melanoma.

BACKGROUND: Neoantigens derived from somatic mutations correlate with therapeutic responses mediated by treatment with immune checkpoint inhibitors. Neoantigens are therefore highly attractive targets for the development of therapeutic approaches in personalized medicine, although many aspects of their quality and associated immune responses are not yet well understood. In a case study of metastatic malignant melanoma, we aimed to perform an in-depth characterization of neoantigens and respective T-cell responses in the context of immune checkpoint modulation. METHODS: Three neoantigens, which we identified either by immunopeptidomics or in silico prediction, were investigated using binding affinity analyses and structural simulations. We isolated seven T-cell receptors (TCRs) from the patient's immune repertoire recognizing these antigens. TCRs were compared in vitro by multiparametric analyses including functional avidity, multicytokine secretion, and cross-reactivity screenings. A xenograft mouse model served to study in vivo functionality of selected TCRs. We investigated the patient's TCR repertoire in blood and different tumor-related tissues over 3 years using TCR beta deep sequencing. RESULTS: Selected mutated peptide ligands with proven immunogenicity showed similar binding affinities to the human leukocyte antigen complex and comparable disparity to their wild-type counterparts in molecular dynamic simulations. Nevertheless, isolated TCRs recognizing these antigens demonstrated distinct patterns in functionality and frequency. TCRs with lower functional avidity showed at least equal antitumor immune responses in vivo. Moreover, they occurred at high frequencies and particularly demonstrated long-term persistence within tumor tissues, lymph nodes and various blood samples associated with a reduced activation pattern on primary in vitro stimulation. CONCLUSIONS: We performed a so far unique fine characterization of neoantigen-specific T-cell responses revealing defined reactivity patterns of neoantigen-specific TCRs. Our data highlight qualitative differences of these TCRs associated with function and longevity of respective T cells. Such features need to be considered for further optimization of neoantigen targeting including adoptive T-cell therapies using TCR-transgenic T cells.

The prognostic and predictive significance of plasma type 1 plasminogen activator inhibitor and endoglin in metastatic colorectal cancer patients treated with bevacizumab-containing chemotherapy.

AIM: This study aims to evaluate the prognostic and predictive value of plasma plasminogen activator inhibitor-1 (PAI-1) and endoglin in metastatic colorectal cancer (mCRC) patients receiving chemotherapy with bevacizumab. MATERIALS AND METHODS: Between April 2012 and September 2013, 47 mCRC patients with a mean age of 58.5 ± 9.6 years were included in the study. Male-to-female ratio was 29/18. The baseline and posttreatment plasma PAI-1 and serum endoglin levels after 3 cycles of bevacizumab-containing chemotherapy were evaluated. The percent change between baseline and posttreatment levels after treatment was also recorded. RESULTS: The median follow-up duration was 26.6 months (range 1.8-70.2 months). The clinical benefit rate was 70% (partial response [32%], stable disease [38%]). Overall survival was 20.8 ± 1.5 months. The patients with progressive disease had statistically significantly higher baseline PAI-1 level (57.9 pg/mL vs. 29.9 pg/mL, P = 0.036). The percent change of the plasma PAI-1 level after the third cycle of treatment was also statistically significantly lower in those with clinical benefit (P = 0.035). However, there was no statistically significant difference in endoglin level and its change after therapy with respect to the response to treatment (P = 0.771 and P = 0.776, respectively). Plasma PAI-1 level had no statistically significant effect on survival (P = 0.709). CONCLUSION: Baseline plasma PAI-1 level and its percent change with bevacizumab were shown to have statistically significant predictive value for the response to therapy whereas serum endoglin had no statistically significant predictive value for the response to therapy. However, neither PAI-1 nor endoglin had prognostic significance in mCRC.

BRAF Targeting Sensitizes Resistant Melanoma to Cytotoxic T Cells.

PURPOSE: BRAF and MEK inhibitors (BRAFi and MEKi) are actively used for the treatment of metastatic melanoma in patients with BRAFV600E mutation in their tumors. However, the development of resistance to BRAFi and MEKi remains a difficult clinical challenge with limited therapeutic options available to these patients. In this study, we investigated the mechanism and potential therapeutic utility of combination BRAFi and adoptive T-cell therapy (ACT) in melanoma resistant to BRAFi. EXPERIMENTAL DESIGN: Investigations were performed in vitro and in vivo with various human melanoma cell lines sensitive and resistant to BRAFi as well as patient-derived xenografts (PDX) derived from patients. In addition, samples were evaluated from patients on a clinical trial of BRAFi in combination with ACT. RESULTS: Herein we report that in human melanoma cell lines, senstitive and resistant to BRAFi and in PDX from patients who progressed on BRAFi and MEKi therapy, BRAFi caused transient upregulation of mannose-6-phosphate receptor (M6PR). This sensitized tumor cells to CTLs via uptake of granzyme B, a main component of the cytotoxic activity of CTLs. Treatment of mice bearing resistant tumors with BRAFi enhanced the antitumor effect of patients' TILs. A pilot clinical trial of 16 patients with metastatic melanoma who were treated with the BRAFi vemurafenib followed by therapy with TILs demonstrated a significant increase of M6PR expression on tumors during vemurafenib treatment. CONCLUSIONS: BRAF-targeted therapy sensitized resistant melanoma cells to CTLs, which opens new therapeutic opportunities for the treatment of patients with BRAF-resistant disease.See related commentary by Goff and Rosenberg, p. 2682.

Serum and knee synovial fluid matrixmetalloproteinase-13 and tumor necrosis factor-alpha levels in patients with late stage osteoarthritis.

OBJECTIVE: To compare the levels of MMP-13 and TNF-α in late stage osteoarthritis, define their predominant pathways and investigate their correlation with McMaster Universities Arthritis Index scores. PATIENTS AND METHODS: A total of 42 patients (mean age 64 ± 8.8) with grade 3 and grade 4 knee osteoarthritis according to Kellegren- Lawrence criteria and who were scheduled for total knee arthroplasty were enrolled in the study. TNF-alpha and MMP-13 levels were measured preoperatively from venous blood samples and intraoperatively from knee synovial fluid via ELISA. Preoperative and 1 month postoperative knee functions were assessed by McMaster Universities Arthritis Index. RESULTS: Grade 4 synovial fluid MMP-13 (4.76 ± 5.82) was elevated compared to grade 3 (3.95 ± 4.45) (p = 0.438), whereas grade 3 serum MMP-13 (1.128 ± 0.308) was found elevated compared to grade 4 (1.038 ± 0.204) (p = 0.430). Grade 4 serum TNF-α (0.253 ± 0.277) was elevated compared to grade 3 (0.206 ± 0.219) whereas grade 3 synovial fluid TNF-α (0.129 ± 0.052) was elevated compared to grade 4 (0.118 ± 0.014). Positive correlation was observed between synovial fluid MMP-13 levels and postoperative WOMAC scores. Mean serum TNF-α level (0.226 ± 0.246 pg/ml) was found higher compared to synovial level (0.124 ± 1.59), synovial MMP-13 level (4.31 ± 1.24) was found higher compared to serum level (1.089 ± 1.519). CONCLUSION: Despite the systemic increase in TNF-α levels concordant with osteoarthritis grade, MMP-13 levels are elevated via local manner with a significant correlation with WOMAC scores. LEVEL OF EVIDENCE: Level IV, Diagnostic study.

Serum and knee synovial fluid matrix metalloproteinase-13 and tumor necrosis factor-alpha levels in patients with late-stage osteoarthritis.

OBJECTIVE: The aim of this study was to compare serum and synovial fluid levels of matrix metalloproteinase-13 (MMP-13) and tumor necrosis factor-alpha (TNF-α) in 2 stages of osteoarthritis, and investigate their correlation with Western Ontario and McMaster Universities Arthritis Index (WOMAC) scores. METHODS: Forty-two patients (mean age: 64±8.8 years) with grade 3 and grade 4 knee osteoarthritis according to Kellgren-Lawrence criteria were enrolled in the study and underwent total knee arthroplasty. TNF-α and MMP-13 levels were measured preoperatively from venous blood samples and intraoperatively from knee synovial fluid via enzyme-linked immunosorbent assay. Preoperative and 1-month postoperative knee functions were assessed by WOMAC. RESULTS: Grade 4 synovial fluid MMP-13 (4.76±5.82 pg/ml) was elevated compared to grade 3 (3.95±4.45), whereas grade 3 serum MMP-13 (1.128±0.308 pg/ml) was found elevated compared to grade 4 (1.038±0.204) (p=0.438, p=0.430, respectively). Grade 4 serum TNF-α (0.253±0.277) was elevated compared to grade 3 (0.206±0.219), whereas grade 3 synovial fluid TNF-α (0.129±0.052) was elevated compared to grade 4 (0.118±0.014). Positive correlation was observed between synovial fluid MMP-13 levels and postoperative WOMAC scores. Mean serum TNF-α (0.226±0.246 pg/ml) was elevated compared to synovial levels (0.124±1.59), and synovial MMP-13 (4.31±1.24) was elevated compared to serum levels (1.089±1.519). CONCLUSION: Despite the systemic increase in TNF-α levels concordant with osteoarthritis grade, MMP-13 levels are elevated via local manner, with a significant correlation with WOMAC scores.

Impact of oxidative stress on early postoperative knee function and muscle injury biochemical markers: Is it possible to create an ischemic preconditioning effect in sequential ischemic surgical procedures?

OBJECTIVE: Simultaneous bilateral total knee arthroplasty (TKA) with prolonged tourniquet time has the potential to trigger ischemia-reperfusion injury, which can adversely affect knee function. Studies suggest that the magnitude of injury is less if it occurs following an ischemic event which takes place in another part of the body, known as ischemic preconditioning (IPC). The purpose of this study was to investigate the impact of oxidative stress on muscle injury and knee function and to elucidate if potential IPC effect can attenuate ischemia-reperfusion injury metabolites and prevent poor functional outcomes in single-stage bilateral TKA. METHODS: Thirty patients who underwent single-stage bilateral TKA under tourniquet were enrolled in the study. All procedures were initiated from the right limb. Upon completion of the procedure, the left tourniquet was inflated 20 minutes after the first tourniquet was deflated. The tourniquet time was noted. Pre- and postoperative levels of malondialdehyde (MDH), creatine kinase (CK), and lactate dehydrogenase (LDH) were evaluated. Knee function was assessed postoperatively at 1 month using WOMAC score. RESULTS: Postoperative levels of MDH, CK, and LDH were significantly increased in both extremities compared to preoperative levels. Serum MDH, CK, and LDH levels were not found to be correlated with tourniquet time for either extremity. Compared to the left extremity, the right extremity revealed increased postoperative oxidative stress, which was indicated by elevated serum MDH, CK, and LDH levels. Although tourniquet time and postoperative serum MDH, CK, and LDH levels were not found to be correlated with WOMAC index in either knee, the average change in WOMAC score at 1 month postoperatively was found to be higher in the left knee compared to the right. CONCLUSION: The biochemical and functional outcomes can be attributed to potential IPC effect. During bilateral TKA, a 20-minute interval between tourniquets can create IPC effect and attenuate the magnitude of ischemia-reperfusion injury, preserving better functional outcomes.

High-fat-diet-mediated dysbiosis promotes intestinal carcinogenesis independently of obesity.

Several features common to a Western lifestyle, including obesity and low levels of physical activity, are known risk factors for gastrointestinal cancers. There is substantial evidence suggesting that diet markedly affects the composition of the intestinal microbiota. Moreover, there is now unequivocal evidence linking dysbiosis to cancer development. However, the mechanisms by which high-fat diet (HFD)-mediated changes in the microbial community affect the severity of tumorigenesis in the gut remain to be determined. Here we demonstrate that an HFD promotes tumour progression in the small intestine of genetically susceptible, K-ras(G12Dint), mice independently of obesity. HFD consumption, in conjunction with K-ras mutation, mediated a shift in the composition of the gut microbiota, and this shift was associated with a decrease in Paneth-cell-mediated antimicrobial host defence that compromised dendritic cell recruitment and MHC class II molecule presentation in the gut-associated lymphoid tissues. When butyrate was administered to HFD-fed K-ras(G12Dint) mice, dendritic cell recruitment in the gut-associated lymphoid tissues was normalized, and tumour progression was attenuated. Importantly, deficiency in MYD88, a signalling adaptor for pattern recognition receptors and Toll-like receptors, blocked tumour progression. The transfer of faecal samples from HFD-fed mice with intestinal tumours to healthy adult K-ras(G12Dint) mice was sufficient to transmit disease in the absence of an HFD. Furthermore, treatment with antibiotics completely blocked HFD-induced tumour progression, suggesting that distinct shifts in the microbiota have a pivotal role in aggravating disease. Collectively, these data underscore the importance of the reciprocal interaction between host and environmental factors in selecting a microbiota that favours carcinogenesis, and they suggest that tumorigenesis is transmissible among genetically predisposed individuals.

Shock the heat shock network.

The targeting of tumors is made possible through establishing protein signatures specific for each cancer type. The recent recognition of the higher expression levels of HSP90 and its accumulation in tumor cell mitochondria has made the HSP90 network a feasible target for neutralization. HSP90 antagonizes the mitochondrial permeability transition,blocking cytochrome c release and apoptosis. In this issue of the JCI, Kang et al. report the synthesis of Gamitrinibs, which target mitochondrially localized HSP90, specifically killing human cancer cell lines, and provide a fresh approach for cancer treatment.

Diagnostic value of blood D-dimer level in acute mesenteric ischaemia in the rat: an experimental study.

OBJECTIVE: The high mortality rate of mesenteric ischaemia is mainly due to delay in diagnosis. For this reason, it is of great importance to find a specific and rapidly elevating marker. The present study investigated the diagnostic value of blood D-dimer level as a potential marker for acute mesenteric ischaemia in a rat model. METHODS: Thirty male Wistar albino rats were divided into three groups. Basal D-dimer and L-lactate levels were determined in the non-operative control group (I). In the operated control group (II), the superior mesenteric artery was simply manipulated, while the artery was ligated in Group III. Blood samples were drawn in all groups for D-dimer and L-lactate assays. RESULTS: Both Group II (p=0.016) and Group III (p=0.001) had significantly higher mean D-dimer levels in the first postoperative hour compared with the basal level in Group I. However, there was no difference between the levels in Groups II and III. The mean level in Group II in the sixth hour had dropped to a statistically insignificant level compared with the basal value, while the mean value in Group III kept rising during this period (p=0.001). Nevertheless, there was no significant difference between Groups II and III. On the other hand, the mean L-lactate level in the first postoperative hour in Group III was significantly higher than the basal level in Group I (p=0.003). No significant rises were recorded in Group II in the first and sixth postoperative hours. The difference between Groups II and III in the first hour was significant (p=0.005). Group III also had significantly higher mean serum L-lactate value in the sixth hour compared with both the basal value in Group I (p=0.001) and the sixth-hour value in Group II (p=0.003). CONCLUSION: These results do not adequately support the use of blood D-dimer level as an independent parameter in the diagnosis of mesenteric ischaemia due to arterial thrombosis. However, this parameter can be used together with other tests in eliminating the possibility of a thromboembolic event.