JDRF Funded Research

JDRF-Funded Research:  Lay Abstract

Name:   Mark Peakman ,Ph.D. Guy's, King's & St Thomas' School of Medicine of King's College London, UK Lab Website:   Project duration:   01-NOV-2005   to   31-OCT-2009 Program Project Grant Project grant award:   $1,674,708.00* * Total Grant award amount may vary depending on budget adjustments and it is contingent upon research progress and availability of JDRF research funds.

File No:   7-2005-877 (Abstract Dt: 31-OCT-2007) Therapeutic Area:   Immune Therapies Grant Status: Active

Project Title

"Epitope discovery and peptide-based therapeutics"

Lay Abstract for Overall Project

Objective

We propose (i) to discover new targets for T cells from patients with T1DM and (ii) establish the optimal use of these for peptide immunotherapy. In the past few years we have developed robust technologies for identifying the peptide targets naturally presented to auto-aggressive T cells that cause islet damage in T1DM. We are employing them in this project to gain a vision of the presentation of such targets across as many different genetic backgrounds as possible, so that the knowledge we gain can be applied to as many patients as possible. The technological approach requires a complex biochemical analysis (Projects 1 and 2), followed by further complex analysis (Core B), followed by validation of targets using blood samples from patients (Project s 1 and 2 and Core A). We are the knowledge gained to conceive and design better assays for measuring T cell responses in patients, which can be of great use in clinical trial monitoring.

Publications/Presentations

Peakman M, Roep BO: Secondary measures of immunologic efficacy in clinical trials. Curr. Opinion in Endocrinology and Diabetes, 13:325-331, 2006. DiLorenzo T, Peakman M, Roep BO: Systematic analysis of T-cell epitopes in autoimmune diabetes. Clin. Exp. Immunol., in press, 2006. Detection of islet autoantigen reactive CD8 T cells in T1DM by Arif, Skowera, Zendegani, Unger, Kester, Dayan, Roep and Peakman, submitted. Staeva-Vieira T, Peakman M, von Herrath M. Immune-based Therapeutic Approaches for Type 1 Diabetes Mellitus. Clin. Exp. Immunol., in press, 2006.

Who benefits from the research

The identification of T cell epitopes is a major driving force behind the development of antigen-specific immune therapy strategies for diabetes prevention, and behind the development of novel assays for T cell responses, an essential prerequisite for future clinical trial monitoring.

Future Plans

Continued epitope identification and validation.

Lay Abstract for project 1

Objective

We propose (i) to discover new epitopes for CD4 T cells by direct elution from antigen presenting cells pulsed with islet autoantigens proinsulin, GAD65 and IA-2 and (ii) validate new CD8 T cell epitopes identified in Project 2 using samples from Core A and functional analyses of T cells from patients. In the past few years we have developed robust technologies for identifying naturally processed and presented CD4 T cell epitopes of islet autoantigens in an antigen- and HLA allele-specific process. This project aims to apply this technology to the 3 major islet autoantigens and the major diabetes HLA-susceptibility molecules (DR4, DR3, DQ8, DQ2) in order to establish a comprehensive portfolio of epitopes relevant to T1DM. These may then be applied in both therapeutic and diagnostic settings. The technological approach requires a complex biochemical analysis (Project 1), followed by chromatography and mass spectrometry (Project 1 and Core B), followed by validation of epitopes by ex vivo analysis of blood samples (Project 1 and Core A). More recently, we have developed technologies for measuring CD8 T cell responses specific for islet autoantigens. These include a sensitive cytokine ELISPOT refined for CD8s (Project 1 and Core C) and peptide-loaded HLA class I tetramers (Project 1, Project 2 and Core B). We have begun to apply these techniques to understand CD8 T cell responses to class I HLA-A2 epitopes identified in Project 2, using T1DM samples from Core A.

Accomplishments

1. Identifying naturally processed and presented epitope targets of CD4 T cells. Identification of novel high efficiency pulsing protocol for autoantigens onto EBV B cells 2. Identifying and validating by T cell assay CD8 epitopes of islet autoantigens Much greater progress than expected has been made in the CD8 component of the work. In relation to CD8 epitopes, as outlined in the report of Project 2, several potentially important CD8 epitopes from proinsulin have been identified as being naturally generated by an in vitro processing assay. We have then probed CD8 T cells from patients to see whether they make a response to these, using the best candidate peptides and additional peptides from islet amyloid peptide, GAD65 and IGRP, with the aim of establishing whether CD8 T cell responses can be detected and whether there is a “best set” of peptides for this approach. Our data show this to be the case, with a manuscript just submitted. Most interestingly, we used HLA-A2 tetramer loaded with one of the main insulin peptides and showed that CD8 T cells could be detected and that their number in the circulation is highly correlated with ELISPOT. We have also identified a novel set of preproinsulin epitopes, for which cytotoxic CD8 T cells have been cloned.

Publications/Presentations

Tree TIM, Roep BO, Peakman M: A mini meta-analysis of studies on CD4+CD25+ T cells in human type 1 diabetes; Report of the Immunology of Diabetes Society T cell Workshop. Ann NY Aca Sci, in press, 2006. Peakman M, Roep BO: Secondary measures of immunologic efficacy in clinical trials. Curr. Opinion in Endocrinology and Diabetes, 13:325-331, 2006. DiLorenzo T, Peakman M, Roep BO: Systematic analysis of T-cell epitopes in autoimmune diabetes. Clin. Exp. Immunol., in press, 2006. Detection of islet autoantigen reactive CD8 T cells in T1DM by Arif, Skowera, Zendegani, Unger, Kester, Dayan, Roep and Peakman, submitted.

Who benefits from the research

The identification of T cell epitopes is a major driving force behind the development of antigen-specific immune therapy strategies for diabetes prevention, and behind the development of novel assays for T cell responses, an essential prerequisite for future clinical trial monitoring.

Future Plans

Continued epitope identification and validation.

Lay Abstract for project 2

Objective

(i) discovery of new targets of autoreactive T-cells (CTLs) capable of direct destruction of pancreatic beta-cells; ii) use of these reagents to develop assays to monitor CTL activity in type 1 diabetic patients; and (iii) explore novel strategies with drug-treated dendritic cells to deviate islet autoreactivity against beta-cell targets towards an anti-inflammatory type, in an attempt to develop new intervention strategies.

Accomplishments

We have accomplisedh all milestones of the first year finishing proteasome digestions, determining HLA peptide binding affinities and discovery of new CD8 epitopes in the first year of our program. A panel of HLA-A2 tetramers has been generated and appled for direct ex vivo cloning of circulating autoreactive CD8 T-cells from new-onset type 1 diabetic patients, and screening of blood samples for autoreactive CD8 T-cells using newly defined CD8 epitopes in the first year of our program. Dendritic cells have been modulated. We compared two drugs for their DC modulating potential, and compare different modes for DC development. Finally, we have assessed stability of the modulated phenotype and evaluated the functional characteristics, employing these modulated DC to deviate T-cell reactivity in a proof-of-principle study.

Publications/Presentations

Pinkse GGM, Tysma OHM, Bergen CAM, Kester MGD, Ossendorp F, Van Veelen PA, Keymeulen B, Pipeleers D, Drijfhout JW, Roep BO: Autoreactive CD8 T cells associated with beta-cell destruction in type 1 diabetes. Proc. Natl. Acad. Sci. USA, 102:18425-30, 2005. Pinkse GGM, Boitard C, Tree TIM, Peakman M, Roep BO: HLA class I epitope discovery in type 1 diabetes: Independent and reproducible identification of proinsulin epitopes of CD8 T-cells. Report of the IDS T-cell workshop committee. Ann NY Aca Sci, in press, 2006. Peakman M, Roep BO: Secondary measures of immunologic efficacy in clinical trials. Curr. Opin. Endocrinol and Diabetes, 13:325-331, 2006. Roep BO: T-cell responses to autoantigens in type 1 diabetes: The search for the Holy Grail. Retrospective: What T-cells see in islets. Diabetes, in press, 2006. Huurman VAL, Decochez K, Mathieu C, Cohen IR, Roep BO: Therapy with the hsp60 peptide p277 (DiaPep277) in c-peptide positive type 1 diabetes patients. Diabetes Metab. Rev. Res., in press, 2006. DiLorenzo T, Peakman M, Roep BO: Systematic analysis of T-cell epitopes in autoimmune diabetes. Clin. Exp. Immunol., in press, 2006.

Who benefits from the research

Our studies have led and will lead to new methods and reagents to monitor beta-cell destructive T-cell autoreactivity in prediabetic subjects, newly-diagnosed type 1 diabetes patients, type 1 diabetic transplant recipients and type 1 diabetes patients participating in therapeutic intervention trials to assess the degree of anti-islet pathogenesis and possibly efficacy of intervention. Furthermore, with the isolation and characterization of these lytic CD8 T-cells, reagents become available to generate and evaluate preclinical humanized animal models of autoimmune diabetes that may be useful to study pathogenesis and intervention. One such intervention is under development and involves the use of professional antigen-presenting cells that have been modulated to induce anti-inflammatory T-cell responses. We aim to use such modulated DC as adjuvant in our clinical vaccination studies using epitopes discovered under Projects 1 and 2 of this program.

Future Plans

We will continue to discover and evaluate new CD8 target epitopes and compare screening measures (Core B) to determine the utility of the CD8 reagents to monitor disease progression (Core A) and possibly intervention. We will translate the DC modulation strategy for clinical application in collaboration with Project 3, to be combined with peptide vaccination using epitopes discovered under Projects 1 and 2 of this program.

Lay Abstract for project 3

Objective

Our goals were to generate preclinical data in Nod mice to aid the current and future clinical trial design for peptide based interventions with NPPEs. To this aim, NOD mice were immunized intranasally and subcutaneously with various CD4 and CD8 peptides, which are known to be processed, presented and recognized in the mouse. The age of immunization was at 10 weeks of age, in order to more realistically model the human situation, where it is difficult to intervene very early, prior to occurrence of anti-islet autoantibodies.

Accomplishments

We observed that intransal immunization in particular could lead to significant disease acceleration in some instances (i.e. B9-23). This stands in contrat to earlier studies, where protection of NOD mice was seen, but only after very early immunization (5-6 weeks of age, Wegmann and others). Such acceleration was not observed, in preliminary investigations, when a combination of peptides was being used. Our hypothesis to explain these findings is that the pre-existing response to autoantigens determines, whether a regulatory T cell response or an augmentation of the aggressive effector cells follows immunization (even when using the ‘tolerogenic’ mucosal route). We intend to corroborate this notion by performing T cell ELISPOTS on diabetic and non-diabetic mice. We will also test various forms of combination therapies with CD4 and CD8 epitopes to optimize clinical protocols that are operational later during disease development without leading to T1D acceleration.

Publications/Presentations

Presented at this year’s NYAS conference in San Francisco in November 2006 – animal models for T1D and MS.

Who benefits from the research

This research will directly hyelp to rationalize and improve the design of ongoing and future trials using naturally processed peptides to induce tolerance to islet antigens in pre-diabetic or recent-onset diabetic patients.

Lay Abstract for project 4

Objective

This core is designed to provide fresh blood samples from new onset T1DM subjects and appropriately matched control subjects for NPPE-related studies at the London laboratory (Projects 1) using a same-day courier service.

Accomplishments

The main focus during the first year of funding has been on further development of a system for provision of fresh blood samples to the London laboratory. The network for collection of samples from patients with newly diagnosed diabetes from hospitals in the area around Bristol and in South Wales has been enhanced by more frequent contact with the clinical teams in the satellite peripheral hospitals, and by recruitment of additional centers. A pool of more than 150 potential control subjects, genotyped for HLA class II, has been established to facilitate collection of fresh blood samples for this and related T cell studies. To date, 50 fresh blood samples from 35 patients with type 1 diabetes and 20 from controls have been collected and delivered by courier to Dr Peakman’s laboratory in London for use in Project 1

Publications/Presentations

No publications have arisen directly from this Core facility

Who benefits from the research

The research presented here supports the work outlined in the Projects described elsewhere. For details on the benefits, see the report of Project 1.

Future Plans

During the next year we propose to further extend our collection network and to explore the possibilities of setting up a register of newly diagnosed T1DM patients across the South West of England potentially interested in taking part in research studies with collection of serum and genetic material to allow genotyping and autoantibody testing. We will also continue to collect samples from newly diagnosed patients and controls using the current network.

Lay Abstract for project 5

Objective

Specific aims of the past period of the project was the generation of peptides that are derived from putative autoantigens in Type I Diabetes and are predicted to bind to HLA-A2. In addition, the aim was to generate long peptides derived from pre-proinsulin (PPI) and from some selected regions in three other putative autoantigens (i.e. GAD65, IGRP and Prepro-IAAP) to serve as starting materials for proteasome digestion. The aim of the proteasome studies was to determine cleavage sites and the peptide fragment pattern in the amino acid sequence of the autoantigen regions. The precise proteasome cleavage sites as well as the peptide fragments produced, are both important to predict HLA class I presented peptides and therewith potential CTL epitopes. In order to be able to test the translocation of proteasome generated peptides a TAP-translocation assay had to be introduced in our lab.

Accomplishments

In total 223 9-mer and 10-mer peptides were synthesized in order to determine their binding to HLA-A2. The binding studies with these peptides are in progress. Pre-pro-insulin was synthesized in overlapping peptides of 30 amino acids. Also some regions of GAD65, IGRP and Prepro-IAAP were synthesized. Two types of 20S proteasomes (i.e. immuno- and constitutive proteasomes) have been purified from the EBV cell line JY and the HELA cell line, respectively. The 8 long peptides were digested with both types of proteasomes and the peptide profile was identified. A TAP-translocation assay was developed in order to test the translocation of the peptides that possess the proper C-terminus.

Publications/Presentations

- Unger WWJ, Pinkse G, Mulder-vd Kracht S, vd Slik A, Ossendorp F, Serreze D, Drijfhout JW and Roep BO. IGRP is a novel target for autoreactive human and pathogenic murine CD8 T cells. Meeting abstract. Ann. NYAS (2006) - Herberts CA, Neijssen JJ, de Haan J, Janssen L, Drijfhout JW, Reits EA and Neefjes JJ. Cutting Edge: HLA-B27 acquires many N-terminal dibasic peptides: Coupling cytosolic peptide stability to antigen presentation. J Immunol 176, 2697-2701 (2006) - Van Hall T, Wolpert EZ, van Veelen P, Laban, S, van der Veer M, Roseboom M, Bres S, Grufman P, deRu A, Meiring H, de Jong A, Franken K, Teixeira A, Valentijn R, Drijfhout JW, Koning F, Camps M, Ossendorp F, Kärre K, Ljunggren H-G, Melief CJM and Offringa R. Selective cytotoxic T-lymphocyte targeting of tumor immune escape variants. Nat Med 12, 417-424 (2006) - Durinovic-Bello I, Rosinger S, Olson JA, Congia M, Ahmad RC, Rickert M, Hampl J, Kalbacher H, Drijfhout JW, Mellins ED, Al Dahouk S, KamradtT, Maeurer MJ, Nhan C, Roep BO, Boehm BO, Polychronakos C, Nepom GT, Karges W, McDevitt HO & Sønderstrup G. DRB1*0401-restricted human T cell clone specific for the major proinsulin73-90 epitope expresses a dow-regulatory T helper 2 phenotype. Proc. Natl. Acad. Sci. USA, 11683-11688 (2006)

Who benefits from the research

In first instance the investigators that work in the Projects that are supported by Core B benefit from this research. Subsequently, these results will contribute to the general knowledge on Type I Diabetes. The outcome of the project is the identification of epitopes that can be used to monitor Type I Diabetes in an early stage and can maybe lead to ways to prevent the progression to disease.

Future Plans

Synthesis of and binding experiments with peptides that are predicted to bind to HLA A1, A3/A11, A24, B7 and B8 are foreseen. In addition, based on the results of the research that is now in progress and will be initiated in the next year, proteasome digestions will be performed to evaluate whether the good binding peptides will and can be generated by proteasomal breakdown of the proteins they are drived from. Then various lenght variant of these peptides will be generated in order to evaluate the TAP-translocation using our newly developed translocation assay. Finally, HLA-tetramers will be generated in order to evaluate whether T cells that recognize these potential epitopes are present in patient material (for details see also the future plans of the Projects).

Lay Abstract for project 6

Objective

This core is designed to provide fresh blood samples from approximately 40 new onset T1DM subjects and 10 siblings per year for T cell studies at the London laboratory (Project 1) using a same-day courier service.

Background/Rationale

T cell studies are complex: importantly, they require regular samples of fresh blood from well-defined cohorts of patients. Single laboratory centers may find this difficult, and sufficient numbers of patients may take several years to accumulate. An additional logistical problem for T cell studies arises when the laboratory expertise (in this case in London) is remote from the clinical experts (in this case in Bristol). In recent years we have addressed this potential limitation to T cell studies through the creation of a case collection network, based around a large, stable UK population that employs a nurse fieldworker to facilitate sample collection and rapid transportation. This Clinical Core is designed to extend our current collection capability to obtain blood samples from a specified number of cases for experimentation in Project 1.

Description of Project

This core is the product of an ongoing collaboration between clinicians and scientists in London and Bristol, UK, with established programs of work relating to the study of Type 1 diabetes. This Clinical Core will take advantage of an existing network of local hospitals covering a large UK population, which would then feed the laboratory centers. These centers include 14 local hospitals in the South West of England and South Wales and cover a potential sampling population of over 4 million people. At current rates of type 1 diabetes incidence in the target age range in this geographical region, the expected total number of new cases in the region covered by the network is in excess of 200 per year. We plan to extend and intensify our recruitment within the network.

Anticipated Outcome

A system for provision of fresh blood samples to the London laboratory from patients with newly diagnosed type 1 diabetes and relatives

Relevance to Type I Diabetes

The immune cell thought to be most important in the development of Type 1 diabetes is the T cell. The systematic study of T cells that react against healthy islets is the major thrust of the Program Project of which this is the Clinical Core.

Lay Abstract for project 7

Objective

The PCCL (Protein Chemistry Core Leiden) will help the projects to perform their research by providing the specialized technolgy and know-how normally not available in immunology laboratories.

Background/Rationale

PCCL has extensive experience in the fields of peptide and protein synthesis as well as in the area of molecular techniques to identify epitopes. Especially the accessibility to expensive state-of-the-art equipment for peptide synthesis and mass spectrometry will serve as important support for the projects.

Description of Project

Various materials such as synthetic peptides (small protein fragments) and whole proteins will be made. These reagents are not commercially available and will be specially made for the projects. In addition, mass spectrometry will be used to identify peptides that might be involved in the onset of type I diabetes. Our new mass spectrometry equipment is ideally suited for this purpose because it is very sensitive and accurate. Also, our experience in the analysis of proteasome digestion of proteins will be made available to the projects. These digestions normally take place within the cell and can be mimicked in our lab in small reaction tubes using the purified enzyme system.

Anticipated Outcome

The outcome of our efforts is the support of the projects by providing them with relevant research materials. In addition, the results of our mass spectrometry analyses and digestion assays will support the projects to focuss on epitopes that are relevant for understanding the molecular basis of the onset of type I diabetes.

Relevance to Type I Diabetes

The products produced and analyses performed focus on ¿ cell autoantigens and are thus highly relevant to type I diabetes. See for further information under the specific projects.