Dorthe Lundsgaard

An Open-Label, Two-Arm, Phase I Trial of Recombinant Human Interleukin-21 in Patients with Metastatic Melanoma

Purpose: Human interleukin-21 (IL-21) is a pleiotropic class I cytokine that activates CD8+ T cells and natural killer cells. We report a phase 1 study of recombinant human IL-21 in patients with surgically incurable metastatic melanoma. The primary objective was to investigate safety and tolerability by determining dose-limiting toxicity (DLT). The secondary objectives were to identify a dose response for various biomarkers in the peripheral blood, estimate the minimum biologically effective dose, determine the pharmacokinetics of IL-21, determine if anti-IL-21 antibodies were induced during therapy, and measure effects on tumor size according to Response Evaluation Criteria in Solid Tumors. Experimental Design: Open-label, two-arm, dose escalation trial of IL-21 administered by i.v. bolus injection at dose levels from 1 to 100 μg/kg using two parallel treatment regimens: thrice weekly for 6 weeks (3/wk) or three cycles of daily dosing for 5 days followed by 9 days of rest (5+9). Results: Twenty-nine patients entered the study. IL-21 was generally well tolerated and no DLTs were observed at the 1, 3, and 10 μg/kg dose levels. In the 3/wk regimen, DLTs were increased in alanine aminotransferase, neutropenia, and lightheadedness with fever and rigors. DLTs in the 5+9 regimen were increased in aspartate aminotransferase and alanine aminotransferase, neutropenia, fatigue, and thrombocytopenia. The maximum tolerated dose was declared to be 30 μg/kg for both regimens. Effects on biomarkers were observed at all dose levels, including increased levels of soluble CD25 and up-regulation of perforin and granzyme B mRNA in CD8+ cells. One partial tumor response observed after treatment with IL-21 for 2 × 6 weeks (3/wk) became complete 3 months later. Conclusions: IL-21 is biologically active at all dose levels administered and is generally well tolerated, and phase 2 studies have commenced using 30 μg/kg in the 5+9 regimen.

Clinical and biological efficacy of recombinant human interleukin-21 in patients with stage IV malignant melanoma without prior treatment : a phase IIa trial

Purpose: Human interleukin-21 (IL-21) is a class I cytokine that mediates activation of CD8+ T cells, natural killer (NK) cells, and other cell types. We report final clinical and biological results of a phase II study of recombinant human IL-21 (rIL-21) in patients with metastatic melanoma. Experimental Design: Open-label, single-arm, two-stage trial. Eligibility criteria: unresectable metastatic melanoma, measurable disease by Response Evaluation Criteria in Solid Tumors, no prior systemic therapy (adjuvant IFN permitted), adequate major organ function, good performance status, no significant autoimmune disease, and life expectancy at least 4 months. Primary objective: antitumor efficacy (response rate). Secondary objectives: safety, blood biomarkers, and generation of anti-rIL-21 antibodies. rIL-21 (30 μg/kg/dose) was administered by intravenous bolus injection in 8-week cycles (5 dosing days followed by 9 days of rest for 6 weeks and then 2 weeks off treatment). Results: Stage I of the study comprised 14 patients. One confirmed complete response (CR) was observed, and as per protocol, 10 more patients were accrued to stage II (total n = 24: 10 female and 14 male). Best tumor response included one confirmed CR and one confirmed partial response, both with lung metastases. Treatment was overall well tolerated. Biomarker analyses showed increases in serum soluble CD25, frequencies of CD25+ NK and CD8+ T cells, and mRNA for IFN-γ, perforin, and granzyme B in CD8+ T and NK cells. Conclusions: rIL-21 administered at 30 μg/kg/d in 5-day cycles every second week is biologically active and well tolerated in patients with metastatic melanoma. Confirmed responses, including one CR, were observed.

Interleukin-21 activates human natural killer cells and modulates their surface receptor expression.

Interleukin (IL)‐21 is a novel cytokine that has been shown to enhance proliferation and activation of CD8+ T cells, enhance natural killer (NK) cell activity and costimulate anti‐CD40‐driven B‐cell proliferation in mice. Several studies have furthermore demonstrated antitumour effects of IL‐21 administration in mouse models. In this study we have investigated how IL‐21 affects the survival and cytotoxicity of human NK cells and modulates their expression of surface receptors and of the effector molecules granzyme B and perforin. In contrast to murine NK cells, where IL‐21 alone cannot sustain survival, IL‐21 and IL‐2 were equally efficient in sustaining survival of human NK cells. In the absence of other cytokines, IL‐21 had little effect on expression of a panel of surface receptors on human NK cells. However, IL‐21 synergized with IL‐2 to up‐regulate several surface receptors, including NKG2A, CD25, CD86 and CD69. The CD25+ CD86+ NK cells were CD56bright and were large and granular. Expression of the effector molecules perforin and granzyme A and B was up‐regulated by IL‐21 at both mRNA and protein levels. Furthermore, IL‐21 increased the cytotoxicity of NK cells against K562 target cells. These findings suggest that IL‐21 modulates NK cell activity through induction of intracellular effector molecules as well as modulation of surface receptor expression.

Inhibition of NKG2D receptor function by antibody therapy attenuates transfer-induced colitis in SCID mice.

A role for the activating NK‐receptor NKG2D has been indicated in several autoimmune diseases in humans and in animal models of type 1 diabetes and multiple sclerosis, and treatment with monoclonal antibodies to NKG2D attenuated disease severity in these models. In an adoptive transfer‐induced model of colitis, we found a significantly higher frequency of CD4+NKG2D+ cells in blood, mesenteric lymph nodes, colon, and spleen from colitic mice compared to BALB/c donor‐mice. We, therefore, wanted to study the effect of anti‐NKG2D antibody (CX5) treatment initiated either before onset of colitis, when the colitis was mild, or when severe colitis was established. CX5 treatment decreased the detectable levels of cell‐surface NKG2D and prophylactic administration of CX5 attenuated the development of colitis significantly, whereas a more moderate reduction in the severity of disease was observed after CX5 administration to mildly colitic animals. CX5 did not attenuate severe colitis. We conclude that the frequency of CD4+NKG2D+ cells increase during development of experimental colitis. NKG2D may play a role in the early stages of colitis in this model, since early administration of CX5 attenuated disease severity.

Flow Sorting from Organ Material by Intracellular Markers

Fluorescence‐activated cell sorting (FACS) is an attractive technique for gene or protein expression studies in rare cell populations. For cell types where specific surface markers are not known, intracellular markers can be used. However, this approach is currently held to be difficult, as the required fixation and permeabilization may cause protein modification and RNA degradation.

An F344 rat congenic for BB/DP rat-derived diabetes susceptibility loci Iddm1 and Iddm2

The diabetes-prone BB rat (BB/DP) spontaneously develops an insulin-dependent diabetes resembling human Type 1 diabetes (previously named IDDM). As in Type 1 diabetes, the insulinproducing b-cells of the islets of Langerhans are targets for autoimmune destruction (Crisa et al. 1992). Two major diabetes susceptibility genes have been genetically positioned from this model: Iddm1 (Lyp) on rat chromosome (Chr) 4 and Iddm2 (RT1) on rat Chr 20 (Colle et al. 1981; Jacob et al. 1992), but several studies have defined additional loci involved in diabetes development (Kloting et al. 1995, 1998; Kloting and Kovacs 1998; Martin et al. 1999; Klaff et al. 1999). In the latter reference, a locus on Chr 2 (Iddm3), where the F344 allele protects against diabetes, was detected. Because the F344 rat also carries protective alleles of Iddm1 and -2, a precise positioning of this locus was not performed. We set out to produce an F344 rat congenic for the Iddm1 and -2 of BB/DP rat origin (F344.lypRT1). Such a strain will make precise positioning of Iddm3 (and other genes affecting diabetes) more feasible, since fewer animals need to be bred. The F344.LypRT1 strain originated in F2(F344 × BB/DP) rats that were backcrossed to F344. These animals were then intercrossed, and offspring were selected for homozygocity for the BB/DP alleles of Lyp and RT1 by FACS (described in Jackerott et al. 1997). At this point, marker-assisted selection was started (Wakeland et al. 1997) in addition to monitoring the two Iddmloci. Four rounds of backcross to F344 followed, with selection of heterozygotes for RT1 by FACS and for Lyp by PCR [via flanking markers D4Rat75 and D4Mit7 (Npy) (described in Hornum and Markholst 1999)]. These rats were designated “N6”F344.LypRT1, since six rounds of introgression had been performed. For marker-assisted selection, 86 microsatellites evenly distributed throughout the genome were typed in each generation, and animals with the highest number of markers of F344 origin were selected for further breeding. The longest distance between markers (or markers and chromosome ends) is 46 cM. “N6”F344.LypRT1 rats, with all 86 microsatellites of F344 origin, were finally intercrossed, and offspring homozygous for the BB/ DP-derived Iddm1and Iddm2-alleles form the basis of the new F344.LypRT1 congenic strain. The segment of BB/DP origin around Iddm1 on Chr 4 is delimited by D4Rat15 and D4Rat37 with map positions 29.4 cM and 46.5 cM on the SHRSP × BN rat genetic map from the Whitehead Institute (www.genome.wi.mit.edu/rat/public), making the maximal size of the BB/DP-derived segment 17.1 cM. The segment of BB/DP origin around Iddm2 on Chr 20 is delimited by the centromere end and D20Rat43 and -70, both of which map to position 15.7 cM on the FHH × ACI rat genetic map from the Whitehead Institute, making the maximal size of the BB/DP derived segment 15.7 cM. While the diabetes incidence in our BB/DP line is >90% with more than 99% of these becoming diabetic before the age of 120 days, none of 22 (0%) F344.LypRT1 congenic rats became diabetic at this age (p << 0.001), although kept under the same specific pathogen-free conditions. Insulitis was not detected among four F344.LypRT1 congenic rats at the age of 120 days. This clearly demonstrates that loci outside Iddm1 and -2 protect

PolyI:C Induction of Diabetes Is Controlled by Iddm4 in Rats with a Full Regulatory T Cell Pool

Abstract:  The Iddm4 gene controls diabetes in rats depleted of regulatory T cells (Treg) and immune‐activated via treatment with the toll‐like receptor 3 (TLR‐3) ligand, polyI:C. Both diabetes‐resistant (BBDR) and diabetes‐prone (BBDP) BB rats carry dominant permissive alleles of Iddm4, while the recessive Wistar Furth (WF) rat allele is protective. Iddm4 is positioned close to Iddm2 on chromosome 4, but when we introgressed BBDP‐derived parts of this region—either containing both genes or Iddm2 alone—into the WF genome, none of these congenic strains developed spontaneous diabetes. Although both strains harbor two copies of the recessive Iddm2 allele of the BBDP rat, making these animals devoid of Treg cells, immune activation in itself via polyI:C treatment did not induce overt diabetes. Interestingly, TLR‐3 ligation without depletion of Tregs resulted in diabetes and insulitis development in nonlymphopenic F1‐offspring of mating the Iddm4+Iddm2 congenic strain to WF. This demonstrates that the diabetogenic allele of Iddm4 is able to confer diabetes susceptibility even in a nonlymphopenic host with a full Treg pool, and that homozygosity for Iddm2—although responsible for an almost total lack of Tregs—delays the disease process. Finally, we have confirmed the position of Iddm4 in truly congenic strains.

Detection of gene expression signatures related to underlying disease and treatment in rheumatoid arthritis patients

ObjectivesGene expression signatures can provide an unbiased view into the molecular changes underlying biologically and medically interesting phenotypes. We therefore initiated this study to identify signatures that would be of utility in studying rheumatoid arthritis (RA).MethodsWe used microarray profiling of peripheral blood mononuclear cells (PBMCs) in 30 RA patients to assess the effect of different biologic agent (biologics) treatments and to quantify the degree of a type-I interferon (IFN) signature in these patients. A numeric score was derived for the quantification step and applied to patients with RA. To further characterize the IFN response in our cohort, we employed type-I IFN treatment of PBMCs in vitro and in reporter assays.ResultsProfiling identified a subset of RA patients with upregulation of type-I IFN-regulated transcripts, thereby corroborating previous reports showing RA to be heterogeneous for an IFN component. A comparison of individuals currently untreated with a biologic with those treated with infliximab, tocilizumab, or abatacept suggested that each biologic induces a specific gene signature in PBMCs.ConclusionsIt is possible to observe signs of type-I IFN pathway activation in a subset of clinically active RA patients without C-reactive protein elevation. Furthermore, biologics-specific gene signatures in patients with RA indicate that looking for a biologic-specific response pattern may be a potential future tool for predicting individual patient response.

Evaluating IL-21 as a Potential Therapeutic Target in Crohn’s Disease

Background and Aim Interleukin-21 (IL-21) is primarily a T cell-derived cytokine; it is upregulated in patients with Crohns Disease (CD) and could be a potential new therapeutic target in CD. Methods In human material, IL-21 and IL-21R expression was investigated by in situ hybridization (ISH) and immunohistochemistry (IHC) in noninflammatory bowel disease (non-IBD) controls and patients with CD. The pathologic role of IL-21 was examined in murine models of T cell-dependent and T cell-independent colitis, either with a neutralizing monoclonal antibody against IL-21 or with the transfer of CD4+CD45RBhighIL-21R−/− T cells. Colonic pathology was examined by endoscopy, histopathology, IHC, ELISA, and Luminex. Results In the human intestine, IL-21 and IL-21R mRNA and protein-expressing cells were observed in the mucosa, in lymphoid aggregates of submucosa in non-IBD controls, and in lymphoid aggregates of muscularis externa in patients with CD. IL-21 expression was most abundant in germinal centers (GCs) of the lymphoid aggregates, and IL-21R expression assessed semiquantitatively, was significantly higher in patients with CD compared to non-IBD controls. Following prophylactic and interventive anti-IL-21 mAb treatment in the adoptive transfer (AdTr) model, clinical and pathological parameters were significantly reduced. The most persistent finding was a reduction in colonic infiltrating neutrophils. As well, Rag2−/− mice receiving CD4+CD45RBhighIL-21R−/− T cells developed less severe colitis compared to Rag2−/− mice receiving CD4+CD45RBhighIL-21R+/+ T cells. No effect of reduced IL-21 signalling was observed in T cell-independent colitis. Conclusion Our study shows that patients with CD have significant expression of IL-21 and IL-21R in the gut. As well, we show that neutralization of IL-21 in experimental T cell-driven colitis is associated with a reduction in clinical and pathological findings. This amelioration seems to be associated with a reduction in colon-infiltrating neutrophils.

Expression of IL-21 receptor in synovial tissue and blood of patients with rheumatoid arthritis

Background and objectives Cytokines regulate a broad range of inflammatory pathways in the pathogenesis of Rheumatoid Arthritis (RA), and cytokine blockade against tumour necrosis factor α and IL-6 has offered substantial advances in the treatment of articular inflammation. However, a large proportion of patients will not respond or exhibit only a partial response to treatment and new therapies are thus required. IL-21 is a member of the four-α-helix bundle family of cytokines that mediates pleiotropic effects through the IL-21 receptor (IL-21R). Since potency of IL-21 is mainly dependent on presence and abundance of its receptor on different cells types, the objective of this study was to characterise expression of IL-21R in the synovium and blood of patients with RA. Materials and methods Immunohistochemistry for IL-21R was carried out on synovial tissue samples derived by arthroplasty from patients with RA (n=5) obtained from the Institute of Infection, Immunity and Inflammation Research Tissue Bank. Mononuclear cells were separated out from peripheral blood (PBMC) of 10 RA patients or 3 healthy controls on a density gradient using Histopaque (Sigma) and analysed by flow cytometry for IL-21R expression on T cells (CD3/CD4/CD8), B cells (CD19/CD27) and NK cells (CD16/CD56). Results Expression of IL-21R was detected in 5/5 synovial RA tissues. The IL-21R+ cells were located in the synovial intimal and sublining layers and in lymphoid aggregates. Flow cytometric analysis on blood PBMC revealed that IL-21R is highly expressed on both CD4+ (73.04%, 12.58 MFI) and CD8+ (50.88%, 13.69 MFI) T cells, as well as on a proportion of NK cells (73.83%, 19.15 MFI) in RA patients. On B cells, IL-21R expression was higher on the CD27- fraction of naïve B cells (95.19%, 37.02 MFI), with lower expression on the CD27+ memory B cells (15.2%, 32.22 MFI). Conclusions Our results show increased expression of IL-21R in established RA synovial tissue and peripheral blood, and indicate that targeting of the IL-21/IL-21R pathway may be a valid therapeutic strategy for the treatment of RA.