Steven D. Hughes

IL-21 Enhances Tumor Rejection through a NKG2D-Dependent Mechanism

IL-21 is a cytokine that can promote the anti-tumor responses of the innate and adaptive immune system. Mice treated with IL-21 reject tumor cells more efficiently, and a higher percentage of mice remain tumor-free compared with untreated controls. In this study, we demonstrate that in certain tumor models IL-21-enhanced tumor rejection is NKG2D dependent. When engagement of the NKG2D receptor was prevented, either due to the lack of ligand expression on the tumor cells or due to direct blocking with anti-NKG2D mAb treatment, the protective effects of IL-21 treatment were abrogated or substantially diminished. Specifically, IL-21 only demonstrated a therapeutic effect in mice challenged with a retinoic acid early inducible-1δ-bearing lymphoma but not in mice bearing parental RMA tumors lacking NKG2D ligands. Furthermore, treatment with a blocking anti-NKG2D mAb largely prevented the therapeutic effect of IL-21 in mice challenged with the 4T1 breast carcinoma, the 3LL lung carcinoma, and RM-1 prostate carcinoma. By contrast, IL-21 did mediate beneficial effects against both the parental DA3 mammary carcinoma and DA3 tumors transfected with H60, a NKG2D ligand. We also observed that IL-21 treatment could enhance RMA-retinoic acid early inducible-1δ tumor rejection in RAG-1−/− deficient mice, thereby demonstrating that the IL-21-induced protective effect can be mediated by the innate immune system and that, in this case, IL-21 does not require the adaptive immune response. Collectively, these findings suggest that IL-21 therapy may work optimally against tumors that can elicit a NKG2D-mediated immune response.

IL-21 induces in vivo immune activation of NK cells and CD8+ T cells in patients with metastatic melanoma and renal cell carcinoma

PurposeHuman interleukin-21 (IL-21) is a class I cytokine previously reported in clinical studies on immune responsive cancers. Here we report the effects of systemic IL-21 therapy on the immune system in two phase 1 trials with this novel cytokine.Experimental designRecombinant IL-21 was administered by intravenous bolus injection at dose levels from 1 to 100xa0μg/kg using two planned treatment regimens: thrice weekly for 6xa0weeks (3/week); or once daily for five consecutive days followed by nine dose-free days (5xa0+xa09). The following biomarkers were studied in peripheral blood mononuclear cells (PBMC) during treatment: phosphorylation of STAT3, alterations in the composition of leukocyte subsets, ex vivo cytotoxicity, expression of effector molecules in enriched CD8+ T cells and CD56+ NK cells by quantitative RT-PCR, and gene array profiling of CD8+ T cells.ResultsEffects of IL-21 were observed at all dose levels. In the 5xa0+xa09 regimen IL-21 induced a dose dependent decrease in circulating NK cells and T cells followed by a return to baseline in resting periods. In both CD8+ T cells and CD56+ NK cells we found up-regulation of perforin and granzyme B mRNA. In addition, full transcriptome analysis of CD8+ T cells displayed changes in several transcripts associated with increased cell cycle progression, cellular motility, and immune activation. Finally, cytotoxicity assays showed that IL-21 enhanced the ability of NK cells to kill sensitive targets ex vivo.ConclusionsIL-21 was biologically active at all dose levels administered with evidence of in vivo NK cell and CD8+ T cell activation.

Exogenous PDGF-D Is a Potent Mesangial Cell Mitogen and Causes a Severe Mesangial Proliferative Glomerulopathy

The PDGF family consists of at least four members, PDGF-A, -B, -C, and -D. All of the PDGF isoforms bind and signal through two known receptors, PDGF receptor-alpha and PDGF receptor-beta, which are constitutively expressed in the kidney and are upregulated in specific diseases. It is well established that PDGF-B plays a pivotal role in the mediation of glomerular mesangial cell proliferation. However, little is known of the roles of the recently discovered PDGF-C and -D in mediating renal injury. In this study, adenovirus constructs encoding PDGF-B, -C, and -D were injected into mice. Mice with high circulating levels of PDGF-D developed a severe mesangial proliferative glomerulopathy, characterized by enlarged glomeruli and a striking increase in glomerular cellularity. The PDGF-B-overexpressing mice had a milder proliferative glomerulopathy, whereas the mice overexpressing PDGF-C and those that received adenovirus alone showed no measurable response. Mitogenicity of PDGF-D and -B for mesangial cells was confirmed in vitro. These findings emphasize the importance of engagement of PDGF receptor-beta in transducing mesangial cell proliferation and demonstrate that PDGF-D is a major mediator of mesangial cell proliferation. Finally, this approach has resulted in a unique and potentially valuable model of mesangial proliferative glomerulopathy and its resolution.

Toxicity as a result of immunostimulation by biologics

The immune system has evolved highly effective mechanisms of surveillance and defense against foreign pathogens, and is also thought to act in surveillance and suppression of cancer. Thus, a predominant goal of immune system-based therapies is to normalize or enhance the host immune response in the areas of infectious disease and oncology. This review considers general approaches used for therapeutic immunostimulation, alterations in immune response mechanisms that occur with these treatments and the syndromes that commonly arise as a result of these changes. Because nonclinical studies of these therapies are conducted in animal models as the basis for predicting potential human toxicities, this review also considers the value of nonclinical testing to predict human toxicity.

Comparison of the ability of wild type and stabilized human IgG4 to undergo Fab arm exchange with endogenous IgG4 in vitro and in vivo

Fab arm exchange by a stabilized anti-IL-31 IgG(4)S228P monoclonal antibody (mAb) was studied using physiologically relevant antibody concentrations and thiol exchange conditions, and directly compared to that of matched wild type IgG(4) (IgG(4)wt) and IgG(1) control antibodies. In vitro arm exchange between the test mAbs and a purified IgG(4)wt exchange partner was monitored using capillary isoelectric focusing and a size-exclusion peak shift assay. Arm exchange between the test mAbs and IgG exchange partners with unknown specificity was monitored using only the shift assay. Studies were performed using single isotype human and mouse mAbs, unfractionated human, mouse, and cynomolgus monkey IgG, and human serum as the sources of the exchange partners. In vitro studies using human serum demonstrated that anti-IL-31 IgG(4)S228P did not undergo significant Fab arm exchange with endogenous human IgG(4) whereas anti-IL-31 IgG(4)wt underwent rapid and extensive Fab arm exchange. The in vitro results were corroborated by in vivo studies in which mice were injected with a mixture of either form of the test mAb and an excess of non-specific human IgG(4) exchange partner.

Interleukin-21 Augments the Efficacy of T-Cell Therapy by Eliciting Concurrent Cellular and Humoral Responses

Interleukin (IL)-21 modulates T-cell-associated, B-cell-associated, and natural killer cell-associated immunity. However, the potential of IL-21 to simultaneously stimulate cellular and humoral antitumor responses and the mechanisms involved have not yet been adequately explored. In this report, we examined the immune-modulating effect of IL-21 when used in vitro and its adjuvant effects when administrated concomitantly with T-cell transfer for cancer therapy. Use of IL-21 in concert with IL-2 in culture up-regulated both type 1 and type 2 cytokine production of activated tumor-draining lymph node cells and enhanced their therapeutic efficacy. Administration of IL-21 and IL-2 as an adjuvant to T-cell transfer resulted in simultaneously elicited cellular and humoral responses. This concurrent response has led to effective regression of established pulmonary metastatic tumors and s.c. tumors. T-cell transfer plus IL-21/IL-2 administration conferred systemic immunity to the treated hosts. This was evident by the induction of protective immunity against tumor rechallenge, expansion of memory T cells, and significantly elevated serum levels of IFN gamma and IL-10. Furthermore, we observed significantly enhanced tumor-associated antibody response after T-cell + IL-2 + IL-21 therapy. Cytotoxic antibody subclass IgG2b increased strikingly in the sera of treated animals; they bound specifically to MCA205 tumor cells, and such immune sera mediated tumor cell lysis in the presence of complement. Use of B-cell-deficient mice provided direct evidence that humoral responses contribute to T-cell + IL-2 + IL-21-elicited antitumor immunity. Collectively, these findings provide a rationale to evaluate the use of IL-21 in T-cell therapy of human cancers.

Immune activation in advanced cancer patients treated with recombinant IL-21: multianalyte profiling of serum proteins

PurposeRecombinant interleukin-21 (rIL-21) is an immune stimulating cytokine recently tested in two Phase 1 trials for immune responsive cancers. A secondary objective of these trials was to characterize pharmacodynamic responses to rIL-21 in patients. Here, we report the effects of systemic rIL-21 on serum markers of immune stimulation.Experimental designRecombinant IL-21 was administered by intravenous bolus injection at dose levels from 1 to 100xa0μg/kg using two distinct treatment regimens: thrice weekly (‘3/w’) for 6xa0weeks; or once daily for five consecutive days followed by nine dose-free days (‘5xa0+xa09’). In the absence of dose limiting toxicity, additional cycles of dosing were initiated immediately following the nine dose-free days. An array of 70 different proteins was profiled in subject serum samples from several time points during the course of the study. Hierarchical clustering analysis was performed on a normalized subset of these data.ResultsSystemic administration of rIL-21 affected the serum levels of several cytokines, chemokines, acute-phase proteins and cell adhesion proteins. The magnitude and duration of response were dose dependent for a subset of these biomarkers. The 5xa0+xa09 dosing regimen generally produced cyclic changes that were of greater magnitude, as compared to a more chronic stimulation with the 3/w dosing regimen. Despite these differences, rIL-21 effects on many analytes were similar between regimens when averaged over the time of treatment. Based on similar temporal, between-subject and dose response changes, groups of analytes were identified that exhibited distinct components of the rIL-21-mediated immune activation. Biomarkers indicative of lymphocyte activation (increased IL-16, decreased RANTES), acute phase response (increased CRP, ferritin), myeloid activation (increased MDC, MIP-1 alpha), and leukocyte chemotaxis/trafficking (increased sCAMs, MCP-1) were strongly modulated in subjects treated with rIL-21.ConclusionsAdministration of rIL-21 resulted in activation of multiple cell types and immune response pathways. The changes observed in serum proteins were consistent with coincident processes of lymphoid and myeloid cell activation and trafficking, and acute phase response.

Topical recombinant thrombin at a concentration of 1000 IU/mL reliably shortens in vivo TTH and delivers durable hemostasis in the presence of heparin anticoagulation and clopidogrel platelet inhibition in a rabbit model of vascular bleeding

BackgroundThis study was designed to evaluate the effect of recombinant human thrombin (rThrombin) concentration on time to hemostasis (TTH), clot durability, and clot strength in settings that replicate the heparinization and platelet inhibition often found in surgical populations.MethodsA modified, anticoagulated rabbit arteriovenous shunt preparation was selected to model vascular anastomotic bleeding. Rabbits were treated with heparin or heparinxa0+xa0clopidogrel and TTH was measured after applying a range of topical rThrombin concentrations or placebo, in combination with absorbable gelatin sponge, USP. Treatments (placebo, rThrombin) were randomly assigned and the investigator was blinded to treatment. TTH was evaluated with the Kaplan-Meier method. After hemostasis was achieved, clot burst assessment was performed for heparinxa0+xa0clopidogrel treated animals. Clot viscoelastic strength and kinetics were measured in ex-vivo samples using thromboelastography (TEG) methods.ResultsTTH decreased with increasing concentrations of rThrombin in heparin-treated animals and was shorter after treatment with 1000xa0IU/mL rThrombin (73xa0seconds) than with 125xa0IU/mL rThrombin (78xa0seconds; pxa0=xa00.007). TTH also decreased with increasing concentrations of rThrombin in heparinxa0+xa0clopidogrel treated animals; again it was significantly shorter after treatment with 1000xa0IU/mL rThrombin (71xa0seconds) than with 125xa0IU/mL rThrombin (177xa0seconds; pxa0<xa00.001). Variability in TTH was significantly smaller after treatment with 1000xa0IU/mL rThrombin than after 125xa0IU/mL rThrombin, indicating greater reliability of clot formation (pxa0<xa00.001 for heparin or heparinxa0+xa0clopidogrel treatments). Clot durability was examined in heparinxa0+xa0clopidogrel treated animals. Clots formed in the presence of 1000xa0IU/mL rThrombin were significantly less likely to rupture during clot burst assessment than those formed in the presence of 125xa0IU/mL rThrombin (0% versus 79%, pxa0<xa00.001). In vitro clot strength and clot kinetics, as determined by TEG in heparinxa0+xa0clopidogrel samples, were positively associated with the amount of rThrombin activity added for clot initiation.ConclusionIn an animal model designed to replicate the anti-coagulation regimens encountered in clinical settings, topical rThrombin at 1000xa0IU/mL more reliably controlled the pharmacological effects of heparin or heparinxa0+xa0clopidogrel on hemostasis than rThrombin at 125xa0IU/mL. Results from in vitro assessments confirmed a positive relationship between the amount of rThrombin activity and both the rate of clot formation and clot strength.

Use of Adenovirus-Mediated Gene Transfer to Facilitate Biological Annotation of Novel Genes

As part of a large program of gene annotation, use of adenovirus-mediated gene transfer facilitated rapid progress in the functional evaluation of more than 100 genes. Localized or systemic exposure to gene products expressed by adenovirus-transduced cells led to the discovery of several novel activities through analysis of resulting physiochemical or histological changes. In this summary of the work, we present examples of two studies in which activities of novel growth factors were initially characterized using this approach. In the first example, intravenous delivery of adenovirus encoding different forms of platelet-derived growth factor (PDGF) allowed us to evaluate effects of systemic exposure to two new members of this family, PDGF-C and PDGF-D, and led to specific new hypotheses regarding their roles in diseases of the liver and kidney, respectively. In the second example, localized delivery of adenovirus encoding fibroblast growth factor (FGF)-18 to mouse pinna led to the discovery that this novel FGF is a trophic factor for mature chondrocytes and their progenitors and might be useful for treating cartilage disease. These examples serve to illustrate the potential of in vivo gene delivery approaches to facilitate functional analysis and focus of secondary investigation in a large screening effort.