Corrigendum: Translatability of findings from cynomolgus monkey to human suggests a mechanistic role for IL-21 in promoting immunogenicity to an anti-PD-1/IL-21 mutein fusion protein.

[This corrects the article DOI: 10.3389/fimmu.2024.1345473.].

Oxidative refolding by Copper-catalyzed air oxidation consistently increases the homogeneity and activity of a Novel Interleukin-2 mutein.

Interleukin-2 (IL-2) has been used in cancer treatment for over 30 years. However, due to its high toxicity, new mutant variants have been developed. These variants retain some of the biological properties of the original molecule but offer other therapeutic advantages. At the Center of Molecular Immunology, the IL-2 no-alpha mutein, an IL-2 agonist with lower toxicity than wtIL-2, has been designed, produced, and is currently being evaluated in a Phase I/II clinical trial. The mutein is produced in E. coli as an insoluble material that must be refolded in vitro to yield a fully active protein. Controlled oxidation steps are essential in the purification process of recombinant proteins produced in E. coli to ensure the proper formation of the disulfide bonds in the molecules. In this case, the new purification process includes a copper-catalyzed air oxidation step to induce disulfide bond establishment. The optimal conditions of pH, copper, protein and detergent concentration for this step were determined through screening. The produced protein demonstrated a conserved 3D structure, higher purity, and greater biological activity than the obtained by established process without the oxidation step. Four batches were produced and evaluated, demonstrating the consistency of the new process.

Translatability of findings from cynomolgus monkey to human suggests a mechanistic role for IL-21 in promoting immunogenicity to an anti-PD-1/IL-21 mutein fusion protein.

AMG 256 is a bi-specific, heteroimmunoglobulin molecule with an anti-PD-1 antibody domain and a single IL-21 mutein domain on the C-terminus. Nonclinical studies in cynomolgus monkeys revealed that AMG 256 administration led to the development of immunogenicity-mediated responses and indicated that the IL-21 mutein domain of AMG 256 could enhance the anti-drug antibody response directed toward the monoclonal antibody domain. Anti-AMG 256 IgE were also observed in cynomolgus monkeys. A first-in-human (FIH) study in patients with advanced solid tumors was designed with these risks in mind. AMG 256 elicited ADA in 28 of 33 subjects (84.8%). However, ADA responses were only robust and exposure-impacting at the 2 lowest doses. At mid to high doses, ADA responses remained low magnitude and all subjects maintained exposure, despite most subjects developing ADA. Limited drug-specific IgE were also observed during the FIH study. ADA responses were not associated with any type of adverse event. The AMG 256 program represents a unique case where nonclinical studies informed on the risk of immunogenicity in humans, due to the IL-21-driven nature of the response.

Potent immunomodulatory and antitumor effect of anti-CD20-IL2no-alpha tri-functional immunocytokine for cancer therapy.

Introduction: The anti-CD20 antibody rituximab (RTX) has substantially improved outcomes of patients with B-cell lymphomas, although more efficient therapies are needed for refractory or relapsing lymphomas. An approach to increase the clinical effectiveness of anti-tumor therapy is the use of antibody-cytokine fusion proteins (immunocytokines (ICKs)) to deliver at the tumor site the antibody effector functions and cytokines that trigger anti-tumor activities. In particular, IL-2-based ICKs have shown significant results in preclinical studies but not in clinical trials due to the toxicity profile associated to high doses IL-2 and the undesired expansion of Tregs. Methods: To improve the efficacy of RTX therapy, we fused a murine (mIgG2a) or a human (hIgG1) version of RTX to a mutated IL-2 (no-alpha mutein), which has a disrupted affinity for the high affinity IL-2 receptor (IL-2R) to prevent the stimulation of Tregs and reduce the binding to endothelial cells expressing CD25, the α chain of high affinity IL-2R. Characterization of anti-CD20-IL2no-alpha ICKs was performed by SDS-PAGE, Western-blotting and SEC-HPLC and also by several functional in vitro techniques like T-cell proliferation assays, apoptosis, CDC and ADCC assays. The in vivo activity was assessed by using murine tumor cells expressing huCD20 in C57/Bl6 mice. Results: Both ICKs exhibited similar in vitro specific activity of their IL2no-alpha mutein moieties and kept CD20-binding capacity. Anti-CD20-IL2no-alpha (hIgG1) retained antibody effector functions as complement-dependent cytotoxicity and enhanced direct apoptosis, NK cell activation and antibody-dependent cellular cytotoxicity relative to RTX. In addition, both ICKs demonstrated a higher antitumor efficacy than parental molecules or their combination in an EL4-huCD20 tumor model in immunocompetent mice. Anti-CD20-IL2no-alpha (hIgG1) strongly expanded NK and CD8+ T cells but not Tregs in tumor-bearing mice. Discussion: These findings suggest that anti-CD20-IL2no-alpha could represent an alternative treatment for B cell lymphoma patients, mainly those refractory to RTX therapy.

The antitumor effect induced by an IL-2 'no-alpha' mutein depends on changes in the CD8+ T lymphocyte/Treg cell balance.

High doses of interleukin-2 (IL-2) have been used for the treatment of melanoma and renal cell carcinoma, but this therapy has limited efficacy, with a ~15% response rate. Remarkably, 7%-9% of patients achieve complete or long-lasting responses. Many patients treated with IL-2 experienced an expansion of regulatory T cells (Tregs), specifically the expansion of ICOS+ highly suppressive Tregs, which correlate with worse clinical outcomes. This partial efficacy together with the high toxicity associated with the therapy has limited the use of IL-2-based therapy. Taking into account the understanding of IL-2 structure, signaling, and in vivo functions, some efforts to improve the cytokine properties are currently under study. In previous work, we described an IL-2 mutein with higher antitumor activity and less toxicity than wtIL-2. Mutein was in silico designed for losing the binding capacity to CD25 and for preferential stimulation of effector cells CD8+ and NK cells but not Tregs. Mutein induces a higher anti-metastatic effect than wtIL-2, but the extent of the in vivo antitumor activity was still unexplored. In this work, it is shown that mutein induces a strong antitumor effect on four primary tumor models, being effective even in those models where wtIL-2 does not work. Furthermore, mutein can change the in vivo balance between Tregs and T CD8+ memory/activated cells toward immune activation, in both healthy and tumor-bearing mice. This change reaches the tumor microenvironment and seems to be the major explanation for mutein efficacy in vivo.

Selective blood-brain barrier permeabilization of brain metastases by a type 1 receptor-selective tumor necrosis factor mutein.

BACKGROUND: Metastasis to the brain is a major challenge with poor prognosis. The blood-brain barrier (BBB) is a significant impediment to effective treatment, being intact during the early stages of tumor development and heterogeneously permeable at later stages. Intravenous injection of tumor necrosis factor (TNF) selectively induces BBB permeabilization at sites of brain micrometastasis, in a TNF type 1 receptor (TNFR1)-dependent manner. Here, to enable clinical translation, we have developed a TNFR1-selective agonist variant of human TNF that induces BBB permeabilization, while minimizing potential toxicity. METHODS: A library of human TNF muteins (mutTNF) was generated and assessed for binding specificity to mouse and human TNFR1/2, endothelial permeabilizing activity in vitro, potential immunogenicity, and circulatory half-life. The permeabilizing ability of the most promising variant was assessed in vivo in a model of brain metastasis. RESULTS: The primary mutTNF variant showed similar affinity for human TNFR1 than wild-type human TNF, similar affinity for mouse TNFR1 as wild-type mouse TNF, undetectable binding to human/mouse TNFR2, low potential immunogenicity, and permeabilization of an endothelial monolayer. Circulatory half-life was similar to mouse/human TNF and BBB permeabilization was induced selectively at sites of micrometastases in vivo, with a time window of ≥24 hours and enabling delivery of agents within a therapeutically relevant range (0.5-150 kDa), including the clinically approved therapy, trastuzumab. CONCLUSIONS: We have developed a clinically translatable mutTNF that selectively opens the BBB at micrometastatic sites, while leaving the rest of the cerebrovasculature intact. This approach will open a window for brain metastasis treatment that currently does not exist.

Purification and Conformational Characterization of a Novel Interleukin-2 Mutein.

Toxicity of high-dose IL-2-based therapies have motivated the development of the IL-2 mutein, which has low expansion properties for regulatory T lymphocytes. The development of two variants (A and B) for the IL-2 mutein purification as well as a conformational comparative study by Circular dichroism (CD) and fluorescence spectroscopy of these products were evaluated. For the first time, in our center, were used of DTT and 2% SDS in the solubilization step to decrease the aggregates on intermediate product, which favors that disulfide bridges are correctly formed during re-folding. A molecular weight of 18 kDa to the monomeric form and of 25-37 kDa to the oligomeric species were estimated by SDS-PAGE. IL-2 mutein showed similar far-UV CD spectral characteristic typical of cytokines with 41% of α-helix content. Batches obtained by Process B showed similar conformational features according near-UV CD and FS studies. However, those obtained by Process A differed in their folding. IL-2 mutein showed that conformational features by near-UV CD were affected by 2% SDS, no variations on secondary structure were observed. Melting temperature values by far-UV CD were higher than 95 °C, indicating a high thermal stability. Finally, the drug product obtained by Process B showed similar conformational characteristics by near-UV CD and FS, and higher biological activity values (7.0 × 103 ng/mL) in the cell proliferation assay with respect to Process A. Also, the recovery was 15% higher than in the Process A and exhibited a 78.48% of purity. Indeed, Process B was selected for the purification.

MK-6, a novel not-α IL-2, elicits a potent antitumor activity by improving the effector to regulatory T cell balance.

IL-2 is a pleiotropic cytokine that regulates immune cell homeostasis. Its immunomodulatory function has been used clinically as an active immunotherapy agent for metastatic cancers. However, severe adverse effects, including the vascular leak syndrome and the preferential stimulation of anti-immunogenic Treg rather than effector T cells, have been obstacles. We newly designed a mutein IL-2, Mutakine-6 (MK-6), with reduced IL-2Rα-binding capability. MK-6 induced comparable cell growth potential toward IL-2Rßγ-positive T cells but was far less efficient in in vitro Treg proliferation and STAT5 activation. Unlike IL-2, in vivo administration of MK-6 produced minimal adverse effects. Using CT26 and B16F10-syngeneic tumor models, we found MK-6 was highly efficacious on tumor regression. Serum albumin conjugation to MK-6 prolonged in vivo half-life and accumulated in CT26 tumors, showing enhanced antitumor effect. Tumor-infiltrating leukocytes analysis revealed that albumin-fused MK-6 increased the ratio of effector CD8+ T cells to CD4+ Treg cells. These results demonstrated that MK-6 is an efficient immunomodulator potentially used for improved immunotherapy with decreased adverse effects and attenuated Treg stimulation.

Emerging Therapeutics for Immune Tolerance: Tolerogenic Vaccines, T cell Therapy, and IL-2 Therapy.

Autoimmune diseases affect roughly 5-10% of the total population, with women affected more than men. The standard treatment for autoimmune or autoinflammatory diseases had long been immunosuppressive agents until the advent of immunomodulatory biologic drugs, which aimed at blocking inflammatory mediators, including proinflammatory cytokines. At the frontier of these biologic drugs are TNF-α blockers. These therapies inhibit the proinflammatory action of TNF-α in common autoimmune diseases such as rheumatoid arthritis, psoriasis, ulcerative colitis, and Crohn's disease. TNF-α blockade quickly became the "standard of care" for these autoimmune diseases due to their effectiveness in controlling disease and decreasing patient's adverse risk profiles compared to broad-spectrum immunosuppressive agents. However, anti-TNF-α therapies have limitations, including known adverse safety risk, loss of therapeutic efficacy due to drug resistance, and lack of efficacy in numerous autoimmune diseases, including multiple sclerosis. The next wave of truly transformative therapeutics should aspire to provide a cure by selectively suppressing pathogenic autoantigen-specific immune responses while leaving the rest of the immune system intact to control infectious diseases and malignancies. In this review, we will focus on three main areas of active research in immune tolerance. First, tolerogenic vaccines aiming at robust, lasting autoantigen-specific immune tolerance. Second, T cell therapies using Tregs (either polyclonal, antigen-specific, or genetically engineered to express chimeric antigen receptors) to establish active dominant immune tolerance or T cells (engineered to express chimeric antigen receptors) to delete pathogenic immune cells. Third, IL-2 therapies aiming at expanding immunosuppressive regulatory T cells in vivo.

Modeling cell-specific dynamics and regulation of the common gamma chain cytokines.

The γ-chain receptor dimerizes with complexes of the cytokines interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21 and their corresponding "private" receptors. These cytokines have existing uses and future potential as immune therapies because of their ability to regulate the abundance and function of specific immune cell populations. Here, we build a binding reaction model for the ligand-receptor interactions of common γ-chain cytokines, which includes receptor trafficking dynamics, enabling quantitative predictions of cell-type-specific response to natural and engineered cytokines. We then show that tensor factorization is a powerful tool to visualize changes in the input-output behavior of the family across time, cell types, ligands, and concentrations. These results present a more accurate model of ligand response validated across a panel of immune cell types as well as a general approach for generating interpretable guidelines for manipulation of cell-type-specific targeting of engineered ligands.

A rationally-engineered IL-2 improves the antitumor effect of anti-CD20 therapy.

Anti-CD20 treatment represents a therapeutic benefit for patients with B-cell lymphomas, although more efficient therapies are needed for refractory or relapsing patients. Among them, the combination of anti-CD20 and IL-2 that induces T cell response has been hampered by the expansion of FoxP3+ Tregs that strongly express the high affinity IL-2 receptor (IL-2R αßγ). We explore here the anti-tumor effect of an anti-CD20 antibody combined with a mutated IL-2 (no-alpha mutein) which has a disrupted affinity for the IL-2R αßγ. We demonstrate that anti-CD20/no-alpha mutein combination significantly augments the survival rate of mice challenged with huCD20+ cells as compared to animals treated with anti-CD20 ± IL-2. Moreover, the combination with no-alpha mutein but not IL-2 provokes an increase of granzyme B and perforin in splenic NK and CD8+ T cells, a reduction of Tregs and an increase in activated macrophages. The former combination also induces a T helper profile different from that obtained with IL-2, with an earlier polarization to Th1 and no increase in Th17. The therapeutic effect of anti-CD20/no-alpha mutein was accompanied by an expansion of peripheral central (TCM) and effector (TEM) memory CD8+ T cell compartments. Last, as opposed to IL-2, no-alpha mutein administered at the beginning of anti-CD20 treatment did not dampen the long-term protection of surviving mice after tumor rechallenge. Thus, this study shows that the combination of anti-tumor antibodies and no-alpha mutein is a promising approach to improve the therapeutic effect of these antibodies by potentiating NK/macrophage-mediated innate immunity and the adaptive T-cell response.

Defining the Threshold IL-2 Signal Required for Induction of Selective Treg Cell Responses Using Engineered IL-2 Muteins.

Among all T and NK cell subsets, regulatory T (Treg) cells typically respond to the lowest concentrations of IL-2 due to elevated surface expression of the IL-2R alpha chain (IL2RA; CD25) and the high affinity IL-2 receptor (IL-2R) complex. This enhanced sensitivity forms the basis for low-dose (LD) IL-2 therapy for the treatment of inflammatory diseases, where efficacy correlates with increased Treg cell number and expression of functional markers. Despite strong preclinical support for this approach, moderate and variable clinical efficacy has raised concerns that adequate Treg selectivity still cannot be achieved with LD IL-2, and/or that doses are too low to stimulate effective Treg-mediated suppression within tissues. This has prompted development of IL-2 variants with greater Treg selectivity, achieved through attenuated affinity for the signaling chains of the IL-2R complex (IL2RB or CD122 and IL2RG or CD132) and, consequently, greater reliance on high CD25 levels for full receptor binding and signaling. While certain IL-2 variants have advanced to the clinic, it remains unknown if the full range of IL-2R signaling potency and Treg-selectivity observed with low concentrations of wildtype IL-2 can be sufficiently recapitulated with attenuated IL-2 muteins at high concentrations. Using a panel of engineered IL-2 muteins, we investigated how a range of IL-2R signaling intensity, benchmarked by the degree of STAT5 phosphorylation, relates to biologically relevant Treg cell responses such as proliferation, lineage and phenotypic marker expression, and suppressor function. Our results demonstrate that a surprisingly wide dynamic range of IL-2R signaling intensity leads to productive biological responses in Treg cells, with negligible STAT5 phosphorylation associating with nearly complete downstream effects such as Treg proliferation and suppressor activity. Furthermore, we show with both in vitro and humanized mouse in vivo systems that different biological responses in Treg cells require different minimal IL-2R signaling thresholds. Our findings suggest that more than minimal IL-2R signaling, beyond that capable of driving Treg cell proliferation, may be required to fully enhance Treg cell stability and suppressor function in vivo.

Characterization of a Long-Acting Site-Specific PEGylated Murine GM-CSF Analog and Analysis of Its Hematopoietic Properties in Normal and Cyclophosphamide-Treated Neutropenic Rats.

Previously we reported that site-specific modification of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) A3C analog with polyethylene glycol (PEG) dramatically improved the pharmacokinetic properties of the protein in rats. However, we could not evaluate the hematological properties of the PEG-A3C protein in rats because human GM-CSF is inactive in rodents. To study the biological effects of PEGylated GM-CSF analogs in rodents we created a homologous site-specific PEGylated murine (mu) GM-CSF (T3C) protein. muGM-CSF and the T3C protein were expressed in Escherichia coli and purified by column chromatography. The purified T3C protein was covalently modified with a linear 20 kDa- or a branched 40 kDa-maleimide-PEG, and the monoPEGylated proteins purified by column chromatography. muGM-CSF, T3C and the two PEG-T3C proteins had comparable in vitro biological activities, as measured by stimulation of proliferation of the murine FDC-P1 cell line. The PEG-T3C proteins had 10- to 25-fold longer circulating half-lives than muGM-CSF and stimulated greater and longer lasting increases in neutrophils and white blood cells than muGM-CSF following a single intravenous or subcutaneous administration to rats. Treatment of rats made neutropenic with cyclophosphamide with the PEG-T3C proteins shortened the time for recovery of neutrophils to normal levels from 9 or 10 days to 5 or 6 days, whereas muGM-CSF showed no benefit versus vehicle solution. Acceleration of neutrophil recovery in cyclophosphamide-treated rats required a minimum of three PEG-T3C treatments over five days. The PEG-T3C proteins should prove useful for evaluating the potential therapeutic benefits of GM-CSF and long-acting GM-CSF proteins in rodent disease models.

Antibody-Based Targeting of Interferon-Beta-1a Mutein in HER2-Positive Cancer Enhances Antitumor Effects Through Immune Responses and Direct Cell Killing.

Type I interferon (IFN) has been approved as an anticancer agent to treat some malignancies. However, IFNs have a short in vivo half-life, systemic toxicity, and poor biophysical properties, which prevent it from being widely used for cancer therapy. This study aimed to construct recombinant IFN-ß-1a mutein immunocytokines that comprise a human epidermal growth factor receptor 2 (HER2)-targeting antibody and IFN-ß muteins with an additional glycosylation, which can overcome the limitation of the cytokine itself. Hence, the molecular design aims to 1) enhance productivity and biophysical properties by adding secondary glycosylation in IFN-ß, 2) increase the therapeutic index of IFN-ß therapy by preferential retention at the tumor by possessing high affinity for HER2-expressing cancer cells, and 3) improve the pharmacokinetics and, thus, the convenience of IFN-ß administration. The yield of trastuzumab-IFN-ß mutein was higher than that of trastuzumab-wild-type IFN-ß in the mammalian cell culture system. Trastuzumab-IFN-ß mutein showed similar IFN activity and HER2-targeting ability equivalent to that of IFN-ß mutein and trastuzumab, respectively. Trastuzumab-IFN-ß mutein directly inhibited the growth of HER2-positive gastric cancer cell lines and was more effective than trastuzumab or IFN-ß mutein alone. Trastuzumab-IFN-ß mutein and IFN-ß mutein displayed enhanced immune cell-mediated cytotoxicity. Collectively, trastuzumab-IFN-ß mutein may have indirect immune cell-mediated antitumor effects and direct cell growth inhibitory effects. Tumor-targeting effect of trastuzumab-IFN-ß mutein was analyzed using in vivo fluorescence imaging. The accumulation of trastuzumab-IFN-ß mutein was observed in HER2-positive tumors rather than other tissues except the liver. To evaluate the both direct tumor growth inhibition effect and indirect immune cell-mediated antitumor effect, we tested the effect of trastuzumab-IFN-ß mutein in HER2-positive cancer xenograft models using nude mice or humanized mice. Trastuzumab-IFN-ß mutein could significantly enhance tumor regression when compared with trastuzumab or IFN-ß mutein. In addition, an increase in tumor-infiltrating lymphocytes was observed in the trastuzumab-IFN-ß mutein-treated group, implying that the tumor-targeting IFN-ß may have an enhanced antitumor effect through increased immune response. Therefore, targeting IFN-ß with an anti-HER2 monoclonal antibody makes the immunocytokine more potent than either agent alone. These novel findings suggest that trastuzumab-IFN-ß mutein merits clinical evaluation as a new candidate of anticancer therapeutics.

A long-lived IL-2 mutein that selectively activates and expands regulatory T cells as a therapy for autoimmune disease.

Susceptibility to multiple autoimmune diseases is associated with common gene polymorphisms influencing IL-2 signaling and Treg function, making Treg-specific expansion by IL-2 a compelling therapeutic approach to treatment. As an in vivo IL-2 half-life enhancer we used a non-targeted, effector-function-silent human IgG1 as a fusion protein. An IL-2 mutein (N88D) with reduced binding to the intermediate affinity IL-2Rßγ receptor was engineered with a stoichiometry of two IL-2N88D molecules per IgG, i.e. IgG-(IL-2N88D)2. The reduced affinity of IgG-(IL-2N88D)2 for the IL-2Rßγ receptor resulted in a Treg-selective molecule in human whole blood pSTAT5 assays. Treatment of cynomolgus monkeys with single low doses of IgG-(IL-2N88D)2 induced sustained preferential activation of Tregs accompanied by a corresponding 10-14-fold increase in CD4+ and CD8+ CD25+FOXP3+ Tregs; conditions that had no effect on CD4+ or CD8+ memory effector T cells. The expanded cynomolgus Tregs had demethylated FOXP3 and CTLA4 epigenetic signatures characteristic of functionally suppressive cells. Humanized mice had similar selective in vivo responses; IgG-(IL-2N88D)2 increased Tregs while wild-type IgG-IL-2 increased NK cells in addition to Tregs. The expanded human Tregs had demethylated FOXP3 and CTLA4 signatures and were immunosuppressive. These results describe a next-generation immunotherapy using a long-lived and Treg-selective IL-2 that activates and expands functional Tregsin vivo. Patients should benefit from restored immune homeostasis in a personalized fashion to the extent that their autoimmune disease condition dictates opening up the possibility for remissions and cures.

The pharmacokinetic and pharmacodynamic properties of site-specific pegylated genetically modified recombinant human interleukin-11 in normal and thrombocytopenic monkeys.

In order to improve the pharmacokinetic and pharmacodynamic properties of recombinant human interleukin-11 mutein (mIL-11) and to reduce the frequency of administration, we examined the feasibility of chemical modification of mIL-11 by methoxy polyethylene glycol succinimidyl carbonate (mPEG-SC). PEG-mIL-11 was prepared by a pH controlled amine specific method. Bioactivity of the protein was determined in a IL-11-dependent in vitro bioassay, its pharmacodynamic and pharmacokinetic properties were investigated by using normal and thrombocytopenic monkey models. N-terminus sequencing and peptide mapping analysis revealed that Lys33 is the PEGylated position for PEG-mIL-11. Bioactivity of PEG-mIL-11 assessed by B9-11 cell proliferation assay was comparable to that of mIL-11. More than 79-fold increase in area-under-the curve (AUC) and 26-fold increase in maximum plasma concentration (Cmax) was observed in pharmacokinetic analysis. Single dose administration of the PEG-mIL-11 induced blood platelets number increase and the effect duration were comparable to that of 7 to 10 consecutive daily administration of mIL-11 to the normal and thrombocytopenic monkey models. PEG-mIL-11 is a promising therapeutic for thrombocytopenia.

Removing a Cystein Group On Interferon Alpha 2b at Position 2 and 99 does Not Diminish Antitumor Activity of the Protein, Even Better.

Interferon alpha 2b is the only standard therapeutic protein for hepatitis virus infections. Further study demonstrated that this protein also posseses antitumor activity in several cancerous organs. One main pathway of this antitumor activity is mediated through antiproliferation as well as proapoptotic effects. Previously, we have successfully developed recombinant human interferon alpha 2b (rhIFNα2b) by using a synthetic gene. In addition, two mutein forms of rhIFNα2b were generated to improve the characteristics of this protein. Two point mutations showed better pharmacokinetic profiles than one point mutation as well as the native form. In the present study, this mutein form was studied for ist antitumor effect in vitro using HepG2 cells. As a comparison, the native form as well as a commercial rIFNα2b were used. Several parameters were investigated including the MTT assay, cell viability test, cell cycle using flow cytometric analysis, and the genes and protein expressions involved in cell growth. The latest was observed to study the mechanism of rhIFNα2b. There was no significant difference in the MTT assay and cell viability after cells were treated with both forms of rhIFNα2b. However, the mutein rhIFNα2b tended to show better proapoptotic activity reflected by flow cytometric data, protein expression of pSTAT1, and DNA expression of caspase 3.

Evidence that leptin-induced weight loss requires activation of both forebrain and hindbrain receptors.

Previous studies with chronic decerebrate rats and rats infused with leptin into the 4th ventricle suggest that hindbrain leptin receptors attenuate the catabolic effect of forebrain leptin receptor activation. To test this further, rats were fitted with both 3rd and 4th ventricle cannulae. They were infused for 12 days with different combinations of saline, low dose leptin or leptin receptor antagonist (leptin mutein protein). Infusion of 0.1 µg leptin/day into the 3rd ventricle or 0.6 µg leptin/day into the 4th ventricle had no significant effect on food intake, energy expenditure or body composition. Infusion of 2 µg mutein/day into either ventricle caused a small, but significant weight gain. When mutein was infused into one ventricle and leptin into the other, the rats lost weight irrespective of which combination was applied. Surprisingly, rats that received leptin infusions into both ventricles showed an initial hypophagia, no change in energy expenditure, but a 75% loss of carcass fat after 12 days. These data suggest that neuronal pathways activated by leptin receptors in either the forebrain or hindbrain modulate each other's effects. In normal conditions hindbrain leptin may attenuate the catabolic effect of forebrain leptin, but if activity in one area is blocked with mutein, then the catabolic response to leptin in the other ventricle is exaggerated. When receptors in both areas are activated there is an integration of response to produce negative energy balance. This may ensure that leptin causes a loss of fat only when leptin is elevated in both the CSF and periphery.