Current State of Type 1 Diabetes Immunotherapy: Incremental Advances, Huge Leaps, or More of the Same?

Thus far, none of the preclinically successful and promising immunomodulatory agents for type 1 diabetes mellitus (T1DM) has conferred stable, long-term insulin independence to diabetic patients. The majority of these immunomodulators are humanised antibodies that target immune cells or cytokines. These as well as fusion proteins and inhibitor proteins all share varying adverse event occurrence and severity. Other approaches have included intact putative autoantigens or autoantigen peptides. Considerable logistical outlays have been deployed to develop and to translate humanised antibodies targeting immune cells, cytokines, and cytokine receptors to the clinic. Very recent phase III trials with the leading agent, a humanised anti-CD3 antibody, call into question whether further development of these biologics represents a step forward or more of the same. Combination therapies of one or more of these humanised antibodies are also being considered, and they face identical, if not more serious, impediments and safety issues. This paper will highlight the preclinical successes and the excitement generated by phase II trials while offering alternative possibilities and new translational avenues that can be explored given the very recent disappointment in leading agents in more advanced clinical trials

Tolerogenic Dendritic Cells and T-Regulatory Cells at the Clinical Trials Crossroad for the Treatment of Autoimmune Disease; Emphasis on Type 1 Diabetes Therapy

Tolerogenic dendritic cells and T-regulatory cells are two immune cell populations with the potential to prevent the onset of clinical stage type 1 diabetes, and manage the beginning of underlying autoimmunity, at the time-at-onset and onwards. Initial phase I trials demonstrated that the administration of a number of these cell populations, generated ex vivo from peripheral blood leukocytes, was safe. Outcomes of some of these trials also suggested some level of autoimmunity regulation, by the increase in the numbers of regulatory cells at different points in a network of immune regulation in vivo. As these cell populations come to the cusp of pivotal phase II efficacy trials, a number of questions still need to be addressed. At least one mechanism of action needs to be verified as operational, and through this mechanism biomarkers predictive of the underlying autoimmunity need to be identified. Efficacy in the regulation of the underlying autoimmunity also need to be monitored. At the same time, the absence of a common phenotype core among the different dendritic cell and T-regulatory cell populations, that have completed phase I and early phase II trials, necessitates a better understanding of what makes these cells tolerogenic, especially if a uniform phenotypic core cannot be identified. Finally, the inter-relationship of tolerogenic dendritic cells and T-regulatory cells for survival, induction, and maintenance of a tolerogenic state that manages the underlying diabetes autoimmunity, raises the possibility to co-administer, or even to serially-administer tolerogenic dendritic cells together with T-regulatory cells as a cellular co-therapy, enabling the best possible outcome. This is currently a knowledge gap that this review aims to address

Identification of a Cardiac Specific Protein Transduction Domain by In Vivo Biopanning Using a M13 Phage Peptide Display Library in Mice

Background: A peptide able to transduce cardiac tissue specifically, delivering cargoes to the heart, would be of significant therapeutic potential for delivery of small molecules, proteins and nucleic acids. In order to identify peptide(s) able to transduce heart tissue, biopanning was performed in cell culture and in vivo with a M13 phage peptide display library. Methods and Results: A cardiomyoblast cell line, H9C2, was incubated with a M13 phage 12 amino acid peptide display library. Internalized phage was recovered, amplified and then subjected to a total of three rounds of in vivo biopanning where infectious phage was isolated from cardiac tissue following intravenous injection. After the third round, 60% of sequenced plaques carried the peptide sequence APWHLSSQYSRT, termed cardiac targeting peptide (CTP). We demonstrate that CTP was able to transduce cardiomyocytes functionally in culture in a concentration and cell-type dependent manner. Mice injected with CTP showed significant transduction of heart tissue with minimal uptake by lung and kidney capillaries, and no uptake in liver, skeletal muscle, spleen or brain. The level of heart transduction by CTP also was greater than with a cationic transduction domain. Conclusions: Biopanning using a peptide phage display library identified a peptide able to transduce heart tissue in vivo efficiently and specifically. CTP could be used to deliver therapeutic peptides, proteins and nucleic acid specifically to the heart. © 2010 Zahid et al

Sequence Variation in Promoter of Ica1 Gene, Which Encodes Protein Implicated in Type 1 Diabetes, Causes Transcription Factor Autoimmune Regulator (AIRE) to Increase Its Binding and Down-regulate Expression

ICA69 (islet cell autoantigen 69 kDa) is a protein implicated in type 1 diabetes mellitus in both the non-obese diabetic (NOD) mouse model and humans. ICA69 is encoded by the Ica1 gene on mouse chromosome 6 A1-A2. We previously reported reduced ICA69 expression in the thymus of NOD mice compared with thymus of several non-diabetic mouse strains. We propose that reduced thymic ICA69 expression could result from variations in transcriptional regulation of the gene and that polymorphisms within the Ica1 core promoter may partially determine this transcriptional variability. We characterized the functional promoter of Ica1 in NOD mice and compared it with the corresponding portions of Ica1 in non-diabetic C57BL/6 mice. Luciferase reporter constructs demonstrated that the NOD Ica1 promoter region exhibited markedly reduced luciferase expression in transiently transfected medullary thymus epithelial (mTEC+) and B-cell (M12)-derived cell lines. However, in a non-diabetic strain, C57BL/6, the Ica1 promoter region was transcriptionally active when transiently transfected into the same cell lines. We concomitantly identified five single nucleotide polymorphisms within the NOD Ica1 promoter. One of these single nucleotide polymorphisms increases the binding affinity for the transcription factor AIRE (autoimmune regulator), which is highly expressed in thymic epithelial cells, where it is known to play a key role regulating self-antigen expression. We conclude that polymorphisms within the NOD Ica1 core promoter may determine AIRE-mediated down-regulation of ICA69 expression in medullary thymic epithelial cells, thus providing a novel mechanistic explanation for the loss of immunologic tolerance to this self-antigen in autoimmunity

Minimum Information about T Regulatory Cells: A Step toward Reproducibility and Standardization.

Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications

The genetic and epigenetic regulation of insulin-like growth factor II gene expression in humans /

The human insulin-like growth factor-II is an important fetal mitogen with demonstrated effects on growth, proliferation and survival of a wide spectrum of cells and tissues in mice and humans. The transcriptional regulation of the gene (IGF2) is under developmental control and is subject to epigenetic and genetic effects. At the epigenetic level, the mouse gene is parentally imprinted with exclusive expression of the paternally-inherited gene copy in most of the tissues examined. In this work, we demonstrate that the human gene is also subject to genomic imprinting and that the trait is tissue-specific and polymorphic whose basis may be genotype-dependent. Additionally, we show that in culture, primary placental fibroblasts lose the ability to functionally imprint IGF2. In instances of biallelic IGF2 gene expression, both in human tissue as well as in culture, we observe that the adjacent, reciprocally-imprinted H19 gene, which is normally expressed from the maternally-transmitted allele, is undetectable.The imprinted IGF2 gene is located in close physical proximity to a variable number of tandem repeats (VNTR) sequence polymorphism that is found upstream of the insulin gene promoter, in humans. Alleles of the VNTR have demonstrated transcriptional effects on insulin gene expression in human fetal and adult pancreas in vivo and in pancreatic beta cells in vitro. In this work, we show that the VNTR also has allelic effects on IGF2 expression in human placenta in vivo and on INS-IGF2 reporter gene activity in human lymphoblasts, in vitro.Alleles of the 5' INS (VNTR) are associated with susceptibility to type I diabetes mellitus. The preferential transmission of paternal susceptibility haplotypes at this locus suggests the functional involvement of a nearby imprinted gene in the susceptibility to type I diabetes. The demonstration of allelic effects of the VNTR on IGF2 mRNA levels, in vivo and in vitro, makes IGF2 an attractive functional candidate