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1.
Angew Chem Int Ed Engl ; 59(22): 8425-8429, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32032465

RESUMO

Chemical protein synthesis allows the construction of well-defined structural variations and facilitates the development of deeper understanding of protein structure-function relationships and new protein engineering strategies. Herein, we report the chemical synthesis of interleukin-2 (IL-2) variants on a multimilligram scale and the formation of non-natural disulfide mimetics that improve stability against reduction. The synthesis was accomplished by convergent KAHA ligations; the acidic conditions of KAHA ligation proved to be valuable for the solubilization of the hydrophobic segments of IL-2. The bioactivity of the synthetic IL-2 and its analogues were shown to be equipotent to recombinant IL-2 and exhibit improved stability against reducing agents.


Assuntos
Dissulfetos/química , Interleucina-2/química , Interleucina-2/síntese química , Técnicas de Química Sintética , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Estabilidade Proteica , Solubilidade
2.
Biomater Sci ; 7(4): 1345-1357, 2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30698174

RESUMO

Interleukin-2 (IL-2) is a potent T-cell mitogen that can adjuvant anti-cancer adoptive T-cell transfer (ACT) immunotherapy by promoting T-cell engraftment. However, the clinical applications of IL-2 in combination with ACT are greatly hindered by the severe adverse effects such as vascular leak syndrome (VLS). Here, we developed a synthetic delivery strategy for IL-2 via backpacking redox-responsive IL-2/Fc nanogels (NGs) to the plasma membrane of adoptively transferred T-cells. The NGs prepared by traceless chemical cross-linking of cytokine proteins selectively released the cargos in response to T-cell receptor activation upon antigen recognition in tumors. We found that IL-2/Fc delivered by T-cell surface-bound NGs expanded transferred tumor-reactive T-cells 80-fold more than the free IL-2/Fc of an equivalent dose administered systemically and showed no effects on tumor-infiltrating regulatory T-cell expansion. Intriguingly, IL-2/Fc NG backpacks that facilitated a sustained and slow release of IL-2/Fc also promoted the CD8+ memory precursor differentiation and induced less T-cell exhaustion in vitro compared to free IL-2/Fc. The controlled responsive delivery of IL-2/Fc enabled the safe administration of repeated doses of the stimulant cytokine with no overt toxicity and improved efficacy against melanoma metastases in a mice model.


Assuntos
Interleucina-2/farmacologia , Melanoma/patologia , Polietilenoglicóis/farmacologia , Polietilenoimina/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Interleucina-2/síntese química , Interleucina-2/química , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Nanogéis , Oxirredução , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Polietilenoimina/síntese química , Polietilenoimina/química , Linfócitos T/imunologia , Linfócitos T/patologia
3.
Bioconjug Chem ; 29(1): 29-34, 2018 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-29231709

RESUMO

The attachment of two different functionalities in a site-selective fashion represents a great challenge in protein chemistry. We report site specific dual functionalizations of peptides and proteins capitalizing on reactivity differences of cysteines in their free (thiol) and protected, oxidized (disulfide) forms. The dual functionalization of interleukin 2 and EYFP proceeded with no loss of bioactivity in a stepwise fashion applying maleimide and disulfide rebridging allyl-sulfone groups. In order to ensure broader applicability of the functionalization strategy, a novel, short peptide sequence that introduces a disulfide bridge was designed and site-selective dual labeling in the presence of biogenic groups was successfully demonstrated.


Assuntos
Compostos Alílicos/química , Cisteína/química , Maleimidas/química , Peptídeos/química , Proteínas/química , Compostos de Sulfidrila/química , Sulfonas/química , Compostos Alílicos/síntese química , Animais , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/química , Linhagem Celular , Cisteína/síntese química , Humanos , Interleucina-2/síntese química , Interleucina-2/química , Proteínas Luminescentes/síntese química , Proteínas Luminescentes/química , Maleimidas/síntese química , Camundongos , Modelos Moleculares , Peptídeos/síntese química , Proteínas/síntese química , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/química , Coloração e Rotulagem/métodos , Compostos de Sulfidrila/síntese química , Sulfonas/síntese química
4.
Angew Chem Int Ed Engl ; 54(28): 8226-30, 2015 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-26012897

RESUMO

The chemical synthesis of human interleukin-2 (IL-2) , having a core 1 sugar, by a ligation method is reported. Although IL-2 is a globular glycoprotein, its C-terminal region, in particular (99-133), is extremely insoluble when synthesized by solid-phase method. To overcome this problem, the side-chain carboxylic acid of the Glu residues was protected by a picolyl ester, thus reversing its polarity from negative to positive. This reverse polarity protection significantly increased the isoelectric point of the peptide segment and made it positive under acidic conditions and facilitated the purification. An efficient method to prepare the prolyl peptide thioester required for the synthesis of the (28-65) segment was also developed. These efforts resulted in the total synthesis of the glycosylated IL-2 having full biological activity.


Assuntos
Glicoproteínas/química , Interleucina-2/análogos & derivados , Peptídeos/química , Humanos , Interleucina-2/síntese química , Interleucina-2/química
5.
Curr Radiopharm ; 7(2): 115-22, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25175622

RESUMO

Dendrimers are branched nanomolecules, with a three dimensional structure, very low polydispersity and high functionality. Poly(amidoamine) (PAMAM) dendrimers are the most investigated class of dendrimers. In this study, PAMAM G4 dendrimer conjugated with HYNIC (hydrazinonicotinamide), an efficient bifunctional chelator, was characterized. Structure of the derivatized dendrimer was confirmed by (1)H-NMR and (13)C-NMR spectra and MALDI-TOF mass spectrometry. HYNIC-dendrimer was labeled with technetium-99m testing three different co-ligands (tricine, nicotinic acid and ethylenediaminodiacetic acid). The radiolabeled complexes were characterized by reverse phase HPLC, as well as their stabilities. Radiolabeling yield was about 99% with all co-ligands and complexes were found stable for 24 h. Biodistribution studies were performed administrating tricine-(99m)Tc-HYNIC-dendrimer, nicotinic acid-(99m)Tc-HYNICdendrimer and EDDA-(99m)Tc-HYNIC-dendrimer to normal mice; results showed blood clearance with hepatic and renal depuration in all cases. In this sense, labeling of PAMAM G4 dendrimer with technetium-99m using HYNIC could be obtained in high yield in a simple method and with high specific activity.


Assuntos
Dendrímeros/farmacologia , Interleucina-2/farmacologia , Niacinamida/análogos & derivados , Ácidos Nicotínicos/farmacologia , Compostos de Organotecnécio/farmacologia , Poliaminas/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Animais , Dendrímeros/síntese química , Interleucina-2/síntese química , Taxa de Depuração Metabólica , Camundongos , Niacinamida/síntese química , Niacinamida/farmacologia , Ácidos Nicotínicos/síntese química , Compostos de Organotecnécio/síntese química , Compostos Radiofarmacêuticos/síntese química
6.
Bioorg Khim ; 38(4): 413-20, 2012.
Artigo em Russo | MEDLINE | ID: mdl-23189555

RESUMO

Synthetic peptides corresponding to the 59-72 (I), 60-72 (II) and 61-72 (III) sequences of human interleukin 2 with their N(alpha) acetylated and C(alpha) methylated termini were shown to exhibit pronounced hepatoprotective properties. These peptides neutralized hepatotoxic effects of such agents as tetrachloromethane and galactosamine in experiments in vivo. The peptide action revealed as normalization of duration of the thiopental narcosis of experimental animals and the level of hepatospecific enzymes in their blood. The effects of peptides (I)-(III) proved to be similar to that of prednisolone (the well-known anti-inflammatory agent), whereas the bestatine cytotoxic dipeptide had no hepatoprotecting effect. The target of the hepatoprotective activity of the peptides was shown to be the preliminary activated macrophages. We proposed that this activity of the peptides was associated with their interaction with the a-subunit of the interleukin 2 receptor (IL-2Ralpha), because the X-Ray analysis pointed to this region as one of binding sites of IL-2 with IL-2Ralpha. Experiments on the influence of the most active (59-72)-peptide on growth of the IL-2 dependent cell line (CTLL) confirmed this proposal. The 3H-labeled peptide corresponding to the 59-72 sequence ofthe human IL-2 was shown to bind to the CTLL cels. We assumed that the binding of this peptide was specific and occurred precisely with IL-2Ra and virtually determined the binding constant. Its value (1.41 x 10(-6) M) was comparable with that of the interaction of IL-2 with IL-2Ralpha (approximately 10(-7) M).


Assuntos
Subunidade alfa de Receptor de Interleucina-2 , Interleucina-2 , Fragmentos de Peptídeos , Substâncias Protetoras , Animais , Sítios de Ligação , Tetracloreto de Carbono/toxicidade , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Galactosamina/toxicidade , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Interleucina-2/síntese química , Interleucina-2/genética , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Subunidade alfa de Receptor de Interleucina-2/química , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Macrófagos/efeitos dos fármacos , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Substâncias Protetoras/síntese química , Substâncias Protetoras/farmacologia
7.
Eur Surg Res ; 39(2): 103-10, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17299267

RESUMO

The immunopharmacological profile of novel biocompatible water-soluble interleukin-2 (IL-2)-conjugated 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer immunosuppressive agents was established. MPC-co-n- butyl methacrylate (BMA)-co-p-nitrophenylcarbonyloxyethyl methacrylate (NPMA) (PMBN) was prepared as a backbone for these novel agents. PMBN contained MPC as a biocompatible unit, BMA as a hydrophobic domain in water, and NPMA as an immobilizable unit with IL-2. This research showed that proliferation of cell lines with high-affinity IL-2 receptors derived from T cell malignancies were suppressed by the PMBN conjugated with IL-2 (PMBN-IL2 conjugate) incorporating paclitaxel (PTX) and cyclosporin A at lower concentrations than used conventionally. PMBN-IL2 conjugates incorporating PTX also inhibited the proliferation of responder cells in a human mixed lymphocyte culture at a lower concentration than unconjugated drug. However, PMBN-IL2 conjugates incorporating FK506 inhibited proliferation no more than FK506 alone. The PMBN-IL2 conjugate with PTX may therefore be useful for selectively eliminating activated lymphocytes that hyperproduce high-affinity IL-2 receptors. As an entirely human 'immunotoxin analogue' it may not be associated with the dose-limiting toxicity and immunogenicity of conventional immunotoxins.


Assuntos
Ciclosporina/farmacocinética , Imunossupressores/farmacocinética , Interleucina-2/farmacocinética , Ativação Linfocitária/efeitos dos fármacos , Metacrilatos/farmacocinética , Polietilenoglicóis/farmacocinética , Linfócitos T/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacocinética , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/farmacocinética , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Interleucina-2/síntese química , Teste de Cultura Mista de Linfócitos , Metacrilatos/síntese química , Paclitaxel/farmacocinética , Polietilenoglicóis/síntese química , Solubilidade , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tacrolimo/farmacocinética
8.
Methods Mol Biol ; 283: 255-66, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15197317

RESUMO

This method describes the conjugation of a synthetic glycopeptide to the N-terminus of a recombinant human interleukin-2 (IL-2) protein fragment. The IL-2 protein fragment is produced as an affinity-tagged fusion protein in Escherichia coli and then cleaved with the highly selective TEV protease to remove the affinity tag and uncover an N-terminal cysteine. The N-terminal cysteine is then used in native chemical ligation reaction to join the IL-2 protein fragment to a glycosylated tripeptide thioester that had been previously synthesized to produce a glycosylated form of IL-2.


Assuntos
Glicopeptídeos/química , Interleucina-2/química , Peptídeos/química , Cisteína/síntese química , Cisteína/genética , Endopeptidases , Glicopeptídeos/síntese química , Humanos , Interleucina-2/biossíntese , Interleucina-2/síntese química , Peptídeos/genética , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
9.
J Biol Chem ; 278(25): 22868-76, 2003 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-12676936

RESUMO

Peptide p1-30, which is composed of the 30 amino-terminal residues (alpha-helix A) of human interleukin-2 (IL-2), binds as a tetramer to the dimeric IL-2Rbeta2 receptor, whereas the entire IL-2 recognizes the tricomponent receptor IL-2Ralphabetagamma. p1-30 is an IL-2 mimetic that activates CD8 low lymphocytes and natural killer cells, because these cells produce IL-2Rbeta constitutively. It also induces a strong lymphokine-activated killer cell response. A series of truncated peptides were analyzed by circular dichroism and analytical centrifugation to elucidate the role of p1-30 residues. We propose a model where residues 10-30 of the p1-30 peptide form an alpha-helix with eight hydrophobic side chains on the same surface buried in a hydrophobic core when four anti-parallel helices combine to form a bundle. IL-2Rbeta dimerization was further studied, and three-dimensional models of the free IL-2Rbeta2 receptor and the p1-304.IL-2Rbeta2 complex were built by comparative modeling based on the crystal structure of the erythropoietin receptor complex, because this belongs to the same hematopoietin family as IL-2. These models suggest that binding of the p1-30 tetramer rotates the COOH-terminal domains and brings both transmembrane regions 50 A closer together, driving the association of the two intracytoplasmic domains that would transduce the signal into the cytoplasm.


Assuntos
Interleucina-2/química , Receptores de Interleucina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Interleucina-2/síntese química , Subunidade beta de Receptor de Interleucina-2 , Cinética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Linfócitos T/imunologia , Transfecção
10.
Int Immunopharmacol ; 2(2-3): 333-44, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11811936

RESUMO

This is the first report of aerosol interleukin 2 (IL-2) liposome administration to individuals with immune deficiency. Parenteral IL-2 therapy has shown beneficial effects in some patients with cancer, common variable immunodeficiency (CVID), and human immunodeficiency virus (HIV) but is problematic because of side effects including fever and malaise as well as local swelling (delayed type hypersensitivity like reaction) after each subcutaneous IL-2 injection. Provision of an IL-2:human albumin liposome formulation via the aerosol route had few side effects in a recent clinical trial in cancer patients. Details of good manufacturing practice (GMP) synthesis and analysis of IL-2 liposomes (N= 6 lots) made without albumin carrier protein and placebo liposomes (three lots) are presented. After centrifugation, IL-2 was closely associated with the liposome pellet (99%). Mean diameter of liposomes was 1.1 microm. Patient acceptance, safety, toxicity, and immune effects of IL-2 liposomes were studied in individuals with primary immune deficiency (N = 15) and subsequently, a larger cohort of patients with hepatitis C. Experience in the immune deficient patients is the subject of this report. Placebo liposomes (12 weeks) and IL-2 liposomes (12 weeks) were provided using a nebulizer. Aerosol placebo liposomes and IL-2 liposomes were well tolerated. No changes in chest X-ray or pulmonary function were seen. Since biologic activity of aerosol IL-2 liposomes has been seen in viral disease (hepatitis C), additional studies of aerosol IL-2 liposomes in individuals with hepatitis C and HIV are planned.


Assuntos
Aerossóis/administração & dosagem , Síndromes de Imunodeficiência/terapia , Interleucina-2/administração & dosagem , Administração por Inalação , Adolescente , Adulto , Aerossóis/síntese química , Aerossóis/farmacologia , Aerossóis/normas , Criança , Pré-Escolar , Estudos Cross-Over , Método Duplo-Cego , Feminino , Humanos , Síndromes de Imunodeficiência/sangue , Interleucina-2/síntese química , Interleucina-2/fisiologia , Interleucina-2/normas , Lipossomos/administração & dosagem , Lipossomos/síntese química , Lipossomos/farmacologia , Lipossomos/normas , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade
11.
Bioconjug Chem ; 12(5): 701-10, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11562188

RESUMO

A guinea pig liver transglutaminase (G-TGase)-mediated procedure for the site-specific modification of chimeric proteins was recently reported. Here, an alternative method with advantages over the recent approach is described. This protocol utilizes a microbial transglutaminase (M-TGase) instead of the G-TGase as the catalyst. M-TGase, which has rather broad structural requirements as compared to the G-TGase, tends to catalyze an acyl transfer reaction between the gamma-carboxamide group of a intact protein-bound glutamine residue and various primary amines. To demonstrate the applicability of the M-TGase-catalyzed protein modification in a drug delivery system, we have utilized recombinant human interleukin 2 (rhIL-2) as the target protein and two synthetic alkylamine derivatives of poly(ethyleneglycol) (PEG12; MW 12 kDa) and galactose-terminated triantennary glycosides ((Gal)(3))) as the modifiers. For the M-TGase-catalyzed reaction with PEG12 and (Gal)(3), 1 mol of alkylamine was incorporated per mole of rhIL-2, respectively. Peptide mapping of (Gal)(3)-modified rhIL-2 ((Gal)(3)-rhIL-2) by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) suggested that the Gln74 residue in rhIL-2 was site specifically modified with (Gal)(3). The PEG12-rhIL-2 and (Gal)(3)-rhIL-2 conjugates retained full bioactivity relative to the unmodified rhIL-2. In pharmacokinetic studies, PEG12-rhIL-2 was eliminated more slowly from the circulation than rhIL-2, whereas (Gal)(3)-rhIL-2 accumulated in the liver via hepatic asialoglycoprotein receptor binding. The results of this study expand the applicability of the TGase-catalyzed methodology for the preparation of protein conjugates for clinical use.


Assuntos
Interleucina-2/análogos & derivados , Interleucina-2/administração & dosagem , Interleucina-2/metabolismo , Transglutaminases/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Linhagem Celular , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/metabolismo , Portadores de Fármacos/farmacocinética , Galactose/análogos & derivados , Galactose/síntese química , Galactose/metabolismo , Glicosilação , Interleucina-2/síntese química , Interleucina-2/farmacocinética , Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Mapeamento de Peptídeos , Polietilenoglicóis
12.
Int J Pept Protein Res ; 43(2): 127-38, 1994 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8200730

RESUMO

General procedures are presented for the site-specific pegylation of peptides at the NH2-terminus, side-chain positions (Lys or Asp/Glu) or COOH-terminus using solid-phase Fmoc/tBu methodologies. A model tridecapeptide fragment of interleukin-2, IL-2(44-56)-NH2, was chosen for this study since it possesses several trifunctional amino acids which serve as potential sites for pegylation. The pegylation reagents were designed to contain either Nle or Orn, which served as diagnostic amino acids for confirming the presence of 1 PEG unit per mole of peptide. NH2-Terminal pegylation was carried out by coupling PEG-CH2CO-Nle-OH to the free NH2-terminus of the peptide-resin. Side-chain pegylation of Lys or Asp was achieved by one of two pathways. Direct side-chain pegylation was accomplished by coupling with Fmoc-Lys(PEG-CH2CO-Nle)-OH or Fmoc-Asp(Nle-NH-CH2CH2-PEG)-OH, followed by solid-phase assemblage of the pegylated peptide-resin and TFA cleavage. Alternatively, allylic protective groups were introduced via Fmoc-Lys(Alloc)-OH or Fmoc-Asp(O-Allyl)-OH, and selectively removed by palladium-catalyzed deprotection after assemblage of the peptide-resin. Solid-phase pegylation of the side-chain of Lys or Asp was then carried out in the final stage with PEG-CH2CO-Nle-OH or H-Nle-NH-(CH2)2-PEG, respectively. COOH-Terminal pegylation was achieved through the initial attachment of Fmoc-Orn(PEG-CH2CO)-OH to the solid support, followed by solid-phase peptide synthesis using the Fmoc/tBu strategy. The pegylated peptides were purified by dialysis and preparative HPLC and were fully characterized by analytical HPLC, amino acid analysis, 1H-NMR spectroscopy and laser desorption mass spectrometry.


Assuntos
Polietilenoglicóis/química , Proteínas/síntese química , Sequência de Aminoácidos , Interleucina-2/análogos & derivados , Interleucina-2/síntese química , Dados de Sequência Molecular
13.
Bioconjug Chem ; 5(1): 88-97, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-8199239

RESUMO

Conjugates of IL-2 with the ribosome-inactivating protein gelonin were prepared using heterobifunctional reagents to link the proteins via disulfide, acid-labile, and noncleavable linkers. In each case, one protein was modified using 2-iminothiolane. The sulfhydryl groups so introduced were then reacted either with 2-nitro-5-dithiobenzoate groups or with iodoacetamido groups which had been introduced into the second protein. In the case of the acid-labile linkage, a reagent which forms a labile bond upon reaction with amino groups, 4-(iodoacetamido)-1-cyclohexene-1,2-dicarboxylic acid anhydride (its synthesis is described in this paper) was used to modify the toxin. The conjugates were separated from nonconjugated proteins by gel filtration on Sephadex G100 (SF). Each was analyzed with respect to its ribosome-inactivating activity, its ability to bind to the IL-2 receptor, and its in vitro cytotoxicity. The ribosome-inactivating activity of gelonin was unaffected by modification with 2-iminothiolane and was retained in conjugates prepared using this reagent. Modification of the toxin with 4-(iodoacetamido)-1-cyclohexene-1,2-dicarboxylic acid anhydride to form the acid-labile link drastically reduced the activity of the toxin. However, the activity of the toxin was recovered following acid treatment to release the native protein. Conjugates containing each type of linkage exhibited both specific binding and selective cytotoxicity toward cells expressing the IL-2 receptor. The most potent of these toxins, that containing the disulfide linkage, exhibited a cytotoxicity which was 2 orders of magnitude greater than that of unconjugated gelonin.


Assuntos
Reagentes de Ligações Cruzadas/química , Interleucina-2/síntese química , Proteínas de Plantas/síntese química , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/fisiologia , Dissulfetos/síntese química , Feminino , Interleucina-2/metabolismo , Interleucina-2/toxicidade , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Plantas/metabolismo , Proteínas de Plantas/toxicidade , Coelhos , Ratos , Ratos Endogâmicos Lew , Receptores de Interleucina-2/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1 , Ribossomos/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologia
14.
Bioorg Khim ; 17(6): 779-88, 1991 Jun.
Artigo em Russo | MEDLINE | ID: mdl-1776962

RESUMO

Artificial DNA fragments encoding human interleukin-2 (133 a.a.) and its analogue (deletion of 14 C-terminal a.a.) were prepared by means of the DNA polymerase I mediated extension of synthetic polynucleotides having short overlapping sequences at their 3'-ends. The fragments were cloned in specially designed pFH-type plasmids and then excised by the FokI and other restriction endonucleases to yield the subfragments with the structurally predetermined 5'-unique cohesive ends. The complete synthetic gene was constructed by one or two-step ligation. The expressed IL-2 was tested by analysing the T-cell proliferation activity of E.coli crude lisates containing the pEXIL2 expression plasmid.


Assuntos
Escherichia coli/genética , Interleucina-2/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Expressão Gênica , Genes Bacterianos , Humanos , Interleucina-2/síntese química , Camundongos , Dados de Sequência Molecular , Plasmídeos , Baço/citologia , Baço/metabolismo , Timidina/metabolismo
15.
Bioorg Khim ; 15(7): 908-21, 1989 Jul.
Artigo em Russo | MEDLINE | ID: mdl-2818650

RESUMO

Synthesis of peptides corresponding to the 59-72 and 25-36 sequences of human IL-2 is reported. The former peptide, which had been shown to be immunogenic in the protein molecule, was prepared to obtain antipeptide antibodies for isolation and purification of the recombinant IL-2. We located the epitope at the C-terminus of this peptide. In accordance with the IL-2 secondary structure and hydrophilicity profile analysis, the 25-36 fragment was chosen as the potential epitope. The peptides were synthesized by conventional methods in solution, conjugated with a protein carrier, and polyclonal rabbit antisera were obtained. Antibodies against both peptides were shown to be specific to human IL-2 in ELISA. Besides, monoclonal antibodies to IL-2 recognized in ELISA the 59-72 peptide, suggesting the epitope located in this region to be main one in the protein molecule.


Assuntos
Interleucina-2/análise , Sequência de Aminoácidos , Anticorpos Monoclonais , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-2/síntese química , Interleucina-2/imunologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/síntese química
16.
J Biol Chem ; 264(2): 816-22, 1989 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-2783421

RESUMO

The structural significance of C-terminal amphiphilic alpha-helix of human interleukin-2 has been investigated using principles of protein design. Employing disulfide-mediated semi-synthesis, several multiple residue substitution patterns were studied in order to provide rapid insight into the most appropriate features to incorporate into fully recombinant proteins. Substitutions directed toward both stabilization and destabilization of the helix resulted in proteins with modulated bioactivity. Circular dichroism verified the conformational integrity and thermal stability of the derivatives. The biologic characteristics of each derivative were evaluated in the standard murine CTLL-2 assay and compared to activities exhibited in both human T-cell bioactivity and binding assay. A strategy for the design of protein ligand agonists and antagonists without knowledge of receptor contact residues is discussed.


Assuntos
Interleucina-2 , Ligação Competitiva , Dicroísmo Circular , Humanos , Interleucina-2/síntese química , Interleucina-2/imunologia , Cinética , Peptídeos/síntese química , Conformação Proteica , Receptores de Interleucina-2/análise , Relação Estrutura-Atividade , Linfócitos T/imunologia
17.
Neurosurgery ; 23(6): 725-32, 1988 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-2851116

RESUMO

Twenty patients with supratentorial, intracerebral lesions defined by computed tomographic scan or magnetic resonance imaging were treated by surgery and adoptive immunotherapy with lymphokine-activated killer (LAK) cells and recombinant Interleukin-2 (rIL-2, Cetus). Seventeen patients had glioblastoma, two had high-grade oligodendroglioma, and one patient had two metastatic sarcoma lesions. LAK cells were produced from blood mononuclear cells (MNC) obtained by 2 to 3 leukapheresis procedures and cultured (2.5 x 10(6) MNC/ml) 3 to 5 days with 1000 units rIL-2/ml. Although LAK cells could be produced from MNC of all patients, those taking steroids or with a low Karnofsky functional status generated, on average, suboptimal LAK cell activity. Age, sex, and serum anticonvulsant levels do not seem to influence a patient's ability to produce LAK cells in vitro. For therapy, cultured MNC (1-15 x 10(9] containing LAK cells were suspended in saline containing 10(6) units rIL-2 and injected into tissue surrounding the tumor cavity during craniotomy. For 3 days after their operations, patients received 10(6) units rIL-2 into the tumor cavity through an Ommaya reservoir. The treatment protocol was tolerated well by all patients, although they all experienced some degree of headache, fever, or lethargy that cleared within a few days of the last rIL-2 injection. When computed tomographic (CT) scans were obtained soon after treatment, areas of low density suggested a greater-than-normal extent of edema around the operative site. At the present time, CT scans indicate that the tumors of seven patients have recurred with an average disease-free interval of 25 +/- 6 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Imunoterapia , Interleucina-2/uso terapêutico , Células Matadoras Naturais/imunologia , Proteínas Recombinantes/uso terapêutico , Adulto , Idoso , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/cirurgia , Feminino , Seguimentos , Glioblastoma/imunologia , Glioblastoma/cirurgia , Humanos , Interleucina-2/síntese química , Interleucina-2/toxicidade , Linfocinas/farmacologia , Masculino , Pessoa de Meia-Idade
18.
J Mol Recognit ; 1(1): 42-7, 1988 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-3273651

RESUMO

A semi-synthetic protein design approach has been employed for the structural investigation of a putative helical region at the C-terminus of Interleukin-2. With crystallographic or NMR derived conformational data as yet unavailable, we have relied only on primary sequence information and computer-assisted modelling to direct the analysis. By employing both chemical peptide synthesis and recombinant DNA methods, the C-terminus of IL-2 was modified according to a strategy designed to stabilize helical secondary structure. A semi-synthetic protein incorporating 12 simultaneous amino acid replacements was constructed, which possessed potentiated biological activity and displayed a far UV circular dichroism spectrum comparable to a hybrid protein with the authentic sequence. By comparison, another hybrid protein containing a C-terminal region designed to contain helix breaking residues was totally devoid of bioactivity. These findings provide evidence that the modelling method correctly identified a helix necessary for the formation of a bioactive tertiary fold. Moreover, by employing semi-synthesis it was possible to circumvent the difficulties associated with the preparation, purification and analysis of multiple recombinant proteins, and also to avoid the unreliability of total chemical synthesis for proteins greater than 100 residues.


Assuntos
Interleucina-2 , Sequência de Aminoácidos , Dicroísmo Circular , Simulação por Computador , Desenho de Fármacos , Humanos , Interleucina-2/síntese química , Interleucina-2/farmacologia , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Estrutura Molecular , Relação Estrutura-Atividade
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