First-in-human study demonstrating the safety and clinical efficacy of novel anti-IL-17A monoclonal antibody CJM112 in moderate to severe plaque psoriasis.

BACKGROUND AND OBJECTIVE: Anti-IL-17A IgG/κ monoclonal antibody CJM112 binds both IL-17A and IL-17AF. The purpose of this First-in-Human study was to assess CJM112 effects on safety and efficacy in patients with moderate to severe plaque psoriasis. METHODS: This study had two parts: single ascending doses of 5-450 mg subcutaneous (s.c.) CJM112 (SAD) and multi-dose parallel groups of CJM112 15 mg, 50 mg and 150 mg s.c. low frequency or high frequency (MD). SAD/MD were double-blind, randomized and placebo-controlled; MD also included a secukinumab 150 mg s.c. arm as an active comparator. Patients 18-65 years with moderate to severe psoriasis were included in this study. The efficacy outcome was the change in Psoriasis Area Severity Index (PASI) from baseline to Week 4 in the SAD part of the study, and from baseline to Week 12 in the MD part. RESULTS: 96 patients were enrolled in this study (SAD, n = 42; MD, n = 54). In SAD, CJM112 doses from 15 mg and above demonstrated higher PASI responses compared with placebo at Week 12. CJM112 450 mg did not add further efficacy, but efficacy duration was prolonged compared with CJM112 150 mg. CJM112 MD resulted in a dose-dependent decrease in PASI over time to Week 12. CJM112 150 mg high frequency did not exceed the effect of CJM112 150 mg low frequency and had similar efficacy to secukinumab 150 mg. The safety profile of CJM112 was as expected for an antibody targeting IL-17A/IL-17AF. CONCLUSIONS: CJM112 had clinical efficacy in moderate to severe psoriasis and was generally safe and well tolerated in the doses tested. Additional neutralization of IL-17AF did not translate to increased clinical efficacy compared with secukinumab.

Role of repetitive antigen patterns for induction of antibodies against antibodies.

Antibody responses against antibodies, such as rheumatoid factors, are found in several immunopathological diseases and may play a role in disease pathogenesis. Experience shows that they are usually difficult to induce experimentally. Antibodies specific for immunoglobulin constant regions (anti-allotypic) or for variable regions (anti-idiotypic) have been investigated in animal models; the latter have even been postulated to regulate antibody and T cell responses via network-like interactions. Why and how such anti-antibodies are induced during autoimmune diseases, has remained largely unclear. Because repetitively arranged epitopes in a paracrystalline structure of a viral envelope cross-link B cell receptors efficiently to induce a prompt T-independent IgM response, this study used immune complexes containing viruses or bacteria to evaluate the role of antigen pattern for induction of anti-antibody responses. We present evidence that antibodies bound to strictly ordered, but not to irregularly arranged, antigens dramatically enhance induction of anti-antibodies, already after a single immunization and without using adjuvants. The results indicate a novel link between anti-antibody responses and infectious agents, and suggest a similar role for repetitive self-antigens such as DNA or collagen involved in chronic immunopathological diseases.

Differentiation of follicular dendritic cells and full antibody responses require tumor necrosis factor receptor-1 signaling.

Using mice double deficient for tumor necrosis factor (TNF) and lymphotoxin alpha (LT alpha), we demonstrated that TNF and/or LT alpha are necessary for development of a normal splenic microarchitecture and for isotype switch after immunization with sheep red blood cells (SRBC). In the present study, we extended these observations by determining which TNF receptor (TNFR) is involved in morphological and functional differentiation of the spleen. Spleen morphology and antibody response were investigated in wild-type, TNFR1-/-, TNFR2-/- and TNF/LT alpha-/- mice immunized with SRBC. TNF/LT alpha-/- mice, which have a complete disruption of the TNF/LT alpha signaling system including the LT beta-receptor pathway, displayed an abnormal microarchitecture, and isotype switch did not take place. TNFR1-/- and TNFR2-/- mice displayed a normal spleen microarchitecture and mounted an IgM and IgG antibody response to SRBC. However, the IgG production in TNFR1-/- mice was minimal, with citers leveling off 6 d after immunization. In this strain, immunofluorescence revealed a lack of follicular dendritic cells (FDC) network, detected with FDC-M1 as well as anti-CR1, and a lack of germinal centers, detected with peanut agglutinin. In conclusion, whereas normal splenic microarchitecture and isotype switch might require the LT beta receptor, differentiation of FDC network, development of germinal centers, and full IgG response depend on signaling via TNFR1.

Regulation of the MEF2 family of transcription factors by p38.

Members of the MEF2 family of transcription factors bind as homo- and heterodimers to the MEF2 site found in the promoter regions of numerous muscle-specific, growth- or stress-induced genes. We showed previously that the transactivation activity of MEF2C is stimulated by p38 mitogen-activated protein (MAP) kinase. In this study, we examined the potential role of the p38 MAP kinase pathway in regulating the other MEF2 family members. We found that MEF2A, but not MEF2B or MEF2D, is a substrate for p38. Among the four p38 group members, p38 is the most potent kinase for MEF2A. Threonines 312 and 319 within the transcription activation domain of MEF2A are the regulatory sites phosphorylated by p38. Phosphorylation of MEF2A in a MEF2A-MEF2D heterodimer enhances MEF2-dependent gene expression. These results demonstrate that the MAP kinase signaling pathway can discriminate between different MEF2 isoforms and can regulate MEF2-dependent genes through posttranslational activation of preexisting MEF2 protein.

Targeting IL-17A in multiple myeloma: a potential novel therapeutic approach in myeloma.

We have previously demonstrated that interleukin-17A (IL-17) producing T helper 17 cells are significantly elevated in blood and bone marrow (BM) in multiple myeloma (MM) and IL-17A promotes MM cell growth via the expression of IL-17 receptor. In this study, we evaluated anti-human IL-17A human monoclonal antibody (mAb), AIN457 in MM. We observe significant inhibition of MM cell growth by AIN457 both in the presence and the absence of BM stromal cells (BMSCs). Although IL-17A induces IL-6 production, AIN457 significantly downregulated IL-6 production and MM cell adhesion in MM-BMSC co-culture. AIN457 also significantly inhibited osteoclast cell differentiation. More importantly, in the SCIDhu model of human myeloma administration of AIN457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth and reduced bone damage compared with isotype control mice. To understand the mechanism of action of anti-IL-17A mAb, we report, here, that MM cells express IL-17A. We also observed that IL-17A knockdown inhibited MM cell growth and their ability to induce IL-6 production in co-cultures with BMSC. These pre-clinical observations suggest efficacy of AIN457 in myeloma and provide the rationale for its clinical evaluation for anti-myeloma effects and for improvement of bone disease.

Coordinate activation of endogenous p38alpha, beta, gamma, and delta by inflammatory stimuli.

The p38 family of mitogen-activated protein kinases is believed to mediate a variety of leukocyte responses to pro-inflammatory stimuli. There are four members of the p38 family, and although activation of the different members has been studied in transiently transfected cells much less is known about activation of the endogenous p38s, particularly in myeloid lineage cells. To investigate activation of endogenous p38s, we have made monoclonal antibodies specific for each p38 and have used these antibodies to study p38 activation by pro-inflammatory stimuli in several human monocytic cell lines. Without stimulation endogenous p38alpha kinase activity was readily detectable, whereas that of p38beta, gamma, and delta was barely measurable. In response to inflammatory stimuli, we observed a time- and dose-dependent activation of all four p38s. The kinetics of activation of each of the p38s were similar for each stimulus used, suggesting a common upstream activation pathway. Simultaneous activation of the p38s suggests that all four may be important in inflammation.

Interleukin-6 is produced by bone and modulated by parathyroid hormone.

Interleukin-6 (IL-6) is a cellular regulatory molecule, the diverse functions of which relate to cells both within and outside the immune system. In this report we demonstrated that bone tissue, specifically osteoblasts, produce interleukin-6 and that this function can be modulated by the osteotrophic hormone parathyroid hormone (PTH). Given that the complex process of bone remodeling is now thought to be regulated not only by systemic hormones but also by locally produced factors, the existence of a parathyroid hormone-stimulated production of interleukin-6 by osteoblasts may have important physiological significance.

Stimulation of lymphocytes by cholinergic receptor in myasthenia gravis.

Lymphocytes from 13 myasthenic patients were incubated in vitro with membranes enriched in nicotinic cholinergic receptor obtained from Torpedo marmorata electric organ. When autologous serum was present in the incubation medium, the lymphocytes were highly stimulated. In contrast, no stimulation occurred when lymphocytes were cultured in presence of AB serum. These results suggest that an immune response against the Ach cholinergic receptor is present in myasthenic patients and give further support to the hypothesis that an autoimmune response against muscular receptors may be involved in Myasthenia Gravis pathogenesis.

Detection and quantification of cyclosporine in body fluids using an interleukin-2 reporter-gene assay.

Different assays are employed to monitor the concentration of immunosuppressive drugs in biological fluids. None of these methods gives direct and precise information on the actual level of immunosuppression in the patient. Here we describe the use of an interleukin-2 (IL-2) reporter-gene assay (IL-2 RGA) to monitor the concentrations of immunosuppressants in body fluids. This assay is based on a chimeric gene construct in which the human IL-2 promoter drives the expression of a reporter gene. Upon mitogenic stimulation the reporter gene is expressed and can be easily quantified. The assay is very sensitive and selective for immunosuppressive compounds inhibiting IL-2 gene expression such as cyclosporine (CsA) and FK506, their active metabolites and derivatives, but not for others such as rapamycin. High reproducibility, fast performance time, and high capacity are additional characteristics of the assay. The assay was developed to monitor immunosuppressive drug levels in human volunteers or in animals receiving CsA analogues as the only immunosuppressive drugs. This assay is sensitive to CsA or ascomycin/FK506 analogues and metabolites, for which there are presently no specific monoclonal antibodies available. The IL-2 reporter-gene assay may be more suitable than other in vitro systems such as MLR or mitogen stimulated PBMC which were previously used to study the immunosuppressive activity of drugs in body fluids.

Tumor necrosis factor receptor-1 signaling is required for differentiation of follicular dendritic cells, germinal center formation, and full antibody responses.

Using mice double deficient for tumor necrosis factor and lymphotoxin alpha (TNF/LT alpha-/-) we have demonstrated that TNF and/or LT alpha are important for morphogenesis of secondary lymphoid organs and for T-cell-dependent antibody responses. In the present study we attempted to identify the receptors involved in those functions of TNF and LT alpha. Spleen morphology and antibody responses were investigated in wild-type, TNFR1-/-, TNFR2-/-, and TNF/LT alpha-/- mice immunized with SRBC. TNF/LT alpha-/- mice, which have a complete disruption of the TNF/LT alpha signaling system including the lymphotoxin beta (LT beta) receptor pathway, displayed an abnormal splenic microarchitecture and isotype switch did not take place. TNFR1-/- and TNFR2-/- mice displayed a normal splenic morphology and mounted an IgM and IgG antibody response to SRBC. However, the IgG production in TNFR1-/- mice was abnormal, with titers leveling off after 6 days following primary immunization, and with a minimal response to a second antigen challenge. Immunofluorescence analysis of spleen sections revealed in this strain a lack of follicular dendritic cell (FDC) network and of germinal centers. In conclusion, while normal splenic microarchitecture and isotype switch might require the LT beta receptor, differentiation of the FDC network, development of germinal centers, a sustained IgG response, and probably the development of memory cells depend on signaling via TNFR1.

Spontaneous and polyclonal Ig secretion by circulating B cells after surgery.

Abnormalities of the immune response are commonly observed after surgery. In many cases, they are part of a physiologic rather than of a pathologic response to trauma. In this study we show that after elective surgery in otherwise healthy subjects the B cell compartment is deeply affected, as documented by the appearance, 7 days after the intervention, of circulating lymphoblastoid B cells spontaneously secreting in vitro IgG and IgA antibodies. Analogous lymphoblastoid B cells have been described after in vivo immunization and represent a sensitive marker of the B cell response against the immunizing antigen. To better understand the origin of the reaction, we have analyzed the specificity of the antibodies secreted in culture supernatants. We show that the antibody response is polyclonal, since low titers of antibodies against several different bacterial antigens--such as tetanus toxoid, pneumococcal capsular polysaccharides (PCPs), and the lipopolysaccharides (LPSs) of several enteropathogenic strains of Escherichia coli--are detected. This response seems to reflect the previous immunologic experience of the single patient and to be caused by antigens released from traumatized tissues or absorbed through breaches in skin or mucous membranes.

p55 and p75 tumor necrosis factor receptors are expressed and mediate common functions in synovial fibroblasts and other fibroblasts.

There is increasing evidence that TNF-alpha is a cytokine of major importance in the pathogenesis of rheumatoid arthritis. Since TNF-alpha mediates its effects via high affinity receptors, we were interested in investigating their expression and function in cells from rheumatoid tissue. Synovial fibroblasts derived from rheumatoid synovial tissue are stimulated by TNF-alpha to proliferate and release cytokines, prostaglandins, proteases and protease inhibitors. We have evaluated through which receptor stimulation of DNA synthesis and the release of the proinflammatory agents, IL-6, IL-8 and PGE2 are induced. It was found that rheumatoid synovial fibroblasts express both the p55 and p75 TNF receptor, in a ratio of 4:1. TNF-alpha-stimulated synovial fibroblast DNA synthesis and the release of IL-6, IL-8 and PGE2 was inhibited by antagonist monoclonal antibodies against either the p55 or the p75 TNF receptor, although the blockade of the p55 TNF receptor had a more potent effect than inhibition of the p75 TNF receptor alone. Similarly, specific monoclonal antibodies, agonistic for either the p55 or p75 TNF receptor stimulated synovial fibroblast DNA synthesis, as well as IL-6, IL-8 and PGE2 release. Both p55 and p75 TNF receptors on dermal and gingival fibroblasts were also involved in TNF-alpha-mediated DNA synthesis and IL-6, IL-8 and PGE2 release, although differences in the levels of DNA synthesis and release of inflammatory cytokines and PGE2 were observed between the three fibroblast types.

S-adenosyl-L-methionine protection against alpha-naphthylisothiocyanate-induced cholestasis in the rat.

The effect of S-adenosyl-L-methionine (SAMe) on cholestasis induced by alpha-naphthylisothiocyanate (ANIT) was studied in rats. SAMe significantly attenuated both bile flow impairment and elevated values of serum bilirubin, glutamic pyruvic transaminase and alkaline phosphatase in ANIT-treated animals. These results suggest that SAMe protects the rat liver against the toxic effects of ANIT.

Influence of autologous serum on in vitro reactivity of peripheral lymphocytes of patients with breast cancer.

The influence of autologous serum on DNA synthesis and phytohemagglutinin (PHA) or pokeweed mitogen (PWM) stimulation of peripheral blood lymphocytes from mammary tumor-bearing women has been studied. Blood samples were collected from patients at the 4 stages of the TNM classification who had not undergone any therapeutic treatment. Lymphocyte functions were significantly reduced only in patients with the largest tumors and in the latest stage of disease. Autologous cancer sera inhibited spontaneous DNA synthesis of lymphocytes and did not inhibit lymphocyte stimulation by mitogens. It is possible that unrecognized inhibiting and stimulating factors, possibly acting in synergism with mitogens, are present in mammary cancer sera.

Persistent immunologic abnormalities in long term survivors of kidney transplantation: role of different immunosuppressive treatments.

Although immunosuppressive treatment with corticosteroids and azathioprine has become a widely employed and highly effective mode of therapy in a variety of conditions, its influence on cellular and humoral immunity is still somewhat intriguing and controversial. In order to further investigate the status of the immune system during various maintenance immunosuppressive treatments, we have studied a group of 32 long term survivors of kidney transplantation who were treated with different immunosuppressive regimens. The present investigation demonstrates the presence of a persistent defect of T and B peripheral lymphocytes in long term kidney transplant recipients. Moreover, it shows that the immune deficits are influenced by the time-dose schedule of the maintenance immunosuppressive therapy but not by the duration of the treatment. In particular methylprednisolone in a daily dose schedule (8 mg per day as a single oral dose in the morning) with or without azathioprine (1.5-3 mg/kg per day as a single oral dose in the morning) produces a global defect of T and B cell populations, while the same dosage of methylprednisolone administered every other day (16 mg every other day as a single oral dose in the morning) in association with daily doses of azathioprine exerts a sparing effect on T cell populations. The humoral parameters do not show definite abnormalities. The factors that may have contributed to the genesis of the immune imbalances we have detected are discussed.

Effect of a concomitant treatment with ethynylestradiol and ascorbic acid on bile secretion in female hamsters.

The administration of large doses (5 mg/kg b.wt./day) of ethynylestradiol to adult female hamsters did not induce cholestasis or modifications of bile lipid composition. These findings are in contrast with the data of other authors who in different experimental conditions described the sensitivity of hamsters to the estrogen-induced hepatobiliary toxicity. Ascorbic acid alone or added to ethynylestradiol did not impair bile secretion. However, it significantly increased the plasma levels of radioactivity tested 24 hours after the oral administration of a tracer dose of radiolabelled ethynylestradiol. These results confirm previous data showing in humans the capability of ascorbic acid to favour the rise of plasma concentrations of ethynylestradiol.

Different survival of long-term kidney transplant recipients treated with daily or alternate day corticosteroids.

To define the effect of different immunosuppressive treatments on survival of long-term kidney transplant recipients, we have followed, for a period of four years, a group of 18 long-term survivors (more than three years) of kidney transplantation who were subjected to two different corticosteroid regimens (daily or alternate day corticosteroids) plus azathioprine. THis study not only confirms that an alternate day corticosteroid treatment reduces the incidence of infectious episodes and of side effects, but also suggests that it may steadily improve the survival of these patients. One of the six patients who performed an alternate day corticosteroid regimen died and only one lost renal graft function. On the contrary, three of the twelve patients who received daily corticosteroids died of causes related to immunosuppressive treatment, while another five patients lost renal graft function. A very low level of peripheral blood lymphocytes, of early E rosette and E rosette-forming cells was detected in patients who died of causes related to immunosuppressive treatment.

[A new approach to immunotherapy: the transfer factor]. / Un nuovo approccio all'immunoterapia: il transfer factor.

The transfer factor is a tiny molecule capable of transferring the function of the T lymphocytes (immunological memory and retarded hypersensitivity) from a sensitized to a non-sensitized individual. The exact structure and action modalities of the molecule have not yet been precisely established. The difficulties involved in the study of the transfer factor are aggravated by the lack of any suitable experimental model. The attention of immunologists is attracted by this factor which opens up new prospects for the treatment of cancer, immunological deficiencies and certain infectious and autoimmune diseases. More profound research would appear useful to evaluate if and in what cases a potentiation of the immune mechanism can represent an alternative to immunosuppression.