Recombinant Human Interferon-gamma Research Articles

Study on the Recombinant Human Interferon α1b, α2b, and Gamma Transient Expression and in Vitro Activities in Tobacco.

Interferons (IFNs) are universally acknowledged for their pivotal role in antiviral and anticancer responses. Thus, the primary aim of our study was to explore the expressions of IFN-α1b, α2b, and gamma in tobacco leaves via agrobacterium-mediated transient transformation and investigate their possible activities. Briefly, fusion with green fluorescent protein tags aided in detecting the expressed IFN proteins in the foliar tissues. The genetic constructs encoding these fusion proteins were inserted into the MagnICON plant transient expression vector, followed by transformation into the Agrobacterium strain GV3101. The transformed bacteria were then used to infiltrate tobacco leaves. After post-infiltration, protein expression was confirmed within 72 h via sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the fusion proteins were subsequently purified using high-performance liquid chromatography for identification. Both the antiviral and anticancer potencies of these IFN fusion proteins were evaluated using the WISH/VSV (WISH cells/Vesicular stomatitis virus) microneutralization and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, respectively. Results indicated robust expression of the targeted IFN genes in plant tissues and significant biological activities against pathogens and cancer cells. Consequently, this study substantiated the viability of producing these therapeutic proteins in plants, potentially revolutionizing the manufacture of interferons biologically.

<i>In vitro</i> activity of human recombinant interferon gamma against SARS-CoV-2 virus

The development of drugs against SARS-CoV-2 continues to be crucial for reducing the spread of infection and associated mortality. The aim of the work is to study the neutralization of the SARS-CoV-2 virus with interferon gamma preparations in vitro. The activity of recombinant human interferon gamma for intramuscular and subcutaneous administration of 500,000 IU and for intranasal administration of 100,000 IU against the SARS-CoV-2 virus in vitro was studied. The methodological approach of this study is based on the phenomenon of a decrease in the number of plaques formed under the action of a potential antiviral drug. The antiviral activity of recombinant interferon gamma has been experimentally confirmed, both in preventive and therapeutic application schemes. The smallest number of plaques was observed with the preventive scheme of application of the tested object at concentrations of 1000 and 333 IU/ml. The semi-maximal effective concentration (EC50) with the prophylactic regimen was 24 IU/ml. The preventive scheme of application of the tested object turned out to be more effective than therapeutic one, which is probably explained by the launch of the expression of various interferon-stimulated genes that affect to a greater extent the steps of virus entry into the cell and its reproduction. Further study of the effect of drugs based on recombinant interferon gamma on the reproduction of the SARS-CoV-2 virus for clinical use for prevention and treatment is highly relevant.

Evaluation of Different Signal Peptides for Secretory Production of Recombinant Human Interferon-gamma: Bioinformatics Approach

Background: The fusion of the secretory signal peptide to the N-terminal of polypeptide’s amino acid sequence is an attractive technique for the secretory production of heterologous proteins. On the other hand, applying computational analysis may be beneficial to overcome the barriers of trial-and-error approaches in detecting proper signal sequences. As the scope of this study, the most probable effective properties of 30 signal sequences for the extracellular production of recombinant human interferon-gamma (rhIFN-γ) were analysed. Methods: The fusion of the secretory signal peptide to the N-terminal of a polypeptide’s amino acid sequence is an attractive technique for the secretory production of heterologous proteins. On the other hand, applying computational analysis may be beneficial in overcoming the barriers of trial-anderror approaches in detecting proper signal sequences. As the scope of this study, the most probable effective properties of 30 signal sequences for the extracellular production of recombinant human interferon-gamma (rhIFN-γ) were analysed. Results: Finally, 12 high probable signal peptides, including OmpC, PhoE, AnsB, and OmpA, were theoretically detected with ideal solubility probabilities and almost balanced physicochemical properties; hopes to be helpful in future experimental studies for the secretion of rhIFN-γ. Conclusion: The experimental analysis is required to validate the in silico results and focus on in-lab affecting factors such as cultivation methods and conditions.

Analysis of neutrophil functional activity in patients with chronic polyposis rhinosinusitis after treatment with interferon gamma.

Chronic polyposis rhinosinusitis (CPRS) is an inflammatory disease of the nose and paranasal sinuses, accompanied by the formation and recurrent growth of polyps. PDRS is an urgent medical problem, because it is difficult to treat and is accompanied by constant exacerbations. The important role of neutrophil granulocytes in the pathogenesis of CPRS has been proved, as they are the first line of defense in response to tissue damage and active participants in the pathological process. There is evidence of successful use of immunocorrectors in the treatment of patients with CPPS, but they are often prescribed without regard to possible pathogenetic mechanisms of the disease. One of the promising immunomodulators of local use is a preparation of human recombinant interferon gamma. It is known that interferon gamma is able to activate neutrophils due to the receptors to this cytokine, which are located on their surface. The aim of the study was to investigate the functional activity of neutrophils in patients with CPPS and the effect of human recombinant interferon gamma on these indicators. Thirty-five patients with CHRS were examined before and after therapy with intranasal interferon gamma. The control group included 30 healthy subjects. Functional activity of neutrophils was studied in whole blood by chemiluminescent method using double stimulation. Patients with CPRS before treatment revealed increased indexes of neutrophils stimulated activity, maximal intensity of cells luminescence, activation coefficient and decreased time of neutrophils output at maximal luminescence. After treatment with intranasal preparation of interferon gamma there was significant decrease of spontaneous and stimulated activity of neutrophils and maximum intensity of cell luminescence. As a result, after the treatment, in patients with CHRS the values of stimulated production of neutrophils and maximum intensity of cell luminescence were reduced to the level of the control group, and the spontaneous activity of neutrophils was even lower than in healthy subjects, while the neutrophils activation factor remained elevated as in patients before therapy. The results obtained testify to normalization of the main indexes of neutrophil functional activity in CHPS patients after treatment with human recombinant interferon gamma.

ImmunoSep (Personalised Immunotherapy in Sepsis) international double-blind, double-dummy, placebo-controlled randomised clinical trial: study protocol

IntroductionSepsis is a major cause of death among hospitalised patients. Accumulating evidence suggests that immune response during sepsis cascade lies within a spectrum of dysregulated host responses. On the one...

Stability-indicative and conformation-specific enzyme linked immunosorbent assay for analysis of recombinant human gamma interferon

Recombinant human interferon gamma (rhIFN-γ) is a promising molecule for the treatment of several diseases. A pair of conformation-specific monoclonal antibodies (mAbs) against rhIFN-γ was selected from generated hybridoma cell lines to design a sensitive, stability-indicative, sandwich-type ELISA. The main assay parameters were optimized by the checkerboard method for the highest signal-to-noise ratio: assay buffer composition, coating buffer pH and composition, coating temperature-incubation time parameters, and coating mAb concentration and conjugate dilution. Detection and quantification limits were estimated between 0.019 and 0.078 ng/mL, respectively, and recovery values were from 92.03% to 98.40%. The coefficient of variation of intra-assay precision parameters ranged from 2.32% to 9.21% while the inter-analyst variation was between 4.70% and 10.63%, supporting the method’s repeatability. The ELISA was specific for correctly folded and non-aggregated molecular species, as compared to intrinsic Trp fluorescence (chemical denaturation) and optical density at 340 nm (thermal aggregation), respectively. However, the method was not sensitive to the small C-terminal degradation of full-length rhIFN-γ1–144 (losses of 6–12 amino acid residues) as compared to results with mass spectrometry and gel electrophoresis. ELISA showed good correlation with rhIFN-γ antiviral biological activity. This method was applied to the stability evaluation of rhIFN-γ in physiological buffer at low concentrations using polypropylene and glass vials also in the presence of adsorption protectant excipients. Furthermore, ELISA could be adapted to other applications such as quantification of IFN-γ in serum samples, Mycobacterium tuberculosis diagnosis, etc.

Antitumor effect of recombinant interferon-gamma in an experimental model of Ehrlich’s bilateral solid carcinoma

Introduction.Immunotherapy, which is part of the complex and combined cancer therapy, is one of the priority areas in the treatment of cancer patients. However, the effectiveness of the use of immunotherapeutic drugs of the latest generation is not so high, and in some patients the effect of therapy was short-lived. Factors that prevent the full realization of the antitumor effect of cytostatics and immunopreparations may be the features of the antigenic composition of the tumor, as well as its cellular and stromal microenvironment. These facts contributed to the development of a new strategy, designated as immunoredaction of cancer by exposure to various biologically active agents that can change the body – tumor ratio in favor of the patient and make the tumor available for the implementation of antitumor effects of the host immune system.The study objective– experimental substantiation of the development of new immunotherapeutic approaches in the treatment of aggressive forms of cancer.Materials and methods.An experimental study of the effect of human recombinant interferon-gamma (IFNγ) on the growth of Ehrlich’s carcinoma during subcutaneous bilateral transplantation of tumor cells to animals was carried out. Transplantation of Ehrlich’s carcinoma to male F1 hybrids (SWAhC57Bl6) was performed by subcutaneous injection of 2.0 × 106 tumor cells (7‑day culture) in 0.1 ml of suspension into the lateral surface of the right and left femur with imitation of multicentric growth.Results.A day after the course of drug administration (day 6 of tumor node growth), the effect of suppressing tumor growth in relation to growth in the control group was noted. The maximum inhibition effect of 19.8 % (p<0.05) of tumor growth was obtained 5 days after the course of the drug (10 days of tumor growth, right node) and 18.5 % (p<0.001) 9 days after administration (14 days of tumor growth, left node).Conclusion.Thus, a distinct, statistically significant antitumor effect of IFNγ was established in relation to a tumor with a multicentric growth pattern.

SLOW QUASIKINETIC CHANGES IN WATER-LACTOSE COMPLEXES DURING STORAGE

Objective: To investigate kinetic changes in the spectral characteristics by Fourier Transform Infrared spectroscopy (FTIR) of water-lactose complexes (SMC), derived during the manufacturing process of the drug, containing release-active forms of antibodies.
 Methods: lactose monohydrate substance, saturated with release-active forms of affinity-purified polyclonal rabbit antibodies to recombinant human interferon-gamma (RA forms of Abs); tablets produced from this substance by direct compression after the addition of excipients (microcrystalline cellulose, magnesium stearate). Powdered and tableted placebo samples saturated with technologically processed water or phosphate-buffered saline, as well as with intact ethanol were used as control. Kinetic changes in SMC were studied using an Agilent Cary 630 FTIR spectrophotometer with a diamond ATR accessory (Agilent Technologies, USA). We used the method of X-ray fluorescence spectroscopy (EDX-7000 Shimadzu energy dispersive X-ray fluorescence spectrometer) to track changes in the fluorescence signal at certain wavelengths. The range of measured elements–11Na-92U.
 Results: Control of some technological characteristics of the obtained active substance (moisture, flowability) and dosage form (mean mass, disintegration rate) was used as indirect indicators of quality, but they did not allow reliably distinguishing intact lactose from the saturated one. Long-period oscillations on FTIR spectra were characteristic for all types of samples; oscillations occur at approximately two-week intervals; S/N indices were more stable for samples of RA forms of Abs than for placebo samples. On some days, the substance saturated with RA forms of Abs significantly differed from the intact lactose powder. The kinetics of the X-ray fluorescence intensity at certain wavelengths indicates the possibility of a periodic cooperative trigger transition of the system. Reversible conformational transitions are observed for powders on the 30th and 130th days (Kα 3.313 keV). For tablets at Kα 3.313 keV and Kα 1.740 keV small changes were visualized on those days (100–110th day) when hysteresis phenomena were recorded in the IR spectra of these samples.
 Conclusion: As a result, the evidence for a long-period dramatic conformational mobility of the water-lactose complex was obtained. Based on the data on the semiannual kinetics of IR spectra, a universal criterion for the identity of lactose powder saturated with RA forms of Abs was obtained. Also, it was confirmed that the lactose conformation state was changed by saturation with RA forms of Abs.

Angiotensin-converting Enzyme 2–containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

Angiotensin-converting Enzyme 2–containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

Type I Interferons Ameliorate Zinc Intoxication of Candida glabrata by Macrophages and Promote Fungal Immune Evasion.

SummaryHost and fungal pathogens compete for metal ion acquisition during infectious processes, but molecular mechanisms remain largely unknown. Here, we show that type I interferons (IFNs-I) dysregulate zinc homeostasis in macrophages, which employ metallothionein-mediated zinc intoxication of pathogens as fungicidal response. However, Candida glabrata can escape immune surveillance by sequestering zinc into vacuoles. Interestingly, zinc-loading is inhibited by IFNs-I, because a Janus kinase 1 (JAK1)-dependent suppression of zinc homeostasis affects zinc distribution in macrophages as well as generation of reactive oxygen species (ROS). In addition, systemic fungal infections elicit IFN-I responses that suppress splenic zinc homeostasis, thereby altering macrophage zinc pools that otherwise exert fungicidal actions. Thus, IFN-I signaling inadvertently increases fungal fitness both in vitro and in vivo during fungal infections. Our data reveal an as yet unrecognized role for zinc intoxication in antifungal immunity and suggest that interfering with host zinc homeostasis may offer therapeutic options to treat invasive fungal infections.

Glucose-methanol-based fed-batch fermentation for the production of recombinant human interferon gamma (rhIFN-γ) and evaluation of its antitumor potential.

Squamous cell carcinoma (SCC) is nonmelanoma skin cancer, which is very common in patients having T-cell immunosuppressant drugs. Anticancerous agents such as cytokines showed effective response on SCC. Human interferon-gamma (hIFN-γ), a type II cytokines, are having potent antiproliferative and immunomodulatory effects. In the current study, the fed-batch cultivation of recombinant Pichia pastoris was carried out, and its effect on cell biomass production, recombinant human interferon-gamma (rhIFN-γ) production, and the overflow metabolites was estimated. P. pastoris GS115 strain coexpressed with 6-phosphogluconolactonase (SOL3) and ribulose-phosphate 3-epimerase (RPE1) gene (GS115/rhIFN-γ/SR) resulted in 60mgL-1 of rhIFN-γ production, which was twofold higher as compared with the production from GS115/rhIFN-γ strain. The antiproliferative potential of rhIFN-γ was examined on the human squamous carcinoma (A431) cell lines. Cells treated with 80ngmL-1 of rhIFN-γ exhibited 50% growth inhibition by enhancing the production of intracellular reactive oxygen species levels and disrupting membrane integrity. Our findings highlight a state of art process development strategy for the high-level production of rhIFN-γ and its potential application as a therapeutic drug in SCC therapy.

Metabolic engineering of Pichia pastoris GS115 for enhanced pentose phosphate pathway (PPP) flux toward recombinant human interferon gamma (hIFN-γ) production.

In the present study, the effects of individual as well as multiple genes of pentose phosphate pathway (PPP) on human interferon gamma (hIFN-γ) production were analyzed. With overexpression of 6-phosphogluconate dehydrogenase (GND2), 1.9-fold increase in hIFN-γ was achieved, while synergetic effect of 6-phosphogluconolactonase (SOL3) and D-ribulose-5-phosphate 3-epimerase (RPE1) resulted in 2.56-fold increase in hIFN-γ as compared to control. Fed batch fermentation using mixed feeding of gluconate and methanol (carbon source) was carried out, resulting in 80 and 123mgL-1 of hIFN-γ enhancement in recombinant Pichia GS115 strain encoding codon optimized hIFN-γ (GS115/hIFN-γ) and Pichia GS115 strain encoding codon optimized hIFN-γ with co-expressed 6-phosphogluconolactonase(SOL3) and D-ribulose-5-phosphate 3-epimerase (RPE1) (GS115/hIFN-γ/SR) respectively. To get more insight of the flux distribution towards hIFN-γ, studies were carried out by applying flux balance analysis during methanol fed batch phase for both strains. In both strains (GS115/hIFN-γ and GS115/hIFN-γ/SR) more than 95% of formaldehyde flux is directed towards assimilatory pathway. The analysis revealed that with the overexpression of SOL3 and RPE1 the flux towards PPP triggering the alleviation in hIFN-γ production.

Re-engineering of an Escherichia coli K-12 strain for the efficient production of recombinant human Interferon Gamma

The Escherichia coli phosphoglucose isomerase (pgi) mutant strain GALG20 was developed previously from wild-type K12 strain MG1655 for increased plasmid yield. To investigate the potential effects of the pgi deletion/higher plasmid levels on recombinant human Interferon Gamma (IFN-γ) production, a detailed network of the central metabolic pathway (100 metabolites, 114 reactions) of GALG20 and MG1655 was constructed. Elementary mode analysis (EMA) was then performed to compare the phenotypic spaces of both the strains and to check the effect of the pgi deletion on flux efficiency of each metabolic reaction. The results suggested that pgi deletion increases amino acid biosynthesis and flux efficiency towards IFN-γ synthesis by 11%. To further confirm the qualitative prediction that the pgi mutation favours recombinant human IFN-γ expression, GALG20 and MG1655 were lysogenised, transformed with a plasmid coding for IFN-γ and tested alongside with BL21(DE3) for their expression capabilities in shake flask experiments using complex media. IFN-γ gene expression was analysed by quantifying plasmid and mRNA copy number per cell and IFN-γ protein production level. Specific IFN-γ yields confirmed the in silico metabolic network predictions, with GALG20(DE3) producing 3.0-fold and 1.5-fold more IFN-γ as compared to MG1655(DE3) and BL21(DE3), respectively. Most of the total IFN-γ was expressed as inclusion bodies across the three strains: 95% in GALG20(DE3), 97% in BL21(DE3) and 72% in MG1655(DE3). The copy number of mRNA coding for IFN-γ was found to be higher in GALG20(DE3) as compared to the other two strains. Overall, these findings show that GALG20(DE3) has the potential to become an excellent protein expression strain.

Application of medium optimization tools for improving recombinant human interferon gamma production from Kluyveromyces lactis

ABSTRACTThe present study is focused upon improving biomass of Kluyveromyces lactis cells expressing recombinant human interferon gamma (hIFN-γ), with the aim of augmenting hIFN-γ concentration using statistical and artificial intelligence approach. Optimization of medium components viz., lactose, yeast extract, and trace elements were performed with Box–Behnken design (BBD) and artificial neural network linked genetic algorithm (ANN-GA) for maximizing biomass of recombinant K. lactis (objective function). The studies resulted over 1.5-fold improvement in the biomass concentration in a medium composed of 80 g/L lactose, 10.353 g/L yeast extract, and 15 mL/L trace elements as compared with initial biomass value. In the same study hIFN-γ concentration reached 881 µg/L which was 2.28-fold higher as compared with initial hIFN-γ concentration obtained in unoptimized medium. Further the batch fermentation study displayed mixed growth associated kinetics with the maximum hIFN-γ production rate of 1.1 mg/L. BBD and ANN-GA, both optimization techniques predicted a higher lactose concentration was clearly beneficial for augmenting K. lactis biomass which in turn increased hIFN-γ concentration.

Engineering folding mechanism through Hsp70 and Hsp40 chaperones for enhancing the production of recombinant human interferon gamma (rhIFN-γ) in Pichia pastoris cell factory

Pichia pastoris is a well-known host for the production of heterologous proteins, but the incorporation of a foreign gene may lead to metabolic burden and sub optimal folding of proteins in the endoplasmic reticulum (ER) and cytoplasm, ultimately resulting in reduced protein secretion. The Hsp70 and Hsp40 chaperone families in the cytoplasm/ER are required to ensure proper folding and translocation of heterologous proteins. In the present study, we have cloned a gene encoding human interferon gamma in Pichia pastoris (GS115) resulting in a very low expression of recombinant human interferon gamma (rhIFN-γ). To evaluate the role of chaperones in protein production we have co-expressed SEC63P, YDJ1P, SSA1P, KAR2P and PDI genes from Saccharomyces cerevisiae and Pichia pastoris (X-33) respectively. The introduction of individual chaperones viz., Ydj1p, PDI and Ssa1p have enhanced rhIFN-γ production about 4 folds, while the synergistic effect of Kar2p + PDI has shown about 6-fold enhancement in rhIFN-γ production. The batch reactor kinetics with complex medium showed that the product is growth associated and maximum production yield of 1.98 mg L−1 was observed at a 72 hr time interval. To evaluate the efficacy of rhIFN-γ on an anti-proliferative property, we have treated oral cancer cell lines with different concentration of rhIFN-γ and found inhibition of cell proliferation up to 25% at 0.5 µg concentration.

Optimizing secretory expression of recombinant human interferon gamma from Kluyveromyces lactis

ABSTRACTBiosimilar/biotherapeutic production is becoming a major area of focus for a big chunk of biotechnology industry. Easy licensing and already approved status for clinical use have given it a boost. In the present study, recombinant human interferon gamma (IFNG) was expressed for the first time in Kluyveromyces lactis expression system and its expression was optimized by varying growth parameters and carbon source concentration with the aim of increasing recombinant protein production level. Human IFNG gene was cloned in the genomic DNA of K. lactis by homologous recombination and under unoptimized conditions in shake flask, IFNγ protein was secreted in the fermentation medium at a level of 175 µg/L quantified by ELISA assay. After the optimization of expression conditions using one-variable-at-a-time technique, expression level was enhanced by 2.2-folds. Substrate inhibition studies revealed that up to 80 g/L of lactose is well tolerated by K. lactis cells for its growth but more than 80 g/L of lactose causes remarkable reduction in biomass production.

Purification of recombinant human interferon gamma from fermentation broth using reverse micellar extraction: A process optimization study

ABSTRACTIn the present investigation, we have shown a single step purification of IFN-γ from the fermentation broth of recombinant Kluyveromyces lactis (K. lactis) strain, using cationic surfactant based reverse micellar extraction (RME) system. The forward and back extraction processes were optimized in order to improve the overall extraction process. 78%, 93% and 98% forward extraction efficiencies (FEE) were obtained after the process by using aqueous phase pH 12, 150 mM CTAB and 0.2 M NaCl respectively. Afterwards, back extraction efficiency (BEE) was maximized by the optimization of stripping phase pH, isopropyl alcohol (IPA) and potassium chloride (KCl) concentration by employing Taguchi’s method of orthogonal array. Stripping phase pH 7, 15% IPA & 0.8 M KCl were found to be the best possible parameters producing 83% BEE. The usage of IPA has proven to reduce the effect of micellar-micellar based interaction, which was reflected as an improvement in BEE. This study demonstrates that the reverse micellar system is a highly promising and efficient tool for the direct extraction of recombinant proteins from fermentation broth. Moreover, this system holds a high potential to be an integral part of the downstream process used in biopharmaceutical industry.

A novel reverse micellar purification strategy for histidine tagged human interferon gamma (hIFN-γ) protein from Pichia pastoris

In the present study, we have demonstrated the process development of human interferon gamma (hIFN-γ) (upstream to downstream). The codon optimized hIFN-γ gene was cloned in Pichia pastoris X-33 and the expression was evaluated in batch reactor study. The purification was carried out with modified nickel chelated reverse micellar system and compared with the existing Nickle- Nitrilotriacetic acid (NI-NTA) method. The parameter optimization for forward extraction demonstrated a significant enhancement of 72% in forward extraction efficiency (FEE). Furthermore, the factors governing back extraction efficiency (BEE) were also optimized with sequential optimization involving Taguchi orthogonal array and Artificial Neural Network linked Simulated Annealing Algorithm (ANN-SA). The optimization resulted in 91.2% back extraction efficiency of recombinant human interferon gamma (rhIFN-γ). The development of this purification system with optimized parameters led to an efficient recovery of 67.3% and improved purity of 79.54%. Alongside, the anti-proliferative activity in MCF-7 cell lines were also investigated and it demonstrated that at 60ngmL−1 concentration of rhIFN-γ more that 25%.

DNA-cytometric parameters in patients with ovarian cancer

Aim. To study DNA flow cytometry parameters in patients with stage III-IV ovarian cancer receiving different treatments. Methods. Surgical material obtained from 93 female patients with verified stage III-IV ovarian cancer was studied. Patients were divided into 4 groups depending on the treatment variant: without neoadjuvant chemotherapy, with neoadjuvant chemotherapy, with neoadjuvant chemotherapy with different recombinant human interferon gamma (Ingaron) injections - intramuscular or intraperitoneal. Cell count was analyzed, and ploidy interpretation and cell cycle analysis were performed on fresh surgical material. Results. Differences in cell distribution depending on the treatment method were found. Increased proliferation rates, proliferation index, aneuploid tumors predominance in the group without drug exposure were demonstrated. Most of diploid tumours were registered in the group receiving neoadjuvant chemoimmunotherapy with intraperitoneal immunomodulator injections (83.4%) that indicates better disease prognosis. Under exposure to neoadjuvant polychemotherapy aneuploid cell number decreases to 38.4%, and chemoimmunotherapy with intramuscular and intraperitoneal recombinant human interferon gamma injections demonstrated even greater decrease - to 27.2 and 16.6%, respectively. Conclusion. Interferon gamma inclusion into the treatment contributes to a decreased number of aneuploid tumors, reduced proportion of aneuploid cells in tumors and reduced proliferation rates and proliferation index characterizing cancer aggressiveness.

FF-10102-01: A Novel, Highly Selective Spleen Tyrosine Kinase Inhibitor, to Be in Clinical Application for Treatment of Autoimmune Diseases and B-Cell Malignancies

Spleen tyrosine kinase (Syk) is essential for downstream pathways in signaling from B-cell receptor (BCR) in B cells and Fc-gamma receptors in macrophages. Because of the essential roles it plays in signaling, Syk has been targeted in drug development for autoimmune and allergic diseases, in which B cells and macrophages have pivotal roles in the pathophysiology. In addition, accumulated data suggest that aberrant BCR signaling is deeply involved in the pathogenesis of B-cell malignancies. Here we report the pharmacological profile of a novel and highly selective Syk inhibitor, FF-10102-01 (FF-10102 hydrochloride).FF-10102-01 was evaluated in the following in vitro assays: Kinase inhibitory activities were performed with ProfilerPro Kit (PerkinElmer, USA). The effects on cell signaling and functions were studied using THP-1, a human monocyte-derived cell line, after differentiation with human recombinant interferon gamma (IFNγ). The effects on CD69, an activated B-cell marker, expression on B cells were evaluated using whole blood samples from healthy volunteers and mice, and on lymphoma cell growth using SU-DHL-6, a human diffuse large B-cell lymphoma-derived cell line. FF-10102-01 was also evaluated in vivo in animal models of antigen-induced antibody production, anti-platelet antibody-induced thrombocytopenia, and collagen-induced arthritis (CIA).FF-10102-01 showed high potency against recombinant human Syk enzyme activity, with a 50% inhibitory concentration (IC50) value of 1.2 nmol/L. Kinase profiling assay in a panel of 216 kinases revealed that FF-10102-01 is highly selective for Syk, with an IC50 value more than 25 times lower than those of the other kinases. FF-10102-01 exhibited suppressive effects on phosphorylation of downstream molecules of Syk, SLP76, and ERK1/2 in THP-1 cells. FF-10102-01 inhibited tumor necrosis factor α secretion under stimulation by immunoglobulin (Ig) G and phagocytosis of IgG-opsonized beads in IFNγ-induced differentiated THP-1 cells. CD69 expression under stimulation with anti-BCR antibodies in human and mouse blood was also inhibited by FF-10102-01, with IC50 values of 314 nmol/L and 307 nmol/L, respectively. FF-10102-01 was the most potent inhibitor of the growth of SU-DHL-6 cells among other known Syk, JAK, and Bruton’s tyrosine kinase inhibitors compared, with an IC50 value of 318 nmol/L.FF-10102-01 is orally available with a good PK profile, and showed inhibitory effect at daily doses 50 mg/kg (as free base) on specific antibody production in ovalbumin-immunized mouse model. Orally administered FF-10102-01 showed significant effects in a mouse model of thrombocytopenia and a rat model of rheumatoid arthritis (CIA) at 25 mg/kg and 10 mg/kg (as free base), respectively. In the thrombocytopenia model, the prevention of platelet decrease by FF-10102-01 was well correlated with suppressions of platelet-phagocytotic macrophages in spleen and CD69-positive B cells in blood.These data indicate that FF-10102-01 is a potent and highly selective orally available Syk inhibitor, with pharmacological effects through inhibition of both B-cell and macrophage activities represented in inhibitions of cell signaling and functions in vitro, and in animal models of autoimmune diseases in vivo. The results suggest that FF-10102-01 is a promising agent for treatment of autoimmune diseases such as immune thrombocytopenia, rheumatoid arthritis, and B-cell malignancies. A clinical trial of FF-10102-01 is planned to commence in 2017. DisclosuresKinouchi:FUJIFILM Corporation: Employment. Taguchi:FUJIFILM Corporation: Employment. Shimoyama:FUJIFILM Corporation: Employment. Ioroi:FUJIFILM Corporation: Employment. Ogura:FUJIFILM Corporation: Employment. Yamamoto:Toyama Chemical Co., Ltd.: Employment. Iino:Toyama Chemical Co., Ltd.: Employment. Maeda:Toyama Chemical Co., Ltd.: Employment. Kato:Toyama Chemical Co., Ltd.: Employment. Fujiwara:FUJIFILM Corporation: Employment. Hagiwara:FUJIFILM Corporation: Employment. Iwamura:FUJIFILM Corporation: Employment. Kuter:Rigel: Consultancy, Research Funding; Genzyme: Consultancy; Bristol-Myers Squibb: Research Funding; GlaxoSmithKline: Consultancy; ONO: Consultancy; Amgen: Consultancy, Paid expert testimony; Protalex: Research Funding; MedImmune: Consultancy; Pfizer: Consultancy; Syntimmune: Consultancy; Shire: Consultancy; Eisai: Consultancy; 3SBios: Consultancy; CRICO: Other: Paid expert testimony; Shionogi: Consultancy. Nakamura:Toyama Chemical Co., Ltd.: Employment. Shimada:FUJIFILM Corporation: Employment.