Soluble cytokine receptors in biological therapy

Due to their fundamental involvement in the pathogenesis of many diseases, cytokines constitute key targets for biotherapeutic approaches. The discovery that soluble forms of cytokine receptors are involved in the endogenous regulation of cytokine activity has prompted substantial interest in their potential application as immunotherapeutic agents. As such, soluble cytokine receptors have many advantages, including specificity, low immunogenicity and high affinity. Potential disadvantages, such as low avidity and short in vivo half-lifes, have been addressed by the use of genetically-designed receptors, hybrid proteins or chemical modifications. The ability of many soluble cytokine receptors to inhibit the binding and biological activity of their ligands makes them very specific cytokine antagonists. Several pharmaceutical companies have generated a number of therapeutic agents based on soluble cytokine receptors and many of them are undergoing clinical trials. The most advanced in terms of clinical development is etanercept (Enbrel?, Immunex), a fusion protein between soluble TNF receptor Type II and the Fc region of human IgG1. This TNF-α antagonist was the first soluble cytokine receptor to receive approval for use in humans. In general, most agents based on soluble cytokine receptors have been safe, well-tolerated and have shown only minor side effects in the majority of patients. Soluble cytokine receptors constitute a new generation of therapeutic agents with tremendous potential for applications in a wide variety of human diseases. Two current areas of research are the identification of their most promising applications and characterisation of their long-term effects.     

Soluble cytokine receptors are present in normal human urine

Affinity chromatography of crude human urinary proteins on either human rIL-6, human rIFN-gamma, or anti-IFN-gamma-R mAb yielded the two respective soluble receptors in significant quantities. A single sequence of 30 amino acid residues was obtained by NH2-terminal microsequencing of the protein peak purified in tandem by affinity chromatography on an IL-6 column and reversed-phase HPLC. This sequence was identical to the predicted NH2-terminal sequence of IL-6-R as previously reported. Analysis of the eluted proteins from both IFN-gamma and anti-IFN-gamma-R columns by inhibition of solid phase RIA, ELISA, SDS-PAGE, and Western blotting proved the existence of soluble IFN-gamma-R in normal urine. Our finding, together with the already known presence of urinary TNF binding proteins and a soluble IL-2-R both in plasma and in urine, indicates that release of soluble cytokine receptors into body fluids is a general phenomenon that occurs under normal physiological conditions.     

Soluble cytokine receptors: basic immunology and clinical applications.

Cytokine activity is tightly regulated at multiple levels. Soluble cytokine receptors (sCR) contribute to the regulation of cytokine activity by modulating the ability of cytokines to bind their membrane receptors and generating a response. Endogenous sCR are generated by proteolytic cleavage or "shedding" of the membrane receptor and/or by translation from alternatively spliced messages different from those encoding the membrane forms. The resulting soluble receptors retain their ligand-binding ability and with some exceptions act as competitive inhibitors of the binding and biologic activity of their ligand, both in vitro and in vivo. However, sCR can also have certain effects on cytokines, such as structural stabilization, protection from proteolysis, and prolonged in vivo half-life, which are consistent with an added role as carrier proteins, and which may under some conditions result in potentiation of cytokine activity in vivo. The exact contribution of endogenous sCR to the regulation of immune or inflammatory responses has not yet been established unequivocally. Nonetheless, evidence indicates that the levels of certain sCR in serum and biological fluids correlate with immunological activation and/or disease activity in a variety of clinical conditions. Hence, sCR levels may have significant value as markers in disease management and prognosis. Moreover, sCR have also shown promising potential as immunotherapeutic agents for a variety of clinical disorders, including sepsis, inflammation, and autoimmune and malignant diseases.     

Soluble receptors of TNF-alpha in rheumatoid arthritis

The serum level of soluble TNF-alpha receptors with molecular mass 55 kDa (sTNF-a55R) was measured by enzyme immunoassay with commercial kits in 30 patients with rheumatoid arthritis (RA) and 38 healthy donors. High sTNF-a55R serum levels were registered in 90% of RA patients. These levels correlated with RA activity by DAS. Thus, assay for sTNF-a55R can be used for assessing RA activity.     

Animal studies on neuropathic pain: the role of cytokines and cytokine receptors in pathogenesis and therapy

When pain becomes chronic this is a process that takes place at several levels of the peripheral and central nervous systems. In recent years, proinflammatory substances like bradykinin, prostaglandins and signal molecules like cytokines have been identified as allogenic factors. In the present paper we examined whether cytokines play a role also in non-inflammatory peripheral nerve lesions, i.e. whether they are of importance in the causation of pain in general and whether their antagonists can be used therapeutically. The development of pain after peripheral nerve lesion in animal models follows the process of Wallerian degeneration. During Wallerian degeneration the expression of proinflammatory cytokines in the nerve is upregulated. Here we studied the temporal course of cytokine expression with several different analytical methods, analyzing tumor necrosis factor-alpha (TNF) and interleukin-beta (IL-beta) in the mouse model of chronic constrictive injury (CCI) of the sciatic nerve. This model is associated with reproducible pain related behavior in the animals. We found an early increase of TNF 12 hours after injury. Neutralizing antibodies to TNF were able to reduce the hyperalgesia that evolved due to the nerve injury. As TNF exerts its effects via two receptors, TNF receptor 1 (TNF-R1) and TNF receptor 2 (TNF R2), we also investigated, which of the receptors is relevant to the causation of pain in this model. It turned out that antibodies to TNF-R1, but not to TNF-R2 reduced hyperalgesia, indicating that TNF-R1 is the receptor concerned. Neutralizing antibodies to IL-1 receptor and to IL-6 receptor also reduced pain related behavior. These results lead to the conclusion that proinflammatory cytokines are involved not only in inflammatory pain but also in neuropathic pain. Therapeutic strategies involving cytokine inhibition have been tested experimentally and are already being used in preliminary clinical studies in immune-mediated diseases. In the future, they might be a useful addition to the range of treatments for patients with neuropathic pain.     

Cytokines and cytokine therapy in gastrointestinal diseases

AIM: To compare cytokine status in gastrointestinal diseases (GID) with reference to etiological factor, course, stage, therapy of the disease. MATERIAL AND METHODS: Enzyme immunoassay was used to examine cytokines in the peripheral blood, tissue homogenates of 560 GID patients. GID were represented by ulcer disease (UD), cholelithiasis, chronic hepatitis (CH), glutenic enteropathy (GE), Crohn's disease (CD), nonspecific ulcer colitis (NUC). RESULTS: In chronic recurrent GID (UD, cholelithiasis, GE) early exacerbation was characterized by elevated concentrations of IL-1 beta, IL-6, IL-8. Concentrations of IL-12, Inf alpha, g, TNF alpha reached maximum on the height of the disease. Intensification of regenerative processes raised concentrations of IL-4, IL-10. An overall level of serum cytokines averaged 190-780 pg/ml, reaching in some patients with active disease 1200-3000 pg/ml, in remission 30-110 pg/ml, in the control 40 pg/ml. In chronic progressive GID the levels of IL-1 beta, IL-2, IL-8, IL-6 reached 30-80 pg/ml, IL-12, Inf gamma, TNF alpha--150-370 pg/ml. A rise in cytokines concentrations in inflammatory viral, bacterial, autoimmune GID was higher than in cancer, alcoholism-related diseases, metabolic disturbances. Basic therapy in patients with chronic recurrent GID led to a significant fall in concentration of serum cytokines. Therapy with monoclonal antibodies to TNF alpha was associated with transitory pronounced favourable changes in peripheral blood cytokine status. CONCLUSION: GID provoke elevation of serum and tissue cytokines, impairment of cytokine balance in correlation with the etiological factor, variants of the course, stage of the disease, on-going therapy.     

Soluble transferrin receptors: significance and diagnostic value in anemia

The study was undertaken for the purpose of demonstrating a diagnostic importance of determining the level of soluble transferrin receptors not only in the diagnosis of iron-deficiency conditions (IDC) or in the differential diagnosis of anemia in a chronic disease (ACD) and of iron-deficiency anemia (IDA) but also in making a diagnosis in case of transfusion-dependent patients with Cooley's anemia. The iron metabolism (including determination of the serum iron (SI), of the total iron-binding ability (TIBA), of saturation of transferrin with iron (STFI), of serum transferrin (ST), and of soluble transferrin receptors (s-STR) was examined in 31 patients with different-genesis anemias, aged 3 to 7. Diagnoses were made for all children on the basis of the standard clinical-and-laboratory examinations and tests. A pattern of iron-metabolism parameters, typical of this pathology, was detected in all IDC patients; it is noteworthy, that the s-STR concentration amounted in this category to 9.4 +/- 1.35 mg/l, which essentially topped the normal values. It can be concluded on the basis of the obtained results that s-STR can be regarded as a key IDC marker. There was an increased SF (up to 324 +/- 64.5 mkg/L) alongside with hypoferremia in the ACD patients; the s-STR content was found to be within the reference values (3.21 +/- 0.55 mg/L), the determination of s-STR can be regarded as a test in the differential diagnosis of IDC and ACD. The results of s-STR research ranged, in patients with Cooley's anemia, drastically from 0.8 mg/L to 17 mg/L; it is noteworthy, that there was a reliable dependence on an adequacy of the conducted therapy in case of such patients. The highest sSTR values were found in children with the hereditary spherocytic hemolytic anemia (11.85 +/- 2.5 mg/L), which is, apparently, explained by a proliferative super-activity of the bone marrow observed in this pathology.     

Soluble transferrin receptor in hemochromatosis patients during phlebotomy therapy

BACKGROUND: The monitoring of phlebotomies in hemochromatosis patients depends on iron status measured by ferritin and transferrin saturation (TS). However, in the presence of inflammation or liver injury, soluble transferrin receptor (sTfR) determination was proposed to replace ferritin for diagnosing iron deficiency (ID). The present study evaluated performances of sTfR for the prediction of iron deficiency in a large number of hemochromatosis patients under phlebotomy therapy. METHODS: We studied 52 patients undergoing therapeutic phlebotomies and obtained 2 samples from 37 patients. Biological parameters were determined before each phlebotomy began. Performances of sTfR and TS in the diagnosis of iron deficiency were compared, according to ferritin levels under 12 microg/l. RESULTS: Ferritin and TS were correlated with removed iron (r=0.473, p<0.005 and r=0.345, p<0.05, respectively) and sTfR was correlated with the decrease in hemoglobin levels induced by phlebotomies (r=-0.678, p<0.0001). Areas under Receiver Operating Characteristics (ROC) curves for sTfR and TS were not statistically different for prediction of iron deficiency and sensitivity/specificity of sTfR at 1.64 mg/l were 67/86%. CONCLUSIONS: sTfR determination could be used to predict iron depletion induced by phlebotomies when ferritin is of limited interest, to avoid the appearance of anemia.     

Soluble interleukin-2 receptors increase during the active periods in cluster headache

OBJECTIVE: To investigate whether cytokines are altered during the active period of cluster headache. BACKGROUND: Patients with cluster headache show activation of the hypothalamus in PET studies and via endocrinologic parameters. Data also suggest an inflammatory process occurs in cluster headache. A connection between the presumed inflammatory cause, an immunological activation, and the hypothalamus could be generated by certain cytokines. DESIGN AND METHODS: ELISA was used to determine the serum levels of soluble interleukin-2 receptors, interleukin-1, interleukin-6, and 2 soluble interleukin-6 receptors (sIL-6R and soluble gp130) in 18 patients with cluster headache (6 women and 12 men) during the cluster period and in 17 healthy controls who were headache-free (3 women and 14 men). RESULTS: Patients with cluster headache had significantly increased soluble interleukin-2 receptors (413.6+/-223 U/mL vs. 290.0+/-112 U/mL; P <.05) compared with controls. Serum levels of interleukin-1 (0.29+/-0.30 pg/mL vs. 0.13+/-0.13 pg/mL, n.s.), interleukin-6 (0.87+/-0.6 pg/mL vs. 0.91+/-0.7 pg/ml; n.s.), soluble interleukin-6 receptors (33,131+/-8,349 pg/mL vs. 35,063+/-7,606 pg/mL; n.s.), or soluble gp130 (289+/-59 pg/mL vs. 283+/-20 pg/mL; n.s.) did not differ between the 2 groups, although patients with cluster tended to have higher interleukin-1 values. CONCLUSIONS: Because elevated soluble interleukin-2 receptors indicate T cell activation, our findings suggest immune activation during cluster headache. Because interleukin-2 can activate the hypothalamus and stimulate the release of Corticotropin-releasing Factor (CRF), interleukin-2 could link a putative immunological cause of cluster headache with the observed hypothalamic activation. Systemic changes of interleukin-1 or the interleukin-6 system do not seem to play a role in cluster headache, as no alterations of serum levels were observed. Even so, unchanged serum levels do not exclude limited local production.     

Identification of cell surface receptors for the Act-2 cytokine

We have identified cell surface receptors for Act-2, a secreted protein expressed upon activation of T cells, B cells, and monocytes. Although 125I-Act-2 showed little, if any, specific binding to resting peripheral blood lymphocytes (PBL) receptors were readily detected on PHA/PMA-activated PBL and a variety of cell lines including MT-2, HL60, DMSO differentiated HL60, HeLa, and K562 cells. The equilibrium dissociation constant (Kd) is 3-12 nM for MT-2, K562, and PBL activated with PHA/PMA for 40-80 h. We have also identified a rabbit polyclonal antiserum that can block Act-2 binding to its receptors. The ability to detect specific Act-2 receptors and the development of a blocking antiserum should prove valuable in efforts to molecularly clone the Act-2 receptor and to dissect the biological actions of Act-2.     

Soluble interleukin-2 receptors in the serum of patients with chronic renal failure

The levels of soluble form of the interleukin-2 receptor (sIL-2R) were evaluated in the peripheral blood of 29 patients with chronic renal failure (CRF) using enzyme-linked immunosorbent assay. All patients had undergone hemodialysis and the mean (+/- S.D.) BUN was before hemodialysis was 80.9 +/- 22.4 mg/100 ml. The mean level of sIL-2R was 1146 +/- 258 U/ml, which was significantly higher than the level (288 +/- 118 U/ml) in 12 individuals with a normal BUN level (p = 0.01). In patients with CRF, the elevated sIL-2R level was not influenced by hemodialysis, and not correlated with creatinine or beta 2-microglobulin levels. The kidneys may play an important role in the catabolism of serum sIL-2R. The elevated sIL-2R level may be related to compromised immunoregulation in CRF.     

Human cancer gene therapy with cytokine gene-modified cells

Cytokines can impede tumour growth and activate innate and adaptive immune responses, leading to elimination of cancer cells. For many years, it was believed that systemic administration of recombinant cytokines might become a standard treatment of different cancer types. However, due to a high toxicity of therapeutic doses and a low efficacy, even in combination with chemotherapy, this strategy is generally not accepted. On the other hand, cancer gene therapy approaches utilising cells modified with cytokine genes seem to represent a novel promising approach. For the last decade, numerous Phase I and II clinical trials evaluating different therapies based on cytokine gene-modified cells have been carried out. In the early studies, several strategies have been shown to improve clinical outcomes and induce strong antitumour immune responses. Recently, a few prospective, randomised, Phase III clinical trials have been initiated in order to finally determine the efficacy of particular cancer immunogene therapy strategies. This article reviews the present status and perspectives of clinical trials of cancer immunotherapies utilising cytokine gene-modified cells.     

Human cancer gene therapy with cytokine gene-modified cells

Cytokines can impede tumour growth and activate innate and adaptive immune responses, leading to elimination of cancer cells. For many years, it was believed that systemic administration of recombinant cytokines might become a standard treatment of different cancer types. However, due to a high toxicity of therapeutic doses and a low efficacy, even in combination with chemotherapy, this strategy is generally not accepted. On the other hand, cancer gene therapy approaches utilising cells modified with cytokine genes seem to represent a novel promising approach. For the last decade, numerous Phase I and II clinical trials evaluating different therapies based on cytokine gene-modified cells have been carried out. In the early studies, several strategies have been shown to improve clinical outcomes and induce strong antitumour immune responses. Recently, a few prospective, randomised, Phase III clinical trials have been initiated in order to finally determine the efficacy of particular cancer immunogene therapy strategies. This article reviews the present status and perspectives of clinical trials of cancer immunotherapies utilising cytokine gene-modified cells.     

Low-dose vaccinia virus-mediated cytokine gene therapy of glioma

Recombinant viruses can produce cytokines in tumors mobilizing an immune response to tumor cells. In this study, the authors investigated gene expression, in vivo antitumor efficacy, and safety of attenuated recombinant vaccinia virus (rVV) carrying murine cytokine genes interleukin (IL)-2 (rVV-mIL-2), IL-12 (rVV-mIL-12), and both IL-2 and IL-12 (rVV-2-12) in an athymic nude mice model. Significant tumor inhibition (p < 0.05) was observed in a preestablished subcutaneously implanted C6 glioma model using rVVs at doses ranging from 10(2) to 10(7) plaque forming units (PFU). An antitumor effect did not depend on the dose of the rVV-mIL-2 and rVV-mIL-12 viruses. All constructed rVVs induced a high level of cytokine expression in vitro and in vivo. Most groups injected with high doses of recombinant viruses encoding cytokine genes (10(5) to 10(7) PFU) showed signs of cytokine toxicity, whereas in the low-dose treatment groups (10(2) to 10(3) PFU) toxicity was greatly reduced. The antitumor activity of rVV-mIL-12 was associated with increases in both the percentage and number of natural killer T cells in the spleen. Local detection of interferon-y and tumor necrosis factor-alpha was also correlated with tumor growth arrest induced by the treatment. High-dose VV control vector per se induced tumor inhibition by activating Mac-1+ cells in blood, but the antitumor effect was less pronounced compared with rVV-carrying cytokine genes (p < 0.05). These results suggest that attenuated recombinant strains of VV at low doses may potentially be efficient vectors for cancer immunotherapy.     

类风湿关节炎的生物治疗进展

类风湿关节炎(R A)是活性T细胞浸润引起滑膜血管增殖及血管翳形成,导致关节软骨和骨的的破坏,30%和70%分别在发病的1年和2年内出现关节的侵蚀犤1犦。40%以上在诊断R A4年内丧失劳动力犤2犦。近年来,以细胞因子如肿瘤坏死因子(TN Fα)、白介素-1(IL-1)及T、B淋巴细胞为靶向的生物...     

Supramolecular approaches to biological therapy

Supramolecular chemistry is a useful methodology for construction of nano- or micro-sized objects and can significantly contribute to nanotechnology through so-called bottom-up processing. In addition, supramolecular self-assembled structures can mimic some aspects of biological systems. Bio-related functions such as molecular sensing, controlled release, signaling and materials separations have been realized. Supramolecular chemistry is a multidisciplinary field that includes subjects such as molecular design and nanosized materials. In this article recent examples of supramolecular chemistry in the context of biological therapy are introduced and classified into five categories: small supramolecular systems; designer polymers; self-assembled structures; predesigned assemblies; and nanomaterials. Finally, hierarchic organization of supramolecular structures for advanced functions is introduced to illustrate future directions of investigation. We hope that scientists studying therapeutic applications receive inspiration from this review to exploit the opportunities offered by supramolecular chemistry in their respective research areas.     

Understanding the dendritic cell lineage through a study of cytokine receptors

Dendritic cells form a system of antigen presenting cells that are specialized to stimulate T lymphocytes, including quiescent T cells. The lineage of dendritic cells is not fully characterized, although prior studies have shown that growth and differentiation are controlled by cytokines, particularly granulocyte/macrophage colony-stimulating factor (GM-CSF). To further elucidate the nature and control of the dendritic cell lineage, we have studied the expression of specific cytokine receptors. Sufficient numbers of dendritic cells were purified from spleen and skin to do quantitative binding studies with radiolabeled M-CSF, GM-CSF, and interleukin 1 (IL-1). To verify the nonlymphoid nature of dendritic cells, we made an initial search for rearrangements in T cell receptor and immunoglobulin genes and none were found. M-CSF binding sites, a property of mononuclear phagocytes, also were absent. In contrast, GM-CSF receptors were abundant on mature dendritic cells, with approximately 3,000 binding sites/cell with a single Kd of 500-1,000 pM. Substantial numbers of high affinity (< 100 pM) IL-1 binding sites were identified as well; cultured epidermal dendritic cells (i.e., epidermal Langerhans cells) had 500/cell and spleen dendritic cells approximately 70/cell. Cross-linking approaches showed the 80-kD species that is expected of high-affinity type 1 IL-1 receptor. Anti-type 1 IL-1 receptor (R) mAbs also visualized these receptors by flow cytometry on freshly isolated epidermal dendritic cells. These results provide new evidence that dendritic cells represent a differentiation pathway distinct from lymphocytes and monocytes. Together with recent findings on the effects of IL-1 and GM- CSF on epidermal dendritic cells in situ (see Results and Discussion), the data lead to a proposal whereby IL-1 signals IL-1R to upregulate GM- CSF receptors and thereby, the observed responsiveness of dendritic cells to GM-CSF for growth, viability, and function.