The lymphatic route. VIII. Distribution and plasma clearance of recombinant human interleukin-2 after SC administration with albumin in patients

It has been postulated that favouring the absorption of interleukin-2 via lymphatics rather than venous capillaries after subcutaneous adminstration may improve its therapeutic index. We have now evaluated in 12 cancer patients the plasma pharmacokinetic of interleukin-2 either dissolved in water or in 20% albumin solution with an internal cross-over after at least three days. Our data show that when albumin is present, the plasma concentrations of interleukin-2 versus time is increased and swelling at the injection sites is reduced. It remains to be seen whether efficacy improves during a prolonged treatment.Abbreviations AUC Area Under Plasma Curve - BRM Biological Response Modifiers - IFN Interferon - IL-2 Interleukin-2 - IV Intravenous - SC Subcutaneous - MU Megaunit     

Purification and renaturation of recombinant human interleukin-2.

Recombinant human interleukin-2 (IL-2) expressed as Escherichia coli was isolated as insoluble aggregates of protein (inclusion bodies) after cell breakage. IL-2 and contaminants were dissolved in 6 M-guanidinium chloride/10 mM-dithiothreitol, pH 8.5, and further purified in reduced and denatured form by gel-permeation chromatography in the same solvent. Renaturation was effected by dilution and autoxidation; IL-2 of native specific activity was isolated at over 95% purity by reversed-phase h.p.l.c.; an additional peak of reduced protein was also observed. Most losses of native IL-2 occurred on refolding, probably because of an aggregation process; concentrations around 1 microgram/ml were necessary to achieve 30% recovery. It was essential to maintain the denatured protein in reduced form before renaturation and autoxidation, which was most efficient at pH 8.5 with 1.5 microM-CuSO4. A procedure based on these observations has been used to prepare IL-2 on the 50 micrograms scale.     

Crystallization studies of glycosylated and unglycosylated human recombinant interleukin-2.

Glycosylated interleukin-2 (glyIL-2) has been crystallized in two crystal forms, and unglycosylated interleukin-2 (uIL-2) has been crystallized in three forms. The glycosylated form of the human recombinant IL-2 has been crystallized from 1.9 M ammonium sulfate, pH 6.5 to 7.0 in the hexagonal space group P6(2)22 or its enantiomorph. The crystals diffract to 2.8 A and contain two or three molecules per asymmetric unit. A second crystal form grows from 1.4 to 1.5 M ammonium sulfate in 0.2 M ammonium acetate, pH 5.0-5.5, as polycrystalline rosettes which are not suitable for even a preliminary crystallographic analysis. The uIL-2 crystallizes from 1.0 to 1.7 M ammonium sulfate, 0.2 M ammonium acetate, pH 4.5-5.6 in the monoclinic space group P2(1), and less frequently in the orthorhombic space group P2(1)2(1)2(1) from 2.5 M ammonium sulfate, pH 4.5 to 5.7. Cross-seeding uIL-2 with seeds from hexagonal crystals of glyIL-2 promotes nucleation of trigonal crystals of unglycosylated IL-2. These trigonal crystals belong to the space group P3(1)21 or its enantiomorph, with similar cell dimensions to the glyIL-2 hexagonal crystals.     

The use of recombinant human interleukin-2 in treating infectious diseases

Data from animal models indicate that interleukin-2 is potentially valuable in the treatment of a variety of infectious diseases of viral, fungal, protozoal, bacterial, and mycobacterial origin. The role of interleukin-2 in resistance to infection with human immunodeficiency virus or Mycobacterium Ieprae (the causative agent of leprosy) has recently been studied in detail. Data from animal models and clinical trials indicate that relatively low doses of interleukin-2 effectively stabilize or reverse the course of these infections. The recent characterization of Thi and Th2 helper T cells, and their relationship to the control of infectious diseases, are revealing the mechanisms involved in producing disease. Increased understanding of these mechanisms may help extend interleukin-2 therapy to other clinical applications.     

Limited proteolysis of recombinant human soluble interleukin-2 receptor. Identification of an interleukin-2 binding core

Limited proteolysis of a recombinant, soluble form of the Tac protein, a human interleukin-2 receptor (rIL-2R), was performed using trypsin, Staphylococcus aureus V8 protease and proteinase K to study the structural requirements of interleukin-2 receptor (IL-2R) for interleukin-2 (IL-2) binding. Sensitive proteolytic sites were found to be clustered in the regions of the polypeptide encoded by exons 3, 5, and 6, with a few semi-sensitive sites located within the two homologous domains encoded by exons 2 and 4. A number of nicked and truncated rIL-2R species generated by proteolysis were assayed for IL-2 binding using recombinant IL-2 (rIL-2) affinity gel and then structurally characterized. The results demonstrated that only the species that consist of the regions encoded by exons 2 and 4, joined by five disulfide bonds, are capable of binding IL-2 and that the presence of semi-sensitive cleavage sites within the two homologous domains had no apparent effect on IL-2 binding. These results suggest that the pattern of the sensitive cleavage sites in rIL-2R is closely related to the structural requirements for IL-2 binding. Based on the experimental results, a highly symmetrical core structure of IL-2R with a total of 135 amino acid residues was identified. This is the smallest protein moiety so far known to be capable of binding IL-2.     

Expression and production of recombinant human interleukin-2 in potato plants

Interleukin-2 is a pharmacologically important cytokine secreted by T lymphocytes. Recombinant interleukin-2 has been produced and found to be useful for many medical applications. Mass production of recombinant interleukin-2 will be prerequisite for a wider application of this molecule. In this study we investigated the possibility of using potato tubers for the production of recombinant human interleukin-2 in large quantity. A binary vector carrying the human interleukin-2 gene under a potato tuber-specific promoter (patatin promoter) was constructed. Several potato transformants expressing the human interleukin-2 gene were generated by Agrobacterium-mediated transformation. Expression of the human interleukin-2 gene was confirmed by Northern blotting and the protein level was determined by Western blot analyses. A bioassay revealed that human interleukin-2 expressed in the potato tuber supported proliferation of interleukin-2-dependent cells, CTLL-2. We found that the recombinant protein in the 2-week-old microtuber has the highest activity (115 units per gram of microtuber) and estimated that an average yield for a potato (average 200 g per potato) was 23,000 units of rhIL-2 activity. The results suggest that the potato tuber is an excellent system for the mass production of biologically active human interleukin-2.     

The structure of lymphatic capillaries in lymph formation.

The lymphatic vascular system consists of endothelial lined vessels which begin as blind-end tubes or saccules that are located within the connective tissue areas. This system serves as a one-way drainage apparatus for the removal of diffusible substances as well as plasma proteins that escape the blood capillaries. If permitted to accumulate, these escaped components would deplete the circulatory system of its plasma colloids and disrupt the balance of forces responsible for the control of fluid movement and the exchange of gases and fluids across the blood vascular wall. The lymphatic capillaries are strategically placed and anatomically constructed to permit a continuous and rapid removal of the transient interstitial fluids, plasma proteins, and cells from the interstitium. Structurally the lymphatic capillaries consist of a continuous endothelium that is extremely attenuated over major aspects of its diameter, except in the perinuclear region which bulges into the lumen. These vessels lack a continuous basal lamina and maintain a close relationship with the adjoining interstitium by way of anchoring filaments. The adjacent cells are extensively overlapped and lack adhesion devices in many areas. When electron-opaque tracers are injected intravenously (i.e., horseradish peroxidase and ferritin), subsequent electron microscopic examination of tissues reveals the presence of tracer particles within the interstitium and the lymphatic capillary lumen. These particles gain access into the lymphatic capillaries via two major pathways: 1) the intercellular clefts of patent junctions and 2) plasmalemmal vesicles (pinocytotic vesicles). Another salient feature of the lymphatic endothelial cell includes the presence of numerous cytoplasmic filaments, which are similar in morphology to the actin filaments observed in a variety of cell types. The ultrastructural features of the lymphatic capillaries are discussed in relation to their role in the removal of interstitial fluids and particulate matter, and in the formation of lymph.     

Crystallization and preliminary X-ray studies of human recombinant interleukin-2

Two different forms of crystals (potentially) suitable for x-ray structure analysis were obtained for recombinant human interleukin-2 (IL-2) using ammonium sulfate as a precipitant in the pH range of 6.3-7.3 (in the case of hexagonal bipyramidal crystals) and 4.5-5.5 (in the case of plate crystals). The hexagonal bipyramidal crystal belongs to a hexagonal space group P6(2)22 or P6(4)22 with a = b = 105.8 A and c = 122.2 A. The crystal diffracts up to 3.4 A resolution and contains 2 or 3 IL-2 molecules in an asymmetric unit. The plate crystal belongs to an orthorhombic space group P2(1)2(1)2 with a = 47.9 A, b = 79.6 A, and c = 31.9 A. The crystal diffracts up to 2.5 A resolution and contains only 1 IL-2 molecule in an asymmetric unit. These facts reconfirmed crystallographically the high homogeneity of the present preparation of human recombinant IL-2.     

Distribution of human recombinant interferon-alpha 2 in rat plasma, liver, and experimental liver metastases

It has been ascertained that one of several possible reasons for negligible interferon activity in solid tumors, namely, hepatic metastases induced in rats after intraportal injection of Walker carcinoma 256 cells, is the significantly lower levels of interferon in the interstitial fluid of metastases in comparison to normal liver and plasma.     

Crystal structure of recombinant human interleukin-4.

The crystal structure of recombinant human interleukin-4 (rhuIL-4) was initially determined at 3.5-A resolution by multiple isomorphous replacement techniques and subsequently refined to a resolution of 2.35 A by simulated annealing. The final crystallographic R-factor, based on all data in the range 6.0-2.35 A (7470 reflections), is 0.232. Bond lengths and bond angles in the molecule have root mean square deviations from ideal values of 0.016 A and 2.4 degrees, respectively. The overall structure is highly compact and globular with a predominantly hydrophobic core. The main structural feature of rhuIL-4 is a four alpha-helix bundle, which composes approximately 58% of the structure. The helices are arranged in a left-handed antiparallel bundle with two overhand connections. Within these connections is a two-stranded antiparallel beta-sheet. Both the tertiary and secondary structures of rhuIL-4 are similar to those of human granulocyte-macrophage colony-stimulating factor. Critical regions for receptor binding are proposed.     

Purification and characterization of recombinant human interleukin-2 produced in Escherichia coli

Recombinant human interleukin-2 (rIL-2) produced in Escherichia coli was purified to apparent homogeneity by cation exchange chromatography and reverse phase high performance liquid chromatography. The amino acid composition, amino terminal amino acid sequence, and carboxyl terminal amino acid were consistent with those deduced from the cDNA sequence. Besides the molecular species with the amino terminal Ala, the purified preparation contained another species having an additional Met residue at the amino terminus corresponding to the initiation codon AUG. The molar absorption coefficient of rIL-2 was determined to be 9.58 X 10(3) M-1 cm-1 at 280nm in water. Ultracentrifugal analyses revealed that it existed as a monomeric form in 0.1 M NaCl. The apparent sedimentation coefficient (S20,w) was calculated to be 1.8 S.     

Autoactivation of human recombinant coagulation factor VII

Single-chain human recombinant factor VII produced by transfected baby hamster kidney cells was purified to homogeneity in the presence of benzamidine. The amidolytic activity of single-chain recombinant factor VII with a peptidylnitroanilide substrate, methoxycarbonyl-D-cyclohexanylglycyl-L-arginine-p-nitroanilide, was less than 1% of that obtained with factor VIIa. Purified single-chain recombinant factor VII spontaneously activated in the absence of inhibitor. The activation reaction was enhanced by at least 2 orders of magnitude in the presence of a positively charged surface, provided either as an anion-exchange matrix or as poly(D-lysine). The progress curve for factor VIIa generation was sigmoidal. Benzamidine inhibits recombinant factor VIIa activity and factor VII activation with identical inhibition constants (Ki) of 11 mM. In contrast, benzamidine inhibition of bovine factor Xa and bovine factor IIa was observed at Ki values equal to 0.3 and 0.5 mM, respectively. Bovine factors Xa and IIa are known activators of factor VII and the most likely contaminants of our recombinant factor VII preparations. Single-chain recombinant factor VII purified from cells cultured in the absence of bovine serum activated at the same rate as factor VII from cells cultured in the presence of bovine serum. This also excluded the possibility that the activation reaction was caused by contaminating bovine proteases. On the basis of these observations, we propose that factor VII is autoactivated in vitro in the presence of a positively charged surface.     

Radiation sensitivity of T-lymphocytes grown with recombinant human interleukin-2

gamma-Ray and UV sensitivities of phytohemagglutinin (PHA)-stimulated T-lymphocytes were examined in the presence of the recombinant human interleukin-2 (IL-2). D0 values for the survival curves after gamma-irradiation varied from 0.90 to 1.25 Gy, and were comparable to those reported for human fibroblast cells. By fractionated exposure of gamma-rays, T-lymphocytes were shown to have the repair capacity for the sublethal damage. UV-survival curves yielded D0 of 6.5 J/m2 for T-lymphocytes from normal donors. T-Lymphocytes from a xeroderma pigmentosum patient with extremely low excision repair were markedly hypersensitive to UV (D0, 1.4 J/m2). T-Lymphocytes may be used to detect individuals who are sensitive to radiation or chemicals, and this method takes less time than that using fibroblast cells.     

Optimized conditions for high-level expression and purification of recombinant human interleukin-2 in E. coli

Interleukin-2 (IL-2), a potent cytokine has been used in anti-cancer therapy for over a decade now. IL-2, originally identified as a growth factor for T lymphocytes is a 15 kDa hydrophobic glycoprotein that induces the activation, clonal proliferation and differentiation of T and B-lymphocytes and enhances the cytotoxicity of monocytes and natural killer (NK) cells. Here, we report a simple method for the cloning, high-level expression and purification of IL-2 protein, which can be easily extended to other bioactive therapeutic proteins. The IL-2 gene was amplified from human spleen cDNA and cloned in a prokaryotic (E. coli) expression system. An optimal expression of the IL-2 protein was determined by varying the expression conditions like temperature, inducer concentration and duration of induction. The protein was expressed as inclusion bodies and a panel of reagents including detergents, urea and guanidine hydrochloride were used to solubilize it. After solubilization, the protein was renatured and subjected to a single step gel-filtration chromatography to yield immunobioactive IL-2 protein with > 99% purity.     

The effect of the recombinant human interleukin-2 Gene in potato (Solanum tuberosum cv. superior)

To examine the effect of the T-cell growth factor (human interleukin-2), we constructed a binary vector, pSSK-1, carrying the recombinant human interleukin-2 (rhlL-2) gene, and transferred it intoAsrobacterium tumefaciens. Using this construct, we then transformed potato explants(Solanum tuberosum cv. Superior), achieving 100% regeneration of shoots on a modified MS medium. Of the putative transformed shoots, 81% rooted and were selected on 200 ms/L kanamycin. Both Southern and northern analyses verified the transformation events. An ELISA test also indicated that the rhlL-2 protein was produced from rhlL-2-transformed potatoes. To determine whether this protein was biologically active in the potato cells, we performed a biological assay using the 11.-2 dependent cell line, CTLL-2. The suspension containing extract from the transformants showed significant proliferation of the 11.-2 dependent CTLL-2 cells, whereas cells did not proliferate in the nontransformed potato. We then grew the verified rhlL-2 transgenic potatoes in soil, and compared their performance with that of nontransgenic potatoes as well as those that had been transformed with GUS. Growth rates, as calculated from plant heights, were up to 50% higher than for either the nontrans-genic or the GUS-transformed potatoes. Similar patterns were found withArabidopsis thaliana plants treated in the same manner. All of these results suggest that rhlLo2 may function as a growth factor in potato.     

Evaluation of production of recombinant human interleukin-2 in fluidized bed bioreactor

The production of recombinant human interleukin-2 in a fluidized bed bioreactor containing porous glass carriers is described. Cultivations were carried out with different medium formulations over 80 days. Maximal cell densities and product yield could be maintained even when protein free medium was perfused, with less than 10% cell washout. Due to this effective immobilization of the cells in the reactor, continuous operation was easy to perform. Final cell densities on the order of 3.8 x 10(8) mL(-1) intrasphere volume were reached while the interleukin-2 production rate was 0.75 mg L(-1) d(-1). The production rate showed a maximum of a 1.9 fold decrease compared with a homogeneous stirred bubble-free aerated system. This result was in contrast to that achieved with hybridoma cell lines, where better performance was obtained with the fluidized bed bioreactor. The situation may reflect the problems caused by the dense cell culture with adherent cells, as previously shown in a hollow-fiber bioreactor with the same cell line.     

Distinct biological activities of recombinant forms of human interleukin-2 in vivo

 The biological activity of all recombinant forms of interleukin-2 (IL-2) is based upon an in vitro lymphocyte proliferation assay and measured in international units (IU). Numerous in vitro investigations have suggested that there may be different cellular effects of recombinant human IL-2 retaining the natural sequence (nIL-2) as compared to another recombinant form containing a serine substitution at amino acid position 125 ([Ser]IL-2). In the present study we investigated whether nIL-2 and [Ser]IL-2 cause similar patterns of systemic toxicities. C57BL/6 mice were treated with identical doses of either nIL-2 or [Ser]IL-2, as measured in IU, for 3 days and had blood and tissues removed for analysis of lymphocyte activation and organ dysfunction. The administration of nIL-2 had considerably greater effects on lymphocyte activation than did [Ser]IL-2, causing much greater up-regulation of the α subunit of the IL-2 receptor and the adhesion molecule lymphocyte function-associated antigen-1. Furthermore, nIL-2 induced more organ edema than did [Ser]IL-2 and caused hepatocellular injury, which was absent in mice treated with [Ser]IL-2. These data demonstrate that equivalent doses, measured in IU, of nIL-2 and [Ser]IL-2 have profoundly different effects on the induction of organ toxicity, suggesting that the IU standard may not be appropriate for the measurement of many in vivo biological activities. Received: 30 August 1996 / Accepted: 8 November 1996     

Endocrine effects of human recombinant interleukin-3 in cancer patients.

It is known that several cytokines can exert hormonal effects. At present, no data are available about the possible influence of IL-3 on the endocrine system. In order to investigate the endocrine effects of IL-3 in humans, we have evaluated serum levels of cortisol, beta-endorphin, GH, PRL, FSH, LH, TSH and melatonin in response to intravenous injection of IL-3 at a dose of 1 mcg/kg b.w. at 6.00 p.m. The study was performed in 5 non-small cell lung cancer patients. GH increased significantly in response to IL-3. PRL showed a progressive decrease after IL-3 injection, but its variations were not statistically significant. All other hormones, including cortisol, were not affected by IL-3. This preliminary study shows that IL-3 may exert endocrine effects in humans, which would seem at variance with previously reported results on most other cytokines.